U.S. patent application number 10/671859 was filed with the patent office on 2005-05-26 for bacillus stearothermophilus tau, delta, and delta prime polymerase subunits and use thereof.
Invention is credited to Bruck, Irina, Jeruzalmi, David, Kuriyan, John, O'Donnell, Michael E., Yurieva, Olga, Yuzhakov, Alexander.
Application Number | 20050112580 10/671859 |
Document ID | / |
Family ID | 34595732 |
Filed Date | 2005-05-26 |
United States Patent
Application |
20050112580 |
Kind Code |
A1 |
O'Donnell, Michael E. ; et
al. |
May 26, 2005 |
Bacillus stearothermophilus tau, delta, and delta prime polymerase
subunits and use thereof
Abstract
The present invention relates to an isolated DNA molecule from a
thermophilic bacterium which encodes a DNA polymerase III-type
enzyme subunit. Also encompassed by the present invention are host
cells and expression system including the heterologous DNA molecule
of the present invention, as well as isolated replication enzyme
subunits encoded by such DNA molecules. Also disclosed is a method
of producing a recombinant thermostable DNA polymerase III-type
enzyme, or subunit thereof, from a thermophilic bacterium, which is
carried out by transforming a host cell with at least one
heterologous DNA molecule of the present invention under conditions
suitable for expression of the DNA polymerase III-type enzyme, or
subunit thereof, and then isolating the DNA polymerase III-type
enzyme, or subunit thereof.
Inventors: |
O'Donnell, Michael E.;
(Hastings-on-Hudson, NY) ; Yuzhakov, Alexander;
(Malden, MA) ; Yurieva, Olga; (New York, NY)
; Jeruzalmi, David; (Cambridge, MA) ; Bruck,
Irina; (New York, NY) ; Kuriyan, John;
(Berkeley, CA) |
Correspondence
Address: |
Nixon Peabody LLP
Clinton Square
P.O. Box 31051
Rochester
NY
14603-1051
US
|
Family ID: |
34595732 |
Appl. No.: |
10/671859 |
Filed: |
September 26, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10671859 |
Sep 26, 2003 |
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09716964 |
Nov 21, 2000 |
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09716964 |
Nov 21, 2000 |
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09642218 |
Aug 18, 2000 |
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09642218 |
Aug 18, 2000 |
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09057416 |
Apr 8, 1998 |
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60043202 |
Apr 8, 1997 |
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Current U.S.
Class: |
435/6.18 ;
435/199; 435/252.31; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12Q 1/689 20130101;
C07H 21/04 20130101; C12N 9/1252 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/199; 435/252.31; 435/320.1; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/22; C12N 001/21; C12N 015/74 |
Goverment Interests
[0002] The present invention was made with funding from National
Institutes of Health Grant No. GM38839. The United States
Government, may have certain rights in this invention.
Claims
What is claimed:
1. An isolated Bacillus delta or delta prime or tau subunit of a
DNA polymerase III-type enzyme, the isolated delta or delta prime
or tau subunit: (i) comprising the amino acid sequence of SEQ ID
NO: 178 or 180 or 182; or (ii) being encoded by a nucleic acid
molecule hybridizing to the complement of SEQ ID NO: 177 or 179 or
181 under hybridization conditions comprising at most about 0.9M
sodium citrate buffer at a temperature of at least about 37.degree.
C.
2. The isolated Bacillus delta or delta prime or tau subunit
according to claim 1 wherein the Bacillus species is Bacillus
stearothermophilus.
3. The isolated Bacillus delta or delta prime or tau subunit
according to claim 1 wherein the delta subunit comprises the amino
acid sequence of SEQ ID NO: 178.
4. The isolated Bacillus delta or delta prime or tau subunit
according to claim 1 wherein the delta prime subunit comprises the
amino acid sequence of SEQ ID NO: 180.
5. The isolated Bacillus delta or delta prime or tau subunit
according to claim 1 wherein the tau subunit comprises the amino
acid sequence of SEQ ID NO: 182.
6. The isolated Bacillus delta or delta prime or tau subunit
according to claim 1 wherein the delta or delta prime or tau
subunit is encoded by a nucleic acid molecule that hybridizes to
the complement of SEQ ID NO: 177 or 179 or 181 under hybridization
conditions comprising at most about 0.9M sodium citrate buffer at a
temperature of at least about 37.degree. C.
7. The isolated Bacillus delta or delta prime or tau subunit
according to claim 1 wherein the subunit is purified.
8. A clamp loader complex comprising at least one of the Bacillus
delta or delta prime or tau subunits according to claim 1.
9. A clamp loader complex comprising the Bacillus delta, delta
prime, and tau subunits according to claim 1.
10. A DNA polymerase III-type enzyme complex comprising the clamp
loader according to claim 8.
11. A DNA polymerase III-type enzyme complex comprising the clamp
loader according to claim 9.
12. A kit comprising: a container that contains therein either a
deoxynucleoside triphosphate or a dideoxynucleoside triphosphate;
and a container that contains therein the DNA polymerase III-type
enzyme complex according to claim 10.
13. A kit comprising: a container that contains therein either a
deoxynucleoside triphosphate or a dideoxynucleoside triphosphate;
and a container that contains therein the DNA polymerase III-type
enzyme complex according to claim 11.
Description
[0001] The present application is a continuation of U.S. patent
application Ser. No. 09/716,964, filed Nov. 21, 2000, which is a
continuation-in-part of U.S. patent application Ser. No.
09/642,218, filed Aug. 18, 2000, as a continuation of U.S. patent
application Ser. No. 09/057,416 filed Apr. 8, 1998, which claims
the benefit of U.S. Provisional Patent Application Ser. No.
60/043,202 filed Apr. 8, 1997, all of which are hereby incorporated
by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to thermostable DNA
polymerases and, more particularly, to such polymerases as can
serve as chromosomal replicases and are derived from thermophilic
bacteria. More particularly, the invention extends to DNA
polymerase III-type enzymes from thermophilic bacteria, including
Aquifex aeolicus, Thermus thermophilus, Thermotoga maritima, and
Bacillus stearothermophilus, as well as purified, recombinant or
non-recombinant subunits thereof and their use, and to isolated DNA
coding for such polymerases and their subunits. Such DNA is
obtained from the respective genes (e.g., dnaX, holA, holB, dnaA,
dnaN, dnaQ, dnaE, ssb, etc.) of various thermophilic eubacteria,
including but not limited to Thermus thermophilus, Aquifex
aeolicus, Thermotoga maritima, and Bacillus stearothermophilus.
BACKGROUND OF THE INVENTION
[0004] Thermostable DNA polymerases have been disclosed previously
as set forth in U.S. Pat. No. 5,192,674 to Oshima et al., U.S. Pat.
Nos. 5,322,785 and 5,352,770 to Comb et al., U.S. Pat. No.
5,545,552 to Mathur, and others. All of the noted references recite
the use of polymerases as important catalytic tools in the practice
of molecular cloning techniques such as polymerase chain reaction
(PCR). Each of the references states that a drawback of the extant
polymerases are their limited thermostability, and consequent
useful life in the participation in PCR. Such limitations also
manifest themselves in the inability to obtain extended lengths of
nucleotides, and in the instance of Taq polymerase, the lack of 3'
to 5' exonuclease activity, and the drawback of the inability to
excise misinserted nucleotides (Perrino, 1990).
[0005] More generally, such polymerases, including those disclosed
in the referenced patents, are of the Polymerase I variety as they
are often 90-95 kDa in size and may have 5' to 3' exonuclease
activity. They define a single subunit with concomitant limits on
their ability to hasten the amplification process and to promote
the rapid preparation of longer strands of DNA.
[0006] Chromosomal replicases are composed of several subunits in
all organisms (Kornberg and Baker, 1992). In keeping with the need
to replicate long chromosomes, replicases are rapid and highly
processive multiprotein machines. Cellular replicases are
classically comprised of three components: a clamp, a clamp loader,
and the DNA polymerase (reviewed in Kelman and O'Donnell, 1995;
McHenry, 1991). For purposes of the present invention, the
foregoing components also serve as a broad definition of a "Pol
III-type enzyme".
[0007] DNA polymerase III holoenzyme (Pol III holoenzyme) is the
multi-subunit replicase of the E. coli chromosome. Pol III
holoenzyme is distinguished from Pol I type DNA polymerases by its
high processivity. (>50 kbp) and rapid rate of synthesis (750
nts/s) (reviewed in Kornberg and Baker, 1992; Kelman and O'Donnell,
1995). The high processivity and speed is rooted in a ring shaped
subunit, called .beta., that encircles DNA and slides along it
while tethering the Pol III holoenzyme to the template (Stukenberg
et al., 1991; Kong et al., 1992). The ring shaped .beta. clamp is
assembled around DNA by the multisubunit clamp loader, called
.gamma. complex. The .gamma. complex couples the energy of ATP
hydrolysis to the assembly of the .beta. clamp onto DNA. This
.gamma. complex, which functions as a clamp loader, is an integral
component of the Pol III holoenzyme particle. A brief overview of
the organization of subunits within the holoenzyme and their
function follows.
[0008] Pol III holoenzyme consists of 10 different subunits, some
of which are present in multiple copies for a total of 18
polypeptide chains (Onrust et al., 1995). The organization of these
subunits in the holoenzyme particle is illustrated in FIG. 1. As
depicted in the diagram, the subunits of the holoenzyme can be
grouped functionally into three components: 1) the DNA polymerase
III core is the catalytic unit and consists of the .alpha. (DNA
polymerase), .epsilon. (3'-5' exonuclease), and .theta. subunits
(McHenry and Crow, 1979), 2) the .beta. "sliding clamp" is the ring
shaped protein that secures the core polymerase to DNA for
processivity (Kong et al., 1992), and 3) the 5 protein .gamma.
complex (.gamma..delta..delta.'.chi..psi.) is the "clamp loader"
that couples ATP hydrolysis to assembly of .beta. clamps around DNA
(O'Donnell, 1987; Maki et al., 1988). A dimer of the .tau. subunit
acts as a "macromolecular organizer" holding together two molecules
of core (Studwell-Vaughan and O'Donnell, 1991; Low et al., 1976)
and one molecule of .gamma. complex forming the Pol III*
subassembly (Onrust et al., 1995). This organizing role of .tau. to
form Pol III* is indicated in the center of FIG. 1. Two .beta.
dimers associate with the two cores within Pol III* to form the
holoenzyme, which is capable of replicating both strands of duplex
DNA simultaneously (Maki et al., 1988).
[0009] The DNA polymerase III holoenzyme assembles onto a primed
template in two distinct steps. In the first step, the .gamma.
complex assembles the .beta. clamp onto the DNA. The .gamma.
complex and the core polymerase utilize the same surface of the
.beta. ring and they cannot both utilize it at the same time
(Naktinis et al., 1996). Hence, in the second step the .gamma.
complex moves away from .beta. thus allowing access of the core
polymerase to the .beta. clamp for processive DNA synthesis. The
.gamma. complex and core remain attached to each other during this
switching process by the .tau.subunit organizer.
[0010] The .gamma. complex consists of 5 different subunits
(.gamma..sub.2-4.delta..sub.1.delta.'.sub.1.chi..sub.1.psi..sub.1).
An overview of the mechanism of the clamp loading process follows.
The .delta. subunit is the major touch point to the .beta. clamp
and leads to ring opening, but .delta. is buried within .gamma.
complex such that contact with .beta. is prevented (Naktinis et
al., 1995). The .gamma. subunit is the ATP interactive protein but
is not an ATPase by itself (Tsuchihashi and Kornberg, 1989). The
.delta.' subunit bridges the .delta. and .gamma. subunits resulting
in a .gamma..delta..delta.' complex that exhibits DNA dependent
ATPase activity and is competent to assemble clamps on DNA (Onrust
et al., 1991). Upon binding of ATP to .gamma., a change in the
conformation of the complex exposes .delta. for interaction with
.beta. (Naktinis et al., 1995). The function of the smaller
subunits, .chi. and .psi., is to contact SSB (through .chi.) thus
promoting clamp assembly and high processivity during replication
(Kelman and O'Donnell, 1995).
[0011] The three component Pol III-type enzyme in eukaryotes
contains a clamp that has the same shape as E. coli .beta., but
instead of a homodimer it is a heterotrimer. This heterotrimeric
ring, called PCNA (proliferating cell nuclear antigen), has 6
domains like .beta., but instead of each PCNA monomer being
composed of 3 domains and dimerizing to form a 6 domain ring (e.g.,
like .beta.), the PCNA monomer has 2 domains and it trimerizes to
form a 6 domain ring (Krishna et al., 1994; Kuriyan and O'Donnell,
1993). The chain fold of the domains are the same in prokaryotes
(.beta.) and eukaryotes (PCNA); thus, the rings have the same
overall 6-domain ring shape. The clamp loader of the eukaryotic Pol
III-type replicase is called RFC (Replication factor C) and it
consists of subunits having homology to the .gamma. and .delta.'
subunits of the E. coli .gamma. complex (Cullmann et al., 1995).
The eukaryotic DNA polymerase III-type enzyme contains either of
two DNA polymerases, DNA polymerase .delta. and DNA polymerase
.epsilon. (Bambara and Jessee, 1991; Linn, 1991; Sugino, 1995). It
is entirely conceivable that yet other types of DNA polymerases can
function with either a PCNA or .beta. clamp to form a Pol III-type
enzyme (for example, DNA polymerase II of E. coli functions with
the .beta. subunit placed onto DNA by the .gamma. complex clamp
loader) (Hughes et al., 1991; Bonner et al., 1992). The
bacteriophage T4 also utilizes a Pol III-type 3-component
replicase. The clamp is a homotrimer like PCNA, called gene 45
protein (Young et al., 1992). The gene 45 protein forms the same
6-domain ring structure as .beta. and PCNA (Moarefi et al., 2000).
The clamp loader is a complex of two subunits called the gene 44/62
protein complex. The DNA polymerase is the gene 43 protein and it
is stimulated by the gene 45 sliding clamp when it is assembled
onto DNA by the 44/62 protein clamp loader. The Pol III-type enzyme
may be either bound together into one particle (e.g., E. coli Pol
III holoenzyme), or its three components may function separately
(like the eukaryotic Pol III-type replicases).
[0012] There is an early report on separation of three DNA
polymerases from T.th. cells, however each polymerase form was
reminiscent of the preexisting types of DNA polymerase isolated
from thermophiles in that each polymerase was in the
110,000-120,000 range and lacked 3'-5' exonuclease activity
(Ruttimann et al., 1985). These are well below the molecular weight
of Pol III-type complexes that contain in addition to the DNA
polymerase subunit, other subunits such as .gamma. and .tau..
Although the three polymerases displayed some differences in
activity (column elution behavior, and optimum divalent cation,
template, and temperatures) it seems likely that these three forms
were either different repair type polymerases or derivatives of one
repair enzyme (e.g., Pol I) that was modified by post translational
modification(s) that altered their properties (e.g.
phosphorylation, methylation proteolytic clipping of residues that
alter activity, or association with different ligands such as a
small protein or contaminating DNA). Despite this previous work, it
remained to be demonstrated that thermophiles harbor a Pol III-type
enzyme that contain multiple subunits such as .gamma. and/or .tau.,
functioned with a sliding clamp accessory protein, or could extend
a primer rapidly and processively over a long stretch (>5 kb) of
ssDNA (Ruttimann et al., 1985).
[0013] Previously, it was not known what polymerase thermophilic
bacteria used to replicate their chromosome since only Pol I type
enzymes have been reported from thermophiles. By distinction,
chromosomal replicases, such as Polymerase III, identified in E.
coli, if available in a thermostable bacterium, with all its
accessory subunits, could provide a great improvement over the
Polymerase I type enzymes, in that they are generally much more
efficient--about 5 times faster--and much more highly processive.
Hence, one may expect faster and longer chain production in PCR,
and higher quality of DNA sequencing ladders. Clearly, the ability
to practice such synthetic techniques as PCR would be enhanced by
these methods disclosed for how to obtain genes and subunits of DNA
polymerase III holoenzyme from thermophilic sources.
[0014] The present invention is directed to achieving these
objectives and overcoming the various deficiencies in the art.
SUMMARY OF THE INVENTION
[0015] In accordance with the present invention, DNA Polymerase.
III-type enzymes as defined herein are disclosed that may be
isolated and purified from a thermophilic bacterial source, that
display rapid synthesis characteristic of a chromosomal replicase,
and that possesses all of the structural and processive advantages
sought and recited above. More particularly, the invention extends
to thermostable Polymerase III-type enzymes derived from
thermophilic bacteria that exhibit the ability to extend a primer
over a long stretch (>5 kb) of ssDNA at elevated temperature,
the ability to be stimulated by a cognate sliding clamp (e.g.,
.beta.) of the type that is assembled on DNA by a `clamp` loader
(e.g., .gamma. complex), and have clamp loading subunits that show
DNA stimulated ATPase activity at elevated temperature and/or ionic
strength. Representative thermophile polymerases include those
isolated from the thermophilic eubacteria Aquifex aeolicus (A.ae.
polymerase) and other members of the Aquifex genus; Thermus
thermophilus (T.th. polymerase), Thermus favus (Tfl/Tub
polymerase), Thermus ruber (Tru polymerase), Thermus brockianus
(DYNAZYME.TM. polymerase), and other members of the Thermus genus;
Bacillus stearothermophilus (B.st. polymerase) and other members of
the Bacillus genus; Thermoplasma acidophilum (Tac polymerase) and
other members of the Thermoplasma genus; and Thermotoga neapolitana
(Tne polymerase; see WO 96/10640 to Chatterjee et al.), Thermotoga
maritima (Tma polymerase; see U.S. Pat. No. 5,374,553 to Gelfand et
al.), and other species of the Thermotoga genus (Tsp polymerase).
In a preferred embodiment, the thermophilic bacteria comprise
species of Aquifex, Thermus, Bacillus, and Thermotoga, and
particularly A.ae., T.th., B.st., and Tma.
[0016] A particular Polymerase III-type enzyme in accordance with
the invention may include at least one of the following
sub-units:
[0017] A. a .gamma. subunit having an amino acid sequence
corresponding to SEQ. ID. Nos. 4 or 5 (T.th.);
[0018] B. a .tau. subunit having an amino acid sequence
corresponding to SEQ. ID. No. 2 (T.th.), SEQ. ID. No. 120 (A.ae.),
SEQ. ID. No. 142 (T.ma.) or SEQ. ID. No. 182 (B.st.);
[0019] C. a .epsilon. subunit having an amino acid sequence
corresponding to SEQ. ID. No. 95 (T.th.), SEQ. ID. No. 128 (A.ae.),
or SEQ. ID. No. 140 (T.ma.);
[0020] D. a .alpha. subunit including an amino acid sequence
corresponding to SEQ. ID. No. 87 (T.th.), SEQ. ID. No. 118 (A.ae.),
SEQ. ID. No. 138 (T.ma.), or SEQ. ID. Nos. 184 (PolC which has both
.alpha. and .epsilon. activity, B.st.);
[0021] E. a .beta. subunit having an amino acid sequence
corresponding to SEQ. ID. No. 107 (T.th.), SEQ. ID. No. 122
(A.ae.), SEQ. ID. No. 144 (T.ma.), or SEQ. ID. No. 174 (B.st.);
[0022] F. a .delta. subunit having an amino acid sequence
corresponding to SEQ. ID. No. 158 (T.th.), SEQ. ID. No. 124
(A.ae.), SEQ. ID. No. 146 (T.ma.) or SEQ. ID. No. 178 (B.st.);
[0023] G. a .delta.' subunit having an amino acid sequence
corresponding to SEQ. ID. No. 156 (T.th.), SEQ. ID. No. 126
(A.ae.), SEQ. ID. No. 148 (T.ma.) or SEQ. ID. No. 180 (B.st.);
[0024] variants, including allelic variants, muteins, analogs and
fragments of any of subparts (A) through (G), and compatible
combinations thereof, capable of functioning in DNA amplification
and sequencing.
[0025] The invention also extends to the genes that correspond to
and can code on expression for the subunits set forth above, and
accordingly includes the following: dnaX, holA, holB, dnaQ, dnaE,
dnaN, and ssb, as well as conserved variants and active fragments
thereof.
[0026] Accordingly, the Polymerase III-type enzyme of the present
invention comprises at least one gene encoding a subunit thereof,
which gene is selected from the group consisting of dnaX, holA,
holB, dnaQ, dnaE and dnaN, and combinations thereof. More
particularly, the invention extends to the nucleic acid molecule
encoding the .gamma. and .tau. subunits, and includes the dnaX gene
which has a nucleotide sequence as set forth herein, as well as
conserved variants, active fragments and analogs thereof. Likewise,
the nucleotide sequences encoding the .alpha. subunit (dnaE gene),
the .epsilon. subunit (dnaQ gene), the .beta. subunit (dnaN gene),
the .delta. subunit (holA gene), and the .delta.' subunit (holB
gene) each comprise the nucleotide sequences as set forth herein,
as well as conserved variants, active fragments and analogs
thereof. Those nucleotide sequences for T.th. are as follows: dnaX
(SEQ. ID. No. 3), dnaE (SEQ. ID. No. 86), dnaQ (SEQ. ID. No. 94),
dnaN (SEQ. ID. No. 106), holA (SEQ. ID. No. 157), and holB (SEQ.
ID. No. 155). Those nucleotide sequences for A.ae. are as follows:
dnaX (SEQ. ID. No. 119), dnaE (SEQ. ID. No. 117), dnaQ (SEQ. ID.
No. 127), dnaN (SEQ. ID No. 121), holA (SEQ. ID. No. 123), and holB
(SEQ. ID. No. 125). Those nucleotide sequences for T.ma. are as
follows: dnaX (SEQ. ID. No. 141), dnaE (SEQ. ID. No. 137), dnaQ
(SEQ. ID. No. 139), dnaN (SEQ. ID. No. 143), holA (SEQ. ID. No.
145), and holB (SEQ. ID. No. 147). Those nucleotide sequences for
B.st. are as follows: dnaX (SEQ. ID. No. 181), polC (SEQ. ID. Nos.
183), dnaN (SEQ. ID. No. 173), holA (SEQ. ID. No. 177), and holB
(SEQ. ID. No. 179).
[0027] The invention also provides methods and products for
identifying, isolating and cloning DNA molecules which encode such
accessory subunits encoded by the recited genes of the DNA
polymerase III-type enzyme hereof.
[0028] Yet further, the invention extends to Polymerase III-type
enzymes prepared by the purification of an extract taken from,
e.g., the particular thermophile under examination, treated with
appropriate solvents and then subjected to chromatographic
separation on, e.g., an anion exchange column, followed by analysis
of long chain synthetic ability or Western analysis of the
respective peaks against antibody to at least one of the
anticipated enzyme subunits to confirm presence of Pol III, and
thereafter, peptide sequencing of subunits that co purify and
amplification to obtain the putative gene and its encoded
enzyme.
[0029] The present invention also relates to recombinant .gamma.,
.tau., .epsilon., .alpha. (as well as PolC), .delta., .delta.' and
.beta. subunits and SSB from thermophiles. In the instance of the
.gamma. and .tau. subunits of T.th., the invention includes the
characterization of a frameshifting sequence that is internal to
the gene and specifies relative abundance of the .gamma. and .tau.
gene products of T.th. dnaX. From this characterization, expression
of either one of the subunits can be increased at the expense of
the other (i.e. mutant frame shift could make all .tau., simple
recloning at the end of the frameshift could make exclusively
.gamma. and no .tau.).
[0030] In a further aspect of the present invention, DNA probes can
be constructed from the DNA sequences coding for, e.g., the T.th.,
A.ae., T.ma., or B.st. dnaX, dnaQ, dnaE, dnaA, dnaN, holA, holB,
and ssb genes, conserved variants and active fragments thereof, all
as defined herein, and may be used to identify and isolate the
corresponding genes coding for the subunits of DNA polymerase III
holoenzyme from other thermophiles, such as those listed earlier
herein. Accordingly; all chromosomal replicases (DNA Polymerase
III-type) from thermophilic sources are contemplated and included
herein.
[0031] The invention also extends to methods for identifying
Polymerase III-type enzymes by use of the techniques of long-chain
extension and elucidation of subunits with antibodies, as described
herein and with reference to the examples.
[0032] The invention further extends to the isolated and purified
DNA Polymerase III from T.th., A.ae., T.ma., and B.st., the amino
acid sequences of the .gamma., .tau., .epsilon., .alpha. (as well
as PolC), .delta., .delta.', and .beta. subunits and SSB, as set
forth herein, and the nucleotide sequences of the corresponding
genes from T.th., A.ae., T.ma., or B.st. set forth herein, as well
as to active fragments thereof, oligonucleotides and probes
prepared or derived therefrom and the transformed cells that may be
likewise prepared. Accordingly, the invention comprises the
individual subunits enumerated above and hereinafter, corresponding
isolated polynucleotides and respective amino acid sequences for
each of the .gamma., .tau., .epsilon., .alpha. (as well as PolC),
.delta., .delta.', and .beta. subunits and SSB, and to conserved
variants, fragments, and the like, as well as to methods of their
preparation and use in DNA amplification and sequencing. In a
particular embodiment, the invention extends to vectors for the
expression of the subunit genes of the present invention.
[0033] The invention also includes methods for the preparation of
the DNA Polymerase III-type enzymes and the corresponding subunit
genes of the present invention, and to the use of the enzymes and
constructs having active fragments thereof, in the preparation,
reconstitution or modification of like enzymes, as well as in
amplification and sequencing of DNA by methods such as PCR, and
like protocols, and to the DNA molecules amplified and sequenced by
such methods. In this regard, a Pol III-type enzyme that is
reconstituted in the absence of .epsilon., or using a mutated
.epsilon. with less 3'-5' exonuclease activity, may be a superior
enzyme in either PCR or DNA sequencing applications, (e.g. Tabor
et. al., 1995).
[0034] The invention is directed to methods for amplifying and
sequencing a DNA molecule, particularly via the polymerase chain
reaction (PCR), using the present DNA polymerase III-type enzymes
or complexes. In particular, the invention extends to methods of
amplifying and sequencing of DNA using thermostable pol III-type
enzyme complexes isolated from thermophilic bacteria such as
Thermotoga and Thermus species, or recombinant thermostable
enzymes. The invention also provides amplified DNA molecules made
by the methods of the invention, and kits for amplifying or
sequencing a DNA molecule by the methods of the invention.
[0035] In this connection, the invention extends to methods for
amplification of DNA that can achieve long chain extension of
primed DNA, as by the application and use of Polymerase III-type
enzymes of the present invention. An illustration of such methods
is presented in Examples 15 and 16, infra.
[0036] Likewise, kits for amplification and sequencing of such DNA
molecules are included, which kits contain the enzymes of the
present invention, including subunits thereof, together with other
necessary or desirable reagents and materials, and directions for
use. The details of the practice of the invention as set forth
above and later on herein, and with reference to the patents and
literature cited herein, are all expressly incorporated herein by
reference and made a part hereof.
[0037] As stated, and in accordance with a principal object of the
present invention, Polymerase III-type enzymes and their sub-units
are provided that are derived from thermophiles and that are
adapted to participate in improved DNA amplification and sequencing
techniques, and the consequent ability to prepare larger DNA
strands more rapidly and accurately.
[0038] It is a further object of the present invention to provide
DNA molecules that are amplified and sequenced using the Polymerase
III-type enzymes hereof.
[0039] It is a still further object of the present invention to
provide enzymes and corresponding methods for amplification and
sequencing of DNA that can be practiced without the participation
of the clamp-loading component of the enzyme.
[0040] It is a still further object of the present invention to
provide kits and other assemblies of materials for the practice of
the methods of amplification and sequencing as aforesaid, that
include and use the DNA polymerase III-type enzymes herein as part
thereof.
[0041] One goal of this invention is to fully reconstitute the
rapid and processive replicase from an extreme thermophilic
eubacterium from fully recombinant protein subunits. One might
think that the extreme heat in which these bacteria grow may have
resulted in a completely different solution to the problem of
chromosome replication. Prior to filing of the
previously-identified priority applications, it is believed that
Pol III had not been identified in any thermophile until the
present inventors found that Thermus thermophilus, which grows at a
rather high temperature of 70-80.degree. C., would appear to
contain a Pol III. Subsequent to this invention, the genome
sequence of A. aeolicus was published which shows dnaE, dnaN, and
dnaX genes. However, previous work did not fully reconstitute the
working replication machinery from fully recombinant subunits. A
holA gene and holB has not been identified previously in T.
thermophilus or A. aeolicus, and studies in the E. coli system show
that delta and delta prime, encoded by holA and holB, respectively,
are essential to loading the beta clamp onto DNA and, thus, is
essential for rapid and processive holoenzyme function (U.S. Pat.
Nos. 5,583,026 and 5,668,004 to O'Donnell, which are hereby
incorporated by reference).
[0042] This invention fully reconstitutes a functional DNA
polymerase III holoenzyme from the extreme thermophiles Thermus
thermophilus and Aquifex aeolicus. Aquifex aeolicus grows at an
even higher temperature than Thermus thermophilus, up to 85.degree.
C. In this invention, the genes of Thermus thermophilus, Aquifex
aeolicus, Thermotoga maritima, and Bacillus stearothermophilus that
are necessary to reconstitute the complete DNA polymerase III
machinery, which acts as a rapid and processive polymerase, are
identified. Indeed, a delta prime (holB) and delta (holA) subunits
are needed.
[0043] The dnaE, dnaN, dnaX, dnaQ, holA, and holB genes are used to
express and purify the protein "gears", and the proteins are used
to reassemble the replication machine. The T.th. Pol III is similar
to E. coli. The A.ae. Pol III is slightly dissimilar from the
machinery of previously studied replicases. The A.ae. dnaX gene
encoded only one protein, tau, and in this fashion is similar to
the dnaX of the gram positive organism, Staphylococcus aureus. In
contrast, the dnaX of the gram negative cell, E. coli, produces two
proteins. The Aquifex aeolicus polymerase subunit, alpha (encoded
by dnaE) does not contain the 3'-5' proofreading exonuclease. In
this regard, A. aeolicus is similar to E. coli, but dissimilar to
the replicase of the gram positive organisms. In Gram positive
organisms, the PolC polymerase subunit of the replicase contains
the exonuclease activity in the same polypeptide chain as the
polymerase (Low et al., 1976, Barnes et al., 1994; Pacitti et al.,
1995). Further, the polymerase III of thermophilic bacteria retains
activity at high temperature.
[0044] Thermostable rapid and processive three component DNA
polymerases can be applied to several important uses. DNA
polymerases currently in use for DNA sequencing and DNA
amplification use enzymes that are much slower and thus could be
improved upon. This is especially true of amplification as the
three component polymerase is capable of speed and high
processivity making possible amplification of very long (tens of Kb
to Mb) lengths of DNA in a time-efficient, manner. These three
component polymerases also function in conjunction with a
replicative helicase (DnaB), and thus are capable of amplification
at a single temperature, using the helicase to melt the DNA duplex.
This property could be useful in some methods of amplification, and
in polymerase chain reaction (PCR) methodology. For example, the
.alpha..tau..delta..delta.'/.beta. form of the E. coli DNA
polymerase III holoenzyme has been shown to function in both DNA
sequencing and PCR (U.S. Pat. Nos. 5,583,026 and 5,668,004 to
O'Donnell).
[0045] Other objects and advantages will become apparent from a
review of the ensuing description which proceeds with reference to
the following illustrative drawings.
DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 is a schematic depiction of the structure and
components of enzymes of the general family to which the enzymes of
the present invention belong.
[0047] FIG. 2 is an alignment of the N-terminal regions of E. coli
(SEQ. ID. No. 19) and B. subtilis (SEQ. ID. No. 20) dnaX gene
product. Asterisks indicate identities. The ATP binding consensus
sequence is indicated. The two regions used for PCR primer design
are shown in bold.
[0048] FIG. 3 is an image showing the Southern analysis of T.
thermophilus genomic DNA. Genomic DNA was analyzed for presence of
the dnaZ gene using the PCR radiolabeled probe. Enzymes used for
digestion are shown above each lane. The numbering to the right
corresponds to the length of DNA fragments (kb).
[0049] FIGS. 4A and 4B depict the full sequence of the dnaX gene of
T. thermophilus. DNA sequence (upper case, and corresponding to SEQ
ID No. 1) and predicted amino acid sequence (lower case, and
corresponding to SEQ ID No. 2) yields a 529 amino acid protein
(.tau.) of 58.0 kDa. A putative frameshifting sequence containing
several A residues 1478-1486 (underlined) may produce a smaller
protein (.gamma.) of 49.8 kDa. The potential Shine-Dalgarno (S.D.)
signal is bold and underlined. The start codon is in bold, and the
stop codon for .tau. is marked by an asterisk. The potential stop
codon for .gamma. is shown in bold after the frameshift site, and
two potential Shine-Dalgarno sequences upstream of the frameshift
site are indicated. Sequences of the primers used for PCR are shown
in italics above the nucleotide sequence of dnaX. The ATP binding
site is indicated, and the asterisks above the four Cys residues
near the ATP site indicate the putative Zn.sup.2+ finger. The
proline rich area is indicated above the sequence. Numbering of the
nucleotide sequence is presented to the right. Numbering of the
amino acid sequence of .tau. is shown in parenthesis to the
right.
[0050] FIG. 4C depicts the isolated DNA coding sequence for the
dnaX gene (also present in FIGS. 3A and 3B) in accordance with the
invention, which corresponds to SEQ. ID. No. 3.
[0051] FIG. 4D depicts the polypeptide sequence of the .gamma.
subunit of the Polymerase III of the present invention, which
corresponds to SEQ. ID. No. 4.
[0052] FIG. 4E depicts the polypeptide sequence of the .gamma.
subunit of the Polymerase III of the present invention defined by a
-1 frameshift, which corresponds to SEQ. ID. No. 4.
[0053] FIG. 4F depicts the polypeptide sequence of the .gamma.
subunit of the Polymerase III of the present invention defined by a
-2 frameshift, which corresponds to SEQ. ID. No. 5.
[0054] FIGS. 5A-B are alignments of the .gamma./.tau. ATP binding
domains for different bacteria. Dots indicate those residues that
are identical to the E. coli dnaX sequence. The ATP consensus site
is underlined; and the conserved cysteine residues that form the
zinc finger are indicated with asterisks. E. coli, Escherichia coli
(SEQ. ID. No. 21); H. inf., Haemophilus influenzae (SEQ. ID. No.
22); B. sub., Bacillus subtilis (SEQ. ID. No. 23); C. cres.,
Caulobacter crescentus (SEQ. ID. No. 24); M. gen., Mycoplasma
genitalium (SEQ; ID. No. 25); T.th., Thermus thermophilus (SEQ. ID.
No. 26). Alignments were produced using Clustal.
[0055] FIG. 6 is a diagram indicating a signal for ribosomal
frameshifting in T.th. dnaX. The diagram shows part of the sequence
of the RNA (SEQ. ID. No. 27.degree. around the frameshifting site
(SEQ. ID. No. 28), including the suspected slippery sequence A9
(bold italic). The stop codon in the -2 reading frame is indicated.
Also indicated are potential step loop structures and the nearest
stop codons in the -1 reading frame.
[0056] FIG. 7 is an image showing a Western analysis of .gamma. and
.tau. in T.th. cells. Whole cells were lysed in SDS and
electrophoresed on a 10% SDS polyacrylamide gel then transferred to
a membrane and probed with polyclonal antibody against E. coli
.gamma./.tau. as described in Experimental Procedures. Positions of
molecular weight size markers are shown to the left. Putative T.th.
.gamma. and .tau. are indicated to the right.
[0057] FIGS. 8A-B are images of E. coli colonies expressing T.th.
dnaX -1 and -2 frameshifts. The region of the dnaX gene slippery
sequence was cloned into the lacZ gene of pUC19 in three reading
frames, then transformed into E. coli cells and plated on LB plates
containing X-gal. The slippery sequence was also mutated by
inserting two G residues into the A9 sequence and then cloned into
pUC19 in all three reading frames. Color of colonies observed are
indicated by the plus signs. The picture shows the colonies, the
type of frameshift required for readthrough (blue color) is
indicted next to the sector.
[0058] FIG. 9 shows the construction of the T.th. .gamma./.tau.
expression vector. A genomic fragment containing a partial sequence
of dnaX was cloned into pALTER-1. This fragment was subcloned into
pUC19 (pUC19_dnaX). Then the N-terminal section of dnaX was
amplified such that the fragment was flanked by NdeI (at the
initiating codon) and the internal BamHI site. This fragment was
inserted to form the entire coding sequence of the dnaX gene in
pUC19 (pUC19_dnaX). The dnaX gene was then cloned behind the
polyhistidine leader in the T7 based expression vector pET16 to
give pET16dnaX. Details are in "Experimental Procedures".
[0059] FIGS. 10A-C illustrate the purification of recombinant T.th.
.gamma. and .tau. subunits. T.th. .gamma. and .tau. subunits were
expressed in E. coli harboring pET16dnaX. Molecular size markers
are shown to the left of the gels, and the two induced proteins are
labeled as g and t to the right of the gel. Panel A) 10% SDS gel of
E. coli whole cell lysates before and after induction with IPTG.
Panel B) 8% SDS gel of the purification two steps after cell lysis.
First lane: the lysate was applied to a HiTrap Nickel
chromatography column. Second lane: the T.th. .gamma./.tau.
subunits were further purified on a Superose 12 gel filtration
column. Third lane, the E. coli .gamma. and .tau. subunits. Panel
C) Western analysis of the pure T.th. .gamma. and .tau. subunits
(first lane) and E. coli .gamma. and .tau. subunits (second
lane).
[0060] FIGS. 11A-B show the gel filtration of T.th. .gamma. and
.tau.. T.th. .gamma. and .tau. were gel filtered on a Superose 12
column. Column fractions were analyzed for ATPase activity and in a
Coomassie Blue stained 10% SDS polyacrylamide gel. Positions of
molecular weight markers are shown to the left of the gel. The
elution position of size standards analyzed in a parallel Superose
12 column under identical conditions are indicated above the gel.
Thyroglobin (670 kDa), bovine gamma globin (150 kDa), chicken
ovalbumin (44 kDa), equine myoglobin (17 kDa).
[0061] FIGS. 12A-C illustrate the characterization of the T.th.
.gamma. and .tau. ATPase activity. The T.th. .gamma./.tau. and E.
coli .tau. subunits are compared in their ATPase activity
characteristics. Due to the greater activity of E. coli .tau., the
values are plotted as percent for ease of comparison. Actual
specific activities for 100% values are given below as pmol ATP
hydrolyzed/30 min./pmol T.th. .gamma./.tau. (or pmol E. coli
.tau.). Panel A) T.th. .gamma. and .tau. ATPase is stimulated by
the presence of ssDNA. T.th. .gamma./.tau. was incubated at
65.degree. C. Specific activity was: 11.5 (+DNA); 2.5 (-DNA); E.
coli .tau. was assayed at 37.degree. C. Specific activity values
were: 112.5 (+DNA); (7.3-DNA). Panel B) Temperature stability of
DNA stimulated ATPase activity. T.th. .gamma./.tau., 11.3
(65.degree. C.); E. coli .tau., 97.5 (37.degree. C.). Panel C)
Stability of T.th. .gamma./.tau. ATPase to NaCl. T.th.
.gamma./.tau., 8.1 (100 mM added NaCl and 65.degree. C.); E. coli
.tau., 52.7 (0 M added NaCl and 37.degree. C.).
[0062] FIGS. 13A-13C are graphs that summarize the purification of
the DNA polymerase III from T.th. extracts. Panel A) shows the
activity and total protein in column fractions from the Heparin
Agarose column. Peak 1 fractions were chromatographed on ATP
agarose. Panel B) depicts the ATP-agarose column step, and Panel C)
shows the total protein and DNA polymerase activity eluted from the
MonoQ column.
[0063] FIGS. 14A-B are SDS polyacrylamide gels of T.th. subunits.
FIG. 14A is a 12% SDS polyacrylamide gel stained with Coomassie
Blue of the MonoQ column. Load stands for the material loaded onto
the column (ATP agarose bound fractions). FT stands for protein
that flowed through the MonoQ column. Fractions are indicated above
the gel. T.th. subunits in fractions 17-19 are indicated by the
labels placed between fractions 18 and 19. Additional small
subunits may be present but difficult to visualize, or may have run
off the gel. E. coli .gamma.,.delta. shows a mixture of the
.alpha., .gamma., and .delta. subunits of DNA polymerase III
holoenzyme (they are labeled to the right in the figure). FIG. 14B
shows the Western results of an SDS gel of the MonoQ fractions
probed with rabbit antiserum raised against the E. coli .alpha.
subunit. Load and FT are as described in Panel A. Fraction numbers
are shown above the gel. The band that comigrates with E. coli
.alpha., and the band in the Coomassie Blue stained gel in Panel A,
is marked with an arrow. This band was analyzed for microsequence
and the results are shown in FIG. 15.
[0064] FIGS. 15A-B show the alignments of the peptides obtained
from T.th. .alpha. subunit, TTH1 (shown in A) and TTH2 (shown in B)
with the amino acid sequences of the .alpha. subunits of other
organisms. The amino acid number of these regions within each
respective protein sequence are shown to the right. The
abbreviations of the organisms are as follows. E. coli--Escherichia
coli (SEQ ID NOS: 72 and 79 in 15A-B, respectively),
V.chol.--Vibrio cholerae (SEQ ID NOS: 73 and 80 in 15A-B,
respectively), H.inf.--Haemophilus influenzae (SEQ ID NOS: 74 and
81 in 15A-B, respectively), R.prow.--Rickettsia prowazekii (SEQ ID
NOS: 75 and 82 in 15A-B, respectively), H.pyl.--Helicobacter pylori
(SEQ ID NOS: 76 and 83 in 15A-B, respectively),
S.sp.--Synechocystis sp. (SEQ ID NOS: 77 and 84 in 15A-B,
respectively), M.tub.--Mycobacterium tuberculosis (SEQ ID NOS: 78
and 85 in 15A-B, respectively), T.th.--Thermus thermophilus (SEQ ID
NOS: 61 and 60 in 15A-B, respectively).
[0065] FIGS. 16A-C show a nucleotide (Panels A-B, SEQ. ID. No. 86)
and amino acid (Panel C, SEQ. ID. No. 87) sequence of the dnaE gene
encoding the .alpha. subunit of DNA polymerase III replication
enzyme.
[0066] FIG. 17 shows an alignment of the amino acid sequence of
.epsilon. subunits encoded by dnaQ of several organisms. The amino
acid sequence of the Thermus thermophilus .epsilon. subunit of dnaQ
is also shown. T.th., Thermus thermophilus (SEQ. ID. No. 88);
D.rad., Deinococcus radiodurans (SEQ. ID. No. 89); Bac.sub.,
Bacillus subtilis (SEQ. ID. No. 90); H.inf., Haemophilus influenzae
(SEQ. ID. No. 91); E.c., Escherichia coli (SEQ. ID. No. 92);
H.pyl., Helicobacter pylori (SEQ. ID. No. 93). The regions used to
obtain the inner part of the dnaQ gene are shown in bold. The
starts used for expression of the T.th. .epsilon. subunit are
marked.
[0067] FIGS. 18A-B show the nucleotide (Panel A, SEQ. ID. No. 94)
and amino acid (Panel B, SEQ. ID. No. 95) sequence of the dnaQ gene
encoding the .epsilon. subunit of DNA polymerase III replication
enzyme.
[0068] FIGS. 19A-B show an alignment of the DnaA protein of several
organisms. The amino acid sequence of the Thermus thermophilus DnaA
protein is also shown. P.mar., Pseudomonas marcesans (SEQ. ID. No.
96); Syn.sp., Synechocystis sp. (SEQ. ID. No. 97); Bac.sub.,
Bacillus subtilis (SEQ. ID. No. 98); M. tub; Mycobacterium
tuberculosis (SEQ. ID. No. 99); T.th., Thermus thermophilus (SEQ.
ID. No. 100); E. coli., Escherichia coli (SEQ. ID. No. 101); T.
mar., Thermatoga maritima (SEQ. ID. No. 102); and H.pyl.,
Helicobacter pylori (SEQ. ID. No. 103).
[0069] FIGS. 20A-B show the nucleotide (Panel A, SEQ. ID. No. 104)
and amino acid (Panel B, SEQ. ID. No. 105) sequence of the dnaA
gene of Thermus thermophilus.
[0070] FIGS. 21A-B show the nucleotide (Panel A, SEQ. ID. No. 106)
and amino acid (Panel B, SEQ. ID. No. 107) sequence of the dnaN
gene encoding the .beta. subunit of DNA polymerase III replication
enzyme.
[0071] FIGS. 22A-B show an alignment of the .beta. subunit of T.th.
to the .beta. subunits of other organisms. T.th., Thermus
thermophilus (SEQ. ID. No. 108); E. coli, Escherichia coli (SEQ.
ID. No. 109); P. mirab, Proteus mirabilis (SEQ. ID. No. 110); H.
infl, Haemophilus influenzae (SEQ. ID. No. 111); P. put,
Pseudomonas putida (SEQ. ID. No. 112); and B. cap., Buchnera
aphidicola (SEQ. ID. No. 113).
[0072] FIG. 23 is a map of the pET24:dnaN plasmid. The functional
regions of the plasmid are indicated by arrows and italic,
restriction sites are marked with bars and symbols. The hatched
parts in the plasmid correspond to T.th. dnaN.
[0073] FIGS. 24A-B show the induction of T.th. .beta. in E. coli
cells harboring the T.th. .beta. expression vector. Panel A is the
cell induction. The first lane shows molecular weight markers (MW).
The second lane shows uninduced E. coli cells, and the third lane
shows induced E. coli. The induced T.th. .beta. is indicated by the
arrow shown to the left. Induced cells were lysed then treated with
heat and the soluble portion was chromatographed on MonoQ. Panel B
shows the results of 1.5 MonoQ purification of T.th. .beta..
[0074] FIG. 25A is a schematic depiction of the use of the use of
the enzymes of the present invention in accordance with an
alternate embodiment hereof. In this scheme the clamp (.beta. or
PCNA) slides over the end of linear DNA to enhance the polymerase
(Pol III-type such as Pol III, Pol.beta. or Pol.delta..) In this
fashion the clamp loader activity is not needed.
[0075] FIG. 25B graphically demonstrates the results of the
practice of the alternate embodiment of the invention described and
set forth in Example 15, infra. Lane 1, E. coli Pol III without
.beta.; Lane 2, E. coli with .beta.; Lane 3, human
Pol.delta.without PCNA; Lane 4, human Pol.delta. with PCNA; Lane 5,
T.th. Pol III without T.th. .beta.; Lane 6, T.th. Pol III with
T.th. .beta.. The respective pmol synthesis in lanes 1-6 are: 6,
35, 2, 24, 0.6 and 1.9.
[0076] FIGS. 26A-B show the use of T.th. Pol III in extending
singly primed M13 mp18 to an RFII form. The scheme in FIG. 26A
shows the primed template in which a DNA 57mer was annealled to the
M13 mp18 ssDNA circle. Then T.th. .beta. subunit (produced
recombinantly) and T.th. Pol III were added to the DNA in the
presence of radioactive nucleoside triphosphates. In FIG. 26B, the
products of the reaction were analyzed in a 0.8% native agarose
gel. The position of ssDNA starting material, the RFII product, and
of intermediate species, are shown to the sides of the gel. Lane 1,
use of Pol III. Lane 2, use of the non-Pol III DNA polymerase.
[0077] FIG. 27 is an SDS polyacrylamide gel of the proteins of the
A. aeolicus replication machinery.
[0078] FIG. 28 is an SDS polyacrylamide gel analysis of the MonoQ
fractions of the method used to reconstitute and purify the A.
aeolicus .tau..delta..delta.' complex.
[0079] FIG. 29 is an SDS polyacrylamide gel analysis of the gel
filtration column fractions used in the preparation of the A.
aeolicus .tau..tau..delta..delta.' complex. The bottom gel analysis
shows the profile obtained using the A. aeolicus .alpha. subunit
(polymerase) in the absence of the other subunits.
[0080] FIG. 30 is an alkaline agarose gel analysis of reaction
products for extension of a single primer around a 7.2 kb M13 mp18
circular ssDNA genome that has been coated with A. aeolicus SSB.
The time course on the left are produced by
.alpha..tau..delta..delta.'/.beta., and the time course on the
right is produced by .alpha..tau..delta..delta.' in the absence of
.beta..
[0081] FIG. 31 is a graph illustrating the optimal temperature for
activity of the alpha subunit of Thermus replicase using a calf
thymus DNA replication assay. Reactions were shifted to the
indicated temperature for 5 minutes before detecting the level of
DNA synthesis activity.
[0082] FIG. 32 is a graph illustrating the optimal temperature for
activity of the alpha subunit of the Aquifex replicase using a calf
thymus DNA replication assay. Reactions were shifted to the
indicated temperature for 5 minutes before detecting the level of
DNA synthesis activity.
[0083] FIGS. 33A-E illustrate the heat stability of Aquifex
components. Assays of either .alpha. (FIG. 33A), .beta. (FIG. 33B),
.tau..delta..delta.' complex (FIG. 33C), SSB (FIG. 33D) and
.alpha..tau..delta..delta.' complex (FIG. 33E) were performed after
heating samples at the indicated temperatures. Components were
heated in buffer containing the following: 0.1% Triton X-100
(filled diamonds); 0.05% Tween-20 and 0.01% NP-40 (filled circles);
4 mM CaCl.sub.2 (filled triangles); 40% Glycerol (inverted filled
triangles); 0.01% Triton X-100, 0.05% Tween-20, 0.01% NP-40, 4 mM
CaCl.sub.2 (half-filled square); 40% Glycerol, 0.1% Triton X-100
(open diamonds); 40% Glycerol, 0.05% Tween-20, 0.01% NP-40 (open
circles); 40% Glycerol, 4 mM CaCl.sub.2 (open triangles); 40%
Glycerol, 0.01% Triton X-100, 0.05% Tween-20, 0.01% NP-40, 4 mM
CaCl.sub.2 (half-filled diamonds).
[0084] FIGS. 34A-B show the nucleotide sequence (SEQ. ID. No. 117)
of the dnaE gene of A. aeolicus.
[0085] FIG. 35 shows the amino acid sequence (SEQ. ID. No. 118) of
the .alpha. subunit of A. aeolicus.
[0086] FIG. 36 shows the nucleotide sequence (SEQ. ID. No. 119) of
the dnaX gene of A. aeolicus.
[0087] FIG. 37 shows the amino acid sequence (SEQ. ID. No. 120) of
the tau subunit of A. aeolicus.
[0088] FIG. 38 shows the nucleotide sequence (SEQ. ID. No. 121) of
the dnaN gene of A. aeolicus.
[0089] FIG. 39 shows the amino acid sequence (SEQ. ID. No. 122) of
the .beta. subunit of A. aeolicus.
[0090] FIG. 40 shows the partial nucleotide sequence (SEQ. ID. No.
123) of the holA gene of A. aeolicus.
[0091] FIG. 41 shows the partial amino acide sequence (SEQ. ID. No.
124) of the .delta. subunit of A. aeolicus.
[0092] FIG. 42 shows the nucleotide sequence (SEQ. ID. No. 125) of
the holB gene of A. aeolicus.
[0093] FIG. 43 shows the amino acid sequence (SEQ. ID. No. 126) of
the .delta.' subunit of A. aeolicus.
[0094] FIG. 44 shows the nucleotide sequence (SEQ. ID. No. 127) of
the dnaQ of A. aeolicus.
[0095] FIG. 45 shows the amino acid sequence (SEQ. ID. No. 128) of
the .epsilon. subunit of A. aeolicus.
[0096] FIG. 46 shows the nucleotide sequence (SEQ. ID. No. 129) of
the ssb gene of A. aeolicus.
[0097] FIG. 47 shows the amino acid sequence (SEQ. ID. No. 130) of
the single-strand binding protein of A. aeolicus.
[0098] FIG. 48 shows the nucleotide sequence (SEQ. ID. No. 131) of
the dnaB gene of A. aeolicus.
[0099] FIG. 49 shows the amino acid sequence (SEQ. ID. No. 132) of
the DnaB helicase of A. aeolicus.
[0100] FIG. 50 shows the nucleotide sequence (SEQ. ID. No. 133) of
the dnaG gene of A. aeolicus.
[0101] FIG. 51 shows the amino acid sequence (SEQ. ID. No. 134) of
the DnaG primase of A. aeolicus.
[0102] FIG. 52 shows the nucleotide sequence (SEQ. ID. No. 135) of
the dnaC gene of A. aeolicus.
[0103] FIG. 53 shows the amino acid sequence (SEQ. ID. No. 136) of
the DnaC protein of A. aeolicus.
[0104] FIG. 54A-B shows the nucleotide sequence (SEQ. ID. No. 137)
of the dnaE gene of T. maritima.
[0105] FIG. 55 shows the amino acid sequence (SEQ. ID. No. 138) of
the .alpha. subunit of T. maritima.
[0106] FIG. 56 shows the nucleotide sequence (SEQ. ID. No. 139) of
the dnaQ gene of T. maritima.
[0107] FIG. 57 shows the amino acid sequence (SEQ. ID. No. 140) of
the .epsilon. subunit of T. maritima.
[0108] FIG. 58 shows the nucleotide sequence (SEQ. ID. No. 141) of
the dnaX gene of T. maritima.
[0109] FIG. 59 shows the amino acid sequence (SEQ. ID. No. 142) of
the tau subunit of T. maritima.
[0110] FIG. 60 shows the nucleotide sequence (SEQ. ID. No. 143) of
the dnaN gene of T. maritima.
[0111] FIG. 61 shows the amino acid sequence (SEQ. ID. No. 144) of
the .beta. subunit of T. maritima.
[0112] FIG. 62 shows the nucleotide sequence (SEQ. ID. No. 145) of
the holA gene of T. maritima.
[0113] FIG. 63 shows the amino acid sequence (SEQ. ID. No. 146) of
the .delta. subunit of T. maritima.
[0114] FIG. 64 shows the nucleotide sequence (SEQ. ID. No. 147) of
the holB gene of T. maritima.
[0115] FIG. 65 shows the amino acid sequence (SEQ. ID. No. 148) of
the .delta.' subunit of T. maritima.
[0116] FIG. 66 shows the nucleotide sequence (SEQ. ID. No. 149) of
the ssb gene of T. maritima.
[0117] FIG. 67 shows the amino acid sequence (SEQ. ID. No. 150) of
the single-strand binding protein of T. maritima.
[0118] FIG. 68 shows the nucleotide sequence (SEQ. ID. No. 151) of
the dnaB gene of T. maritima.
[0119] FIG. 69 shows the amino acid sequence (SEQ. ID. No. 152) of
the DnaB helicase of T. maritima.
[0120] FIG. 70 shows the nucleotide sequence (SEQ. ID. No. 153) of
the dnaG gene of T. maritima.
[0121] FIG. 71 shows the amino acid sequence (SEQ. ID. No. 154) of
the DnaG primase of T. maritima.
[0122] FIG. 72 shows the nucleotide sequence (SEQ. ID. No. 155) of
the holB gene of T. thermophilus.
[0123] FIG. 73 shows the amino acid sequence (SEQ. ID. No. 156) of
the .delta.' subunit of T. thermophilus.
[0124] FIG. 74 shows the nucleotide sequence (SEQ. ID. No. 157) of
the holA gene of T. thermophilus.
[0125] FIG. 75 shows the amino acid sequence (SEQ. ID. No. 158) of
the .delta. subunit of T. thermophilus.
[0126] FIG. 76 shows the nucleotide sequence (SEQ. ID. No. 171) of
the ssb gene of T. thermophilus.
[0127] FIG. 77 shows the amino acid sequence (SEQ. ID. No. 172) of
the single-strand binding protein of T. thermophilus.
[0128] FIG. 78 shows the partial nucleotide sequence (SEQ. ID. No.
173) of the dnaN gene of B. stearothermophilus.
[0129] FIG. 79 shows the partial amino acid sequence (SEQ. ID. No.
174) of the .beta. subunit of B. stearothermophilus.
[0130] FIG. 80 shows the nucleotide sequence (SEQ. ID. No. 175) of
the ssb gene of B. stearothermophilus.
[0131] FIG. 81 shows the amino acid sequence (SEQ. ID. No. 176) of
the single-strand binding protein of B. stearothermophilus.
[0132] FIG. 82 shows the nucleotide sequence (SEQ. ID. No. 177) of
the holA gene of B. stearothermophilus.
[0133] FIG. 83 shows the amino acid sequence (SEQ. ID. No. 178) of
the .delta. subunit of B. stearothermophilus.
[0134] FIG. 84 shows the nucleotide sequence (SEQ. ID. No. 179) of
the holB gene of B. stearothermophilus.
[0135] FIG. 85 shows the amino acid sequence (SEQ. ID. No. 180) of
the .delta.' subunit of B. stearothermophilus.
[0136] FIGS. 86A-B show the partial nucleotide sequence (SEQ. ID.
No. 181) of the dnaX gene of B. stearothermophilus.
[0137] FIG. 87 shows the partial amino acid sequence (SEQ. ID. No.
182) of the tau subunit of B. stearothermophilus.
[0138] FIGS. 88A-B show the nucleotide sequence (SEQ. ID. No. 183)
of the polC gene of B. stearothermophilus.
[0139] FIG. 89 shows the amino acid sequence (SEQ. ID. No. 184) of
the PolC or .alpha.-large subunit of B. stearothermophilus.
DETAILED DESCRIPTION OF THE INVENTION
[0140] In accordance with the present invention there may be
employed conventional molecular biology, microbiology; and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook et al., "Molecular Cloning: A Laboratory Manual" (1989);
"Current Protocols in Molecular Biology" Volumes I-III (Ausubel, R.
M., ed.) (1994); "Cell Biology: A Laboratory Handbook" Volumes
I-III (Celis, J. E., ed.) (1994); "Current Protocols in Immunology"
Volumes I-III (Coligan, J. E., ed.) (1994); "Oligonucleotide
Synthesis" (M. J. Gait, ed.) (1984); "Nucleic Acid Hybridization"
(B. D. Hames & S. J. Higgins, eds.) (1985); "Transcription And
Translation" (B. D. Hames & S. J. Higgins, eds.) (1984);
"Animal Cell Culture" (R. I. Freshney, ed.) (1986); "Immobilized
Cells And Enzymes" (IRL Press) (1986); B. Perbal, "A Practical
Guide To Molecular Cloning" (1984), each of which is hereby
incorporated by reference.
[0141] Therefore, if appearing herein, the following terms shall
have the definitions set out below.
[0142] The terms "DNA Polymerase III," "Polymerase III-type
enzyme(s)", "Polymerase III enzyme complex(s)", "T.th. DNA
Polymerase III", "A.ae. DNA Polymerase III", "T.ma. DNA Polymerase
III", and any variants not specifically listed, may be used herein
interchangeably, as are .beta. subunit and sliding clamp and clamp
as are also .gamma. complex, clamp loader, and RFC, as used
throughout the present application and claims refer to
proteinaceous material including single or multiple proteins, and
extends to those proteins having the amino acid sequence data
described herein and presented in the Figures and corresponding
Sequence Listing entries, and the corresponding profile of
activities set forth herein and in the claims. Accordingly,
proteins displaying substantially equivalent or altered activity
are likewise contemplated. These modifications may be deliberate,
for example, such as modifications obtained through site-directed
mutagenesis, or may be accidental, such as those obtained through
mutations in hosts that are producers of the complex or its named
subunits. Also, the terms "DNA Polymerase III," "T.th. DNA
Polymerase III," and ".gamma. and .tau. subunits", ".beta.
subunit", ".alpha. subunit", ".epsilon. subunit", ".delta.
subunit", ".delta.' subunit", "SSB protein", "sliding clamp" and
"clamp loader" are intended to include within their scope proteins
specifically recited herein as well as all substantially homologous
analogs and allelic variations. As used herein .gamma. complex
refers to a particular type of clamp loader that includes a .gamma.
subunit.
[0143] Also as used herein, the term "thermolabile enzyme" refers
to a DNA polymerase which is not resistant to inactivation by heat.
For example, T5 DNA polymerase, the activity of which is totally
inactivated by exposing the enzyme to a temperature of 90.degree.
C. for 30 seconds, is considered to be a thermolabile DNA
polymerase. As used herein, a thermolabile DNA polymerase is less
resistant to heat inactivation than in a thermostable DNA
polymerase. A thermolabile DNA polymerase typically will also have
a lower optimum temperature than a thermostable DNA polymerase.
Thermolabile DNA polymerases are typically isolated from mesophilic
organisms, for example mesophilic bacteria or eukaryotes, including
certain animals.
[0144] As used herein, the term "thermostable enzyme" refers to an
enzyme which is stable to heat and is heat resistant and catalyzes
(facilitates) combination of the nucleotides in the proper manner
to form the primer extension products that are complementary to
each nucleic acid strand. Generally, the synthesis will be
initiated at the 3' end of each primer and will proceed in the 5'
direction along the template strand, until synthesis terminates,
producing molecules of different lengths.
[0145] The thermostable enzyme herein must satisfy a single
criterion to be effective for the amplification reaction, i.e., the
enzyme must not become irreversibly denatured (inactivated) when
subjected to the elevated temperatures for the time necessary to
effect denaturation of double-stranded nucleic acids. Irreversible
denaturation for purposes herein refers to permanent and complete
loss of enzymatic activity. The heating conditions necessary for
denaturation will depend, e.g., on the buffer salt concentration
and the length and nucleotide composition of the nucleic acids
being denatured, but typically range from about 90 C..degree. to
about 96.degree. C. for a time depending mainly on the temperature
and the nucleic acid length, typicality about 0.5 to four minutes.
Higher temperatures may be tolerated as the buffer salt
concentration and/or GC composition of the nucleic acid is
increased. Preferably, the enzyme will not become irreversibly
denatured at about 90.degree.-100.degree. C.
[0146] The thermostable enzymes herein preferably have an optimum
temperature at which they function that is higher than about
40.degree. C., which is the temperature below which hybridization
of primer to template is promoted, although, depending on (1)
magnesium and salt concentrations and (2) composition and length of
primer hybridization can occur at higher temperature (e.g.,
45'-70.degree. C.). The higher the temperature optimum for the
enzyme, the greater the specificity and/or selectivity of the
primer-directed extension process. However, enzymes that are active
below 40.degree. C., e.g., at 37.degree. C., are also within the
scope of this invention provided they are heat-stable. Preferably,
the optimum temperature ranges from about 50.degree. to about
90.degree. C., more preferably about 60.degree. to about 80.degree.
C. In this connection, the term "elevated temperature" as used
herein is intended to cover sustained temperatures of operation of
the enzyme that are equal to or higher than about 60.degree. C.
[0147] The term "template" as used herein refers to a
double-stranded or single-stranded DNA molecule which is to be
amplified, synthesized, or sequenced. In the case of a
double-stranded DNA molecule, denaturation of its strands to form a
first and a second strand is performed before these molecules may
be amplified, synthesized or sequenced. A primer, complementary to
a portion of a DNA template is hybridized under appropriate
conditions and the DNA polymerase of the invention may then
synthesize a DNA molecule complementary to said template or a
portion thereof. The newly synthesized DNA molecule, according to
the invention, may be equal or shorter in length than the original
DNA template. Mismatch incorporation during the synthesis or
extension of the newly synthesized DNA molecule may result in one
or a number of mismatched base pairs. Thus, the synthesized DNA
molecule need not be exactly complementary to the DNA template.
[0148] The term "incorporating" as used herein means becoming a
part of a DNA molecule or primer.
[0149] As used herein "amplification" refers to any in vitro method
for increasing the number of copies of a nucleotide sequence, or
its complimentary sequence, with the use of a DNA polymerase.
Nucleic acid amplification results in the incorporation of
nucleotides into a DNA molecule or primer thereby forming a new DNA
molecule complementary to a DNA template. The formed DNA molecule
and its template can be used as templates to synthesize additional
DNA molecules. As used herein, one amplification reaction may
consist of many rounds of DNA replication. DNA amplification
reactions include, for example, polymerase chain reactions (PCR).
One PCR reaction may consist of about 20 to 100 "cycles" of
denaturation and synthesis of a DNA molecule. In this connection,
the use of the term "long stretches of DNA" as it refers to the
extension of primer along DNA is intended to cover such extensions
of an average length exceeding 7 kilobases. Naturally, such length
will vary, and all such variations are considered to be included
within the scope of the invention.
[0150] As used herein, the term "holoenzyme" refers to a
multi-subunit DNA polymerase activity comprising and resulting from
various subunits which each may have distinct activities but which
when contained in an enzyme reaction operate to carry out the
function of the polymerase (typically DNA synthesis) and enhance
its activity over use of the DNA polymerase subunit alone. For
example, E. coli DNA polymerase III is a holoenzyme comprising
three components of one or more subunits each: (1) a core component
consisting of a heterotrimer of .alpha., .epsilon. and .theta.
subunits; (2) a .beta. component consisting of a .beta. subunit
dimer; and (3) a .gamma. complex component consisting of a
heteropentamer of .gamma., .delta., .delta.', .chi. and .psi.
subunits (see Studwell and O'Donnell, 1990). These three
components, and the various subunits of which they consist, are
linked non-covalently to form the DNA polymerase III holoenzyme
complex. However, they also function when not linked in
solution.
[0151] As used herein, "enzyme complex" refers to a protein
structure consisting essentially of two or more subunits of a
replication enzyme, which may or may not be identical,
noncovalently linked to each other to form a multi-subunit
structure. An enzyme complex according to this definition ideally
will have a particular enzymatic activity, up to and including the
activity of the replication enzyme. For example, a "DNA pol III
enzyme complex" as used herein means a multi-subunit protein
activity comprising two or more of the subunits of the DNA pol III
replication enzyme as defined above, and having DNA polymerizing or
synthesizing activity. Thus, this term encompasses the, native
replication enzyme, as well as an enzyme complex lacking one or
more of the subunits of the replication enzyme (e.g., DNA pol III
exo-, which lacks the .epsilon. subunit).
[0152] The amino acid residues described herein are preferred to be
in the "L" isomeric form. However, residues in the "D" isomeric
form can be substituted for any L-amino acid residue, as long as
the desired functional property of immunoglobulin-binding is
retained by the polypeptide. NH.sub.2 refers to the free amino
group present at the amino terminus of a polypeptide. COOH refers
to the free carboxy group present at the carboxy terminus of a
polypeptide. In keeping with standard polypeptide nomenclature, J.
Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid
residues are shown in the following Table of Correspondence:
1 TABLE OF CORRESPONDENCE SYMBOLS 1-Letter 3-Letter AMINO ACID Y
Tyr tyrosine G Gly glycine F Phe phenylalanine M Met methionine A
Ala alanine S Ser serine I Ile isoleucine L Leu leucine T Thr
threonine V Val valine P Pro proline K Lys lysine H His histidine Q
Gln glutamine E Glu glutamic acid W Trp tryptophan R Arg arginine D
Asp aspartic acid N Asn asparagine C Cys cysteine
[0153] It should be noted that all amino-acid residue sequences are
represented herein by formulae whose left and right orientation is
in the conventional direction of amino-terminus to
carboxy-terminus. Furthermore, it should be noted that a dash at
the beginning or end of an amino acid residue sequence indicates a
peptide bond to a further sequence of one or more amino-acid
residues. The above Table is presented to correlate the
three-letter and one-letter notations which may appear alternately
herein.
[0154] A "replicon" is any genetic element (e.g., plasmid,
chromosome, virus) that functions as an autonomous unit of DNA
replication in vivo; i.e., capable of replication under its own
control.
[0155] A "vector" is a replicon, such as plasmid, phage or cosmid,
to which another DNA segment may be attached so as to bring about
the replication of the attached segment.
[0156] A "DNA molecule" refers to the polymeric form of
deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in
its either single stranded form, or a double-stranded helix. This
term refers only to the primary and secondary structure of the
molecule, and does not limit it to any particular tertiary forms.
Thus, this term includes double-stranded DNA found, inter alia, in
linear DNA molecules (e.g., restriction fragments), viruses,
plasmids, and chromosomes. In discussing the structure of
particular double-stranded DNA molecules, sequences may be
described herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the nontranscribed
strand of DNA (i.e., the strand having a sequence homologous to the
mRNA).
[0157] An "origin of replication" refers to those DNA sequences
that participate in DNA synthesis.
[0158] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide in vivo when
placed under the control of appropriate regulatory sequences. The
boundaries of the coding sequence are determined by a start codon
at the 5' (amino) terminus and a translation stop codon at the 3'
(carboxyl) terminus. A coding sequence can include, but is not
limited to, prokaryotic sequences, cDNA from eukaryotic mRNA,
genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and
even synthetic DNA sequences. A polyadenylation signal and
transcription termination sequence will usually be located 3' to
the coding sequence.
[0159] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, polyadenylation
signals, terminators, and the like, that provide for the expression
of a coding sequence in a host cell.
[0160] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site (conveniently defined by mapping with
nuclease S1), as well as protein binding domains (consensus
sequences) responsible for the binding of RNA polymerase.
Eukaryotic promoters will often, but not always, contain "TATA"
boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno
sequences in addition to the -10 and -35 consensus sequences.
[0161] An "expression control sequence" is a DNA sequence that
controls and regulates the transcription and translation of another
DNA sequence. A coding sequence is "under the control" of
transcriptional and translational control sequences in a cell when
RNA polymerase transcribes the coding sequence into mRNA, which is
then translated into the protein encoded by the coding
sequence.
[0162] A "signal sequence" can be included before the coding
sequence. This sequence encodes a signal peptide, N-terminal to the
polypeptide, that communicates to the host cell to direct the
polypeptide to the cell surface or secrete the polypeptide into the
media, and this signal peptide is clipped off by the host cell
before the protein leaves the cell. Signal sequences can be found
associated with a variety of proteins native to prokaryotes and
eukaryotes.
[0163] The term "oligonucleotide," as used generally herein, such
as in referring to probes prepared and used in the present
invention, is defined as a molecule comprised of two or more
(deoxy)ribonucleotides, preferably more than three. Its exact size
will depend upon many factors which, in turn, depend upon the
ultimate function and use of the oligonucleotide.
[0164] The term "primer" as used herein refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product, which
is complementary to a nucleic acid strand, is induced, i.e., in the
presence of nucleotides and an inducing agent such as a DNA
polymerase and at a suitable temperature and pH. The primer may be
either single-stranded or double-stranded and must be sufficiently
long to prime the synthesis of the desired extension product in the
presence of the inducing agent. The exact length of the primer will
depend upon many factors including temperature, source of primer
and use of the method. For example, for diagnostic applications,
depending on the complexity of the target sequence, the
oligonucleotide primer typically contains 15-25 or more
nucleotides, although it may contain fewer nucleotides.
[0165] The primers herein are selected to be "substantially"
complementary to different strands of a particular target DNA
sequence. This means that the primers must be sufficiently
complementary to hybridize with their respective strands.
Therefore, the primer sequence need not reflect the exact sequence
of the template. For example, a non-complementary nucleotide
fragment may be attached to the 5' end of the primer, with the
remainder of the primer sequence being complementary to the strand.
Alternatively, non-complementary bases or longer sequences can be
interspersed into the primer, provided that the primer sequence has
sufficient complementarity with the sequence of the strand to
hybridize therewith and thereby form the template for the synthesis
of the extension product.
[0166] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double-stranded DNA at or near a specific nucleotide sequence.
[0167] A cell has been "transformed" by exogenous or heterologous
DNA when such DNA has been introduced inside the cell. The
transforming DNA may or may not be integrated (covalently linked)
into chromosomal DNA making up the genome of the cell. In
prokaryotes, yeast, and mammalian cells for example, the
transforming DNA may be maintained on an episomal element such as a
plasmid. With respect to eukaryotic cells, a stably transformed
cell is one in which the transforming DNA has become integrated
into a chromosome so that it is inherited by daughter cells through
chromosome replication. This stability is demonstrated by the
ability of the eukaryotic cell to establish cell lines or clones
comprised of a population of daughter cells containing the
transforming DNA. A "clone" is a population of cells derived from a
single cell or common ancestor by mitosis. A "cell line" is a clone
of a primary cell that is capable of stable growth in vitro for
many generations.
[0168] Two DNA sequences are "substantially homologous" when at
least about 75% (preferably at least about 80%, and most preferably
at least about 90 or 95%) of the nucleotides match over the defined
length of the DNA sequences. Sequences that are substantially
homologous can be identified by comparing the sequences using
standard software available in sequence data banks, or in a
Southern hybridization experiment under, for example, stringent
conditions as defined for that particular system. Suitable
conditions include those characterized by a hybridization buffer
comprising 0.9M sodium citrate ("SSC") buffer at a temperature of
about 37.degree. C. and washing in SSC buffer at a temperature of
about 37.degree. C.; and preferably in a hybridization buffer
comprising 20% formamide in 0.9M SSC buffer at a temperature, of
about 42.degree. C. and washing with 0.2.times.SSC buffer at about
42.degree. C. Stringency conditions can be further varied by
modifying the temperature and/or salt content of the buffer, or by
modifying the length of the hybridization probe as is known to
those of skill in the art. Defining appropriate hybridization
conditions is within the skill of the art. See, e.g., Maniatis et
al., 1982; Glover, 1985; Hames and Higgins, 1984.
[0169] It should be appreciated that also within the scope of the
present invention are degenerate DNA sequences. By "degenerate" is
meant that a different three-letter codon is used to specify a
particular amino acid. It is well known in the art that the
following codons can be used interchangeably to code for each
specific amino acid:
2 Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG
or CUU or CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUC or AUA
Methionine (Met or M) AUG Valine (Val or V) GUU or GUC of GUA or
GUG Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC
Proline (Pro or P) CCU or CCC or CCA or CCG Threonine (Thr or T)
ACU or ACC or ACA or ACG Alanine (Ala or A) GCU or GCG or GCA or
GCG Tyrosine (Tyr or Y) UAU or UAC Histidine (His or H) CAU or CAC
Glutamine (Gln or Q) CAA or CAG Asparagine (Asn or N) AAU or AAC
Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAU or GAC
Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU or UGC
Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine
(Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGG
Termination codon UAA (ochre) or UAG (amber) or UGA (opal)
[0170] It should be understood that the codons specified above are
for RNA sequences. The corresponding codons for DNA have a T
substituted for U.
[0171] Mutations can be made, e.g., in SEQ. ID. No. 1, or any of
the nucleic acids set forth herein, such that a particular codon is
changed to a codon which codes for a different amino acid. Such a
mutation is generally made by making the fewest nucleotide changes
possible. A substitution mutation of this sort can be made to
change an amino acid in the resulting protein in a non-conservative
manner (i.e., by changing the codon from an amino acid belonging to
a grouping of amino acids having a particular size or
characteristic to an amino acid belonging to another grouping) or
in a conservative manner (i.e., by changing the codon from an amino
acid belonging to a grouping of amino acids having a particular
size or characteristic to an amino acid belonging to the same
grouping). Such a conservative change generally leads to less
change in the structure and function of the resulting protein. A
non-conservative change is more likely to alter the structure,
activity or function of the resulting protein. The present
invention should be considered to include sequences containing
conservative changes which do not significantly alter the activity
or binding characteristics of the resulting protein.
[0172] The following is one example of various groupings of amino
acids:
[0173] Amino Acids with Nonpolar R Groups
[0174] Alanine
[0175] Valine
[0176] Leucine
[0177] Isoleucine
[0178] Proline
[0179] Phenylalanine
[0180] Tryptophan
[0181] Methionine
[0182] Amino Acids with Uncharged Polar R Groups
[0183] Glycine
[0184] Serine
[0185] Threonine
[0186] Cysteine
[0187] Tyrosine
[0188] Asparagine
[0189] Glutamine
[0190] Amino Acids with Charged Polar R Groups (Negatively Charged
at pH 6.0)
[0191] Aspartic acid
[0192] Glutamic acid
[0193] Basic Amino Acids (Positively Charged at pH 6.0)
[0194] Lysine
[0195] Arginine
[0196] Histidine (at pH 6.0)
[0197] Amino Acids with Phenyl Groups:
[0198] Phenylalanine
[0199] Tryptophan
[0200] Tyrosine
[0201] Another grouping may be according to molecular weight (i.e.,
size of R groups):
3 Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine
119 Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic
acid 133 Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149
Histidine (at pH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine
181 Tryptophan 204
[0202] Particularly preferred substitutions are:
[0203] Lys for Arg and vice versa such that a positive charge may
be maintained;
[0204] Glu for Asp and vice versa such that a negative charge may
be maintained;
[0205] Ser for Thr such that a free --OH can be maintained; and
[0206] Gln for Asn such that a free NH.sub.2 can be maintained.
[0207] Amino acid substitutions may also be introduced to
substitute an amino acid with a particularly preferable property.
For example, a Cys may be introduced into a potential site for
disulfide bridges with another Cys. A His may be introduced as a
particularly "catalytic" site (i.e., His can act as an acid or base
and is the most common amino acid in biochemical catalysis). Pro
may be introduced because of its particularly planar structure,
which induces .beta.-turns in the protein's structure.
[0208] Two amino acid sequences are "substantially homologous" when
at least about 70% of the amino acid residues (preferably at least
about 80%, and most preferably at least about 90 or 95%) are
identical, or represent conservative substitutions.
[0209] A "heterologous" region of the DNA construct is an
identifiable segment of DNA within a larger DNA molecule that is
not found in association with the larger molecule in nature. Thus,
when the heterologous region encodes a mammalian gene, the gene
will usually be flanked by DNA that does not flank the mammalian
genomic DNA in the genome of the source organism. Another example
of a heterologous coding sequence is a construct where the coding
sequence itself is not found in nature (e.g., a cDNA where the
genomic coding sequence contains introns, or synthetic sequences
having codons different than the native gene). Allelic variations
or naturally-occurring mutational events do not give rise to a
heterologous region of DNA as defined herein.
[0210] An "antibody" is any immunoglobulin, including antibodies
and fragments thereof, that binds a specific epitope. The term
encompasses polyclonal, monoclonal, and chimeric antibodies, the
last mentioned described in further detail in U.S. Pat. No.
4,816,397 to Boss et al. and U.S. Pat. No. 4,816,567 to Cabilly et
al.
[0211] An "antibody combining site" is that structural portion of
an antibody molecule comprised of heavy and light chain variable
and hypervariable regions that specifically binds antigen.
[0212] The phrase "antibody molecule" in its various grammatical
forms as used herein contemplates both an intact immunoglobulin
molecule and an immunologically active portion of an immunoglobulin
molecule. Exemplary antibody molecules are intact immunoglobulin
molecules, substantially intact immunoglobulin molecules and those
portions of an immunoglobulin molecule that contains the paratope,
including those portions known in the art as Fab, Fab',
F(ab').sub.2 and F(v), which portions are preferred for use in the
therapeutic methods described herein. Fab and F(ab').sub.2 portions
of antibody molecules are prepared by the proteolytic reaction of
papain and pepsin, respectively, on substantially intact antibody
molecules by methods that are well-known. See for example, U.S.
Pat. No. 4,342,566 to Theofilopolous et al. Fab' antibody molecule
portions are also well-known and are produced from F(ab').sub.2
portions followed by reduction of the disulfide bonds linking the
two heavy chain portions as with mercaptoethanol, and followed by
alkylation of the resulting protein mercaptan with a reagent such
as iodoacetamide. An antibody containing intact antibody molecules
is preferred herein.
[0213] The phrase "monoclonal antibody" in its various grammatical
forms refers to an antibody having only one species of antibody
combining site capable of immunoreacting with a particular antigen.
A monoclonal antibody thus typically displays a single binding
affinity for any antigen with which it immunoreacts. A monoclonal
antibody may therefore contain an antibody molecule having a
plurality of antibody combining sites, each immunospecific for a
different antigen; e.g., a bispecific (chimeric) monoclonal
antibody.
[0214] A DNA sequence is "operatively linked" to an expression
control sequence when the expression control sequence controls and
regulates the transcription and translation of that DNA sequence.
The term "operatively linked" includes having an appropriate start
signal (e.g., ATG) in front of the DNA sequence to be expressed and
maintaining the correct reading frame to permit expression of the
DNA sequence under the control of the expression control sequence
and production of the desired product encoded by the DNA sequence.
If a gene that one desires to insert into a recombinant DNA
molecule does not contain an appropriate start signal, such a start
signal can be inserted in front of the gene.
[0215] The term "standard hybridization conditions" refers to salt
and temperature conditions substantially equivalent to 5.times.SSC
and 65.degree. C. for both hybridization and wash. However, one
skilled in the art will appreciate that such "standard
hybridization conditions" are dependent on particular conditions
including the concentration of sodium and magnesium in the buffer,
nucleotide sequence length and concentration, percent mismatch,
percent formamide, and the like. Also important in the
determination of "standard hybridization conditions" is whether the
two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such
standard hybridization conditions are easily determined by one
skilled in the art according to well known formulae, wherein
hybridization is typically 10-20.degree. C. below the predicted or
determined T.sub.m with washes of higher stringency, if
desired.
[0216] In its primary aspect, the present invention concerns the
identification of a class of DNA Polymerase III-type enzymes or
complexes found in thermophilic bacteria such as Thermus
thermophilus (T.th.), Aquifex aeolicus (A.ae.), Thermotoga maritima
(T.ma.), Bacillus stearothermophilus (B.st.) and other eubacteria
which exhibit the following characteristics, among their
properties: the ability to extend a primer over a long stretch of
ssDNA at elevated temperature, stimulation by its cognate sliding
clamp of the type that is assembled on DNA by a clamp loader,
accessory subunits that exhibit DNA-stimulated ATPase activity at
elevated temperature and/or ionic strength, and an associated 3'-5'
exonuclease activity. In a particular aspect, the invention extends
to Polymerase III-type enzymes derived from a broad class of
thermophilic eubacteria that include polymerases isolated from the
thermophilic bacteria Aquifex aeolicus (A.ae. polymerase) and other
members of the Aquifex genus; Thermus thermophilus (T.th.
polymerase), Thermus favus (Tfl/Tub polymerase), Thermus ruber (Tru
polymerase), Thermus brockianus (DYNAZYME.TM. polymerase) and other
members of the Thermus genus; Bacillus stearothermophilus (Bst
polymerase) and other members of the Bacillus genus; Thermoplasma
acidophilum (Tac polymerase) and other members of the Thermoplasma
genus; and Thermotoga neapolitana (Tne polymerase; See WO 96/10640
to Chatterjee et al.), Thermotoga maritima (Tma polymerase; See
U.S. Pat. No. 5,374,553 to Gelfand et al.), and other members of
the Thermotoga genus. The particular polymerase discussed herein by
way of illustration and not limitation, is the enzyme derived from
T.th., A.ae., T.ma., or B.st.
[0217] Polymerase III-type enzymes covered by the invention include
those that may be prepared by purification from cellular material,
as described in detail in the Examples infra, as well as enzyme
assemblies or complexes that comprise the combination of
individually prepared enzyme, subunits or components. Accordingly,
the entire enzyme may be prepared by purification from cellular
material, or may be constructed by the preparation of the
individual components and their assembly into the functional
enzyme. A representative and non-limitative protocol for the
preparation of an enzyme by this latter route is set forth in U.S.
Pat. No. 5,583,026 to O'Donnell, and the disclosure thereof is
incorporated herein in its entirety for such purpose.
[0218] Likewise, individual subunits may be modified, e.g. as by
incorporation therein of single residue substitutions to create
active sites therein, for the purpose of imparting new or enhanced
properties to enzymes containing the modified subunits. (see, e.g.,
Tabor, 1995). Likewise, individual subunits prepared in accordance
with the invention, may be used individually and for example, may
be substituted for their counterparts in other enzymes, to improve
or particularize the properties of the resultant modified enzyme.
Such modifications are within the skill of the art and are
considered to be included within the scope of the present
invention.
[0219] Accordingly, the invention includes the various subunits
that may comprise the enzymes, and accordingly extends to the genes
and corresponding proteins that may be encoded thereby, such as the
.alpha. (as well as PolC), .beta., .gamma., .epsilon., .tau.,
.delta. and .delta.' subunits, respectively. More particularly, in
Thermus thermophilus the .alpha. subunit corresponds to dnaE, the
.beta. subunit corresponds to dnaN, the .epsilon. subunit
corresponds to dnaQ, and the .gamma. and .tau. subunits correspond
to dnaX, the .delta. subunit corresponds to holA, and the .delta.'
subunit corresponds to holB. In Aquifex aeolicus and Thermotoga
maritima, the .alpha. subunit corresponds to dnaE, the .beta.
subunit corresponds to dnaN, the .epsilon. subunit corresponds to
dnaQ, the .tau. subunit corresponds to dnaX, the .delta. subunit
corresponds to holA, and the .delta.' subunit corresponds to holB.
In Bacillus stearothermophilus, the PolC which has both .alpha. and
.epsilon. activities corresponds to polC, the .beta. subunit
corresponds to dnaN, the .epsilon. subunit corresponds to dnaQ, the
.tau. subunit corresponds to dnaX, the .delta. subunit corresponds
to holA, and the .delta.' subunit corresponds to holB.
[0220] Accordingly, the Polymerase III-type enzyme of the present
invention comprises at least one gene encoding a subunit thereof,
which gene is selected from the group consisting of dnaX, dnaQ,
dnaE, dnaN, holA, holB, and combinations thereof. More
particularly, the invention extends to the nucleic acid molecule
encoding them and their encoded subunits.
[0221] In the T.th. Pol III enzyme, this includes the following
nucleotide sequences: dnaX (SEQ. ID. No. 3), dnaE (SEQ. ID. No.
86), dnaQ (SEQ. ID. No. 94), dnaN (SEQ. ID. No. 106), holA (SEQ.
ID. No. 157), and holB (SEQ. ID. No. 155).
[0222] In the A.ae. Pol III enzyme, this includes the following
nucleotide sequences: dnaX (SEQ. ID. No. 119), dnaE (SEQ. ID. No.
117), dnaQ (SEQ. ID. No. 127), dnaN (SEQ. ID. No. 121), holA (SEQ.
ID. No. 123), and holB (SEQ. ID. No. 125).
[0223] In the T.ma. Pol III enzyme, this includes the following
nucleotide sequences: dnaX (SEQ. ID. No. 141), dnaE (SEQ. ID. No.
137), dnaQ (SEQ. ID. No. 139), dnaN (SEQ. ID. No. 143), holA (SEQ.
ID. No. 145), and holB (SEQ. ID. No. 147).
[0224] In the B.st. Pol III enzyme, this includes the following
nucleotide sequences: dnaX (SEQ. ID. No. 181), dnaN (SEQ. ID. No.
173), holA (SEQ. ID. No. 177), holB (SEQ. ID. No. 179), and polC
(SEQ. ID. Nos. 183).
[0225] In each of the Pol III type enzymes of the present
invention, not only are each of the above-identified coding
sequences contemplated, but, also conserved variants, active
fragments and analogs thereof.
[0226] A particular T.th. Polymerase III-type enzyme in accordance
with the invention may include at least one of the following
sub-units: a .gamma. subunit having an amino acid sequence
corresponding to SEQ. ID. Nos. 4 and 5; a .tau. subunit having an
amino acid sequence corresponding to SEQ. ID. No. 2; a .epsilon.
subunit having an amino acid sequence corresponding to SEQ. ID. No.
95; a .alpha. subunit including an amino acid sequence
corresponding SEQ. ID. No. 87; a .beta. subunit having an amino
acid sequence corresponding to SEQ. ID. No. 107; a .delta. subunit
having an amino acid sequence corresponding to SEQ. ID. No. 158; a
.delta.' subunit having an amino acid sequence corresponding to
SEQ. ID. No. 156; as well as variants, including allelic variants,
muteins, analogs and fragments of any of the subunits, and
compatible combinations thereof, capable of functioning in DNA
amplification and sequencing.
[0227] A particular A.ae. Polymerase III-type enzyme in accordance
with the invention may include at least one of the following
sub-units: a .tau. subunit having an amino acid sequence
corresponding to SEQ. ID. No. 120; a .epsilon. subunit having an
amino acid sequence corresponding to SEQ. ID. No. 128; a .alpha.
subunit including amino acid sequence corresponding to SEQ. ID. No.
118; a .beta. subunit having an amino acid sequence corresponding
to SEQ. ID. No. 122; a .delta. subunit having an amino acid
sequence corresponding to SEQ. ID. No. 124; .delta.' subunit having
an amino acid sequence corresponding to SEQ. ID. No. 126; as well
as variants, including allelic variants, muteins, analogs and
fragments of any of the subunits, and compatible combinations
thereof, capable of functioning in DNA amplification and
sequencing.
[0228] A particular T.ma. Polymerase III-type enzyme in accordance
with the invention may include at least one of the following
sub-units: a .tau. subunit having an amino acid sequence
corresponding to SEQ. ID. No. 142; a .epsilon. subunit having an
amino acid sequence corresponding to SEQ. ID. No. 140; a .alpha.
subunit including an amino acid sequence corresponding to SEQ. ID.
No. 138; a .beta. subunit having an amino acid sequence
corresponding to SEQ. ID. No. 144; a .delta. subunit having an
amino acid sequence corresponding to SEQ. ID. No. 146; a .delta.'
subunit having an amino acid sequence corresponding to SEQ. ID. No.
148; as well as variants, including allelic variants, muteins,
analogs and fragments of any of the subunits, and compatible
combinations thereof, capable of functioning in DNA amplification
and sequencing.
[0229] A particular B.st. Polymerase III-type enzyme in accordance
with the invention may include at least one of the following
subunits: a .tau. subunit having a partial amino acid sequence
corresponding to SEQ. ID. No. 182; .beta. subunit having an amino
acid sequence corresponding to SEQ ID. No. 174; a .delta. subunit
having an amino acid sequence corresponding to SEQ. ID. No. 178; a
.delta.' subunit having an amino acid sequence corresponding to
SEQ. ID. No. 180; a PolC subunit having an amino acid sequence
corresponding to SEQ. ID. Nos. 184; as well as variants, including
allelic variants, muteins, analogs and fragments of any of the
subunits, and compatible combinations thereof, capable of
functioning in DNA amplification and sequencing.
[0230] The invention also includes and extends to the use and
application of the enzyme and/or one or more of its components for
DNA molecule amplification and sequencing by the methods set forth
hereinabove, and in greater detail later on herein.
[0231] One of the subunits of the invention is the T.th.
.gamma./.tau. subunit encoded by a dnaX gene, which frameshifts as
much as -2 with high efficiency, and that, upon frameshifting,
leads to the addition of more than one extra amino acid residue to
the C-terminus (to form the .gamma. subunit). Further, the
invention likewise extends to a dnaX gene derived from a
thermophile such as T.th., that possesses the frameshift defined
herein and that codes for expression of the .gamma. and .tau.
subunits of DNA Polymerase III.
[0232] The present invention provides methods for amplifying or
sequencing a nucleic acid molecule comprising contacting the
nucleic acid molecule with a composition comprising a DNA
polymerase III enzyme (DNA pol III) complex (for sequencing,
preferably a DNA pol III complex that is substantially reduced in
3'-5' exonuclease activity). DNA pol III complexes used in the
methods of the present invention are thermostable.
[0233] The invention also provides DNA molecules amplified by the
present methods, methods of preparing a recombinant vector
comprising inserting a DNA molecule amplified by the present
methods into a vector, which is preferably an expression vector,
and recombinant vectors prepared by these methods.
[0234] The invention also provides methods of preparing a
recombinant host cell comprising inserting a DNA molecule amplified
by the present methods into a host cell, which preferably a
bacterial cell, most preferably an Escherichia coli cell; a yeast
cell; or an animal cell, most preferably an insect cell, a nematode
cell or a mammalian cell. The invention also provides and
recombinant host cells prepared by these methods.
[0235] In additional preferred embodiments, the present invention
provides kits for amplifying or sequencing a nucleic acid molecule.
DNA amplification kits according to the invention comprise a
carrier means having in close confinement therein two or more
container means, wherein a first container means contains a DNA
polymerase III enzyme complex and a second container means contains
a deoxynucleoside triphosphate. DNA sequencing kits according to
the present invention comprise a multi-protein Pol III-type enzyme
complex and a second container means contains a dideoxynucleoside
triphosphate. The DNA pol III contained in the container means of
such kits is preferably substantially reduced in 5'-3' exonuclease
activity, may be thermostable; and may be isolated from the
thermophilic cellular sources described above.
[0236] DNA pol III-type enzyme complexes for use in the present
invention may be isolated from any organism that produced the DNA
pol III-type enzyme complexes naturally or recombinantly. Such
enzyme complexes may be thermostable, isolated from a variety of
thermophilic organisms.
[0237] The thermostable DNA polymerase III-type enzymes or
complexes that are an important aspect of this invention, may be
isolated from a variety of thermophilic bacteria that are available
commercially (for example, from American Type Culture Collection,
Rockville, Md.). Suitable for use as sources of thermostable
enzymes are the thermophilic eubacteria Aquifex aeolicus and other
species of the Aquifex genus; Thermus aquaticus, Thermus
thermophilus, Thermus flavus, Thermus ruber, Thermus brockianus,
and other species of the Thermus genus; Bacillus
stearothermophilus, Bacillus subtilis, and other species of the
Bacillus genus; Thermoplasma acidophilum and other species of the
Thermoplasma genus; Thermotoga neapolitana, Thermotoga maritima and
other species of the Thermotoga genus; and mutants of each of these
species. It will be understood by one of ordinary skill in the art,
however, that any thermophilic microorganism might be used as a
source of thermostable DNA pol III-type enzymes and polypeptides
for use in the methods of the present invention. Bacterial cells
may be grown according to standard microbiological techniques,
using culture media and incubation conditions suitable for growing
active cultures of the particular thermophilic species that are
well-known to one of ordinary skill in the art (see, e.g., Brock et
al., 1969; Oshima et al., 1974). Thermostable DNA pol III complexes
may then be isolated from such thermophilic cellular sources as
described for thermolabile complexes above.
[0238] Several methods are available for identifying homologous
nucleic acids and protein subunits in other thermophilic
eubacteria, either those listed above or otherwise. These methods
include the following:
[0239] (1) The following procedure was used to obtain the genes
encoding T.th. .epsilon. (dnaQ), .tau./.gamma. (dnaX), DnaA (dnaA),
and .beta. (dnaN). Protein sequences encoded by genes of
non-thermophilic bacteria (i.e., mesophiles) are aligned to
identify highly conserved amino acid sequences. PCR primers at
conserved positions are designed using the codon usage of the
organism of interest to amplify an internal section of the gene
from genomic DNA extracted from the organism. The PCR product is
sequenced. New primers are designed near the ends of the sequence
to obtain new sequence that flanks the ends using circular PCR
(also called inversed PCR) on genomic DNA that has been cut with
the appropriate restriction enzyme and ligated into circles. These
new PCR products are sequenced. The procedure is repeated until the
entire gene sequence has been obtained. Also, dnaN (encoding
.beta.) is located next to dnaA in bacteria and, therefore, dnaN
can be obtained by cloning DNA flanking the dnaA gene by the
circular PCR procedure starting within dnaA. Once the gene is
obtained, it is cloned into an expression vector for protein
production.
[0240] (2) The following procedure was used to obtain the genes
encoding T.th .alpha. polymerase (dnaE gene). The DNA polymerase
III can be purified directly from the organism of interest and
amino acid sequence of the subunit(s) obtained directly. In the
case of T.th., T.th. cells were lysed and proteins were
fractionated. An antibody against E. coli .alpha. was used to probe
column fractions by Western analysis, which reacted with T.th.
.alpha.. The T.th. .alpha. was transferred to a membrane,
proteolyzed, and fragments were sequenced. The sequence was used to
design PCR primers for amplification of an internal section of the
dnaE gene. Remaining flanking sequences are then obtained by
circular PCR.
[0241] (3) The following procedure can be used to identify
published nucleotide sequences which have not yet been identified
as to their function. This method was used to obtain T.th. .delta.
(holA) and .delta.' (holB), although they could presumably also
have been obtained via Methods 1 and 2 above. Discovery of T.th.
dnaE (.alpha.), dnaN (.beta.) and dnaX (.tau./.gamma.) indicates
that thermophiles use a class III type of DNA polymerase (.alpha.)
that utilize a clamp (.beta.) and must also use a clamp loader
since they have .tau./.gamma.. Also, the biochemical experiments in
the Examples infra show that the T.th. polymerase functions with
the T.th. .beta. clamp. Having demonstrated that a thermophile
(e.g., T.th.) does indeed utilize a class III type of polymerase
with a clamp and clamp loader, it can be assumed that they may have
.delta. and .delta.40 subunits needed to form a complex with
.tau./.gamma. for functional clamp loading activity (i.e., as shown
in E. coli, .delta. and .delta.' bind either .tau. or .gamma. to
form .tau..delta..delta.' or .gamma..delta..delta.' complex, both
of which are functional clamp loaders). The .delta. subunit is not
very well conserved, but does give a match in the sequence
databases for A.ae., T.ma, and T.th. The T.th. database provided
limited information on the aminoacid sequence of .delta. subunit,
although one can easily obtain the complete sequence of T.th. holA
by PCR and circular PCR as outlined above in Method 1. The A.ae.
and T.ma. databases are complete and, therefore, the entire holA
sequence from these genomes are identified. Neither database
recognized these sequences as .delta. encoded by holA. The .delta.'
subunit (holB) is fairly well conserved. Again the incomplete T.th.
database provided limited .delta.' sequence, but as with .delta.,
it is a straight forward process for anyone experienced in the area
to obtain the rest of the holB sequence using PCR and circular PCR
as described in Method 1. Neither the A.ae. nor T.ma. databases
recognized holB encoding .delta.'. Nevertheless, holB was
identified as encoding .delta.' by searching the databases with
.delta.' sequence. In each case, the Thermatoga maritima and
Aquifex aeolicus holB gene and .delta.' sequence were obtained in
their entirety. Neither database had previously annotated holA or
holB encoding .delta. and .delta.'.
[0242] As stated above and in accordance with the present
invention, once nucleic acid molecules have been obtained, they may
be amplified according to any of the literature-described manual or
automated amplification methods. Such methods includes, but are not
limited to, PCR (U.S. Pat. No. 4,683,195 to Mullis et al. and U.S.
Pat. No. 4,683,202 to Mullis), Strand Displacement Amplification
(SDA) (U.S. Pat. No. 5,455,166 to Walker), and Nucleic Acid
Sequence-Based. Amplification (NASBA) (U.S. Pat. No. 5,409,818 to
Davey et al.; EP 329,822 to Davey et al.). Most preferably, nucleic
acid molecules are amplified by the methods of the present
invention using PCR-based amplification techniques.
[0243] In the initial steps of each of these amplification methods,
the nucleic acid molecule to be amplified is contacted with a
composition comprising a DNA polymerase belonging to the
evolutionary "family A" class (e.g., Taq DNA pol I or E. coli pol
I) or the "family "B" class (e.g., Vent and Pfu DNA
polymerases--see Ito and Braithwaite, 1991). All of these DNA
polymerases are present as single subunits and are primarily
involved in DNA repair. In contrast, the DNA pol III-type enzymes
are multisubunit complexes that mainly function in the replication
of the chromosome, and the subunit containing the DNA polymerase
activity, is in the "family C" class.
[0244] Thus, in amplifying a nucleic acid molecule according to the
methods of the present invention, the nucleic acid molecule is
contacted with a composition comprising a thermostable DNA pol
III-type enzyme complex.
[0245] Once the nucleic acid molecule to be amplified is contacted
with the DNA pol III-type complex, the amplification reaction may
proceed according to standard protocols for each of the
above-described techniques. Since most of these techniques comprise
a high-temperature denaturation step, if a thermolabile. DNA pol
III-type enzyme complex is used in nucleic acid amplification by
any of these techniques the enzyme would need to be added at the
start of each amplification cycle, since it would be
heat-inactivated at the denaturation step. However, a thermostable
DNA pol III-type complex used in these methods need only be added
once at the start of the amplification (as for Taq DNA polymerase
in traditional PCR amplifications), as its activity will be
unaffected by the high temperature of the denaturation step. It
should be noted, however, that because DNA pol III-type enzymes may
have a much more rapid rate of nucleotide incorporation than the
polymerases commonly used in these amplification techniques, the
cycle times may need to be adjusted to shorter intervals than would
be standard.
[0246] In an alternative preferred embodiment, the invention
provides methods of extending primers for several kilobases, a
reaction that is central to amplifying large nucleic acid
molecules, by a technique commonly referred to, as "long chain PCR"
(Barnes, 1994; Cheng, 1994).
[0247] In such a method the target primed DNA can contain a single
strand stretch of DNA to be copied into the double strand form of
several or tens of kilobases. The reaction is performed in a
suitable buffer, preferably Tris, at a pH of between 5.5-9.5,
preferably 7.5. The reaction also contains MgCl.sub.2 in the range
1 mM to 10 mM, preferably 8 mM, and may contain a suitable salt
such as NaCl, KCl or sodium or potassium acetate. The reaction also
contains ATP in the range of 20 .mu.M to 1 mM, preferably 0.5 mM,
that is needed for the clamp loader to assemble the clamp onto the
primed template, and a sufficient concentration of deoxynucleoside
triphosphates in the range of 50 .mu.M to 0.5 mM, preferably 60
.mu.M for chain extension. The reaction contains a sliding clamp,
such as the .beta. subunit, in the range of 20 ng to 200 ng,
preferably 100 ng, for action as a clamp to stimulate the DNA
polymerase. The chain extension reaction contains a DNA polymerase
and a clamp loader, that could be added either separately or as a
single Pol III*-like particle, preferably as a Pol III* like
particle that contains the DNA polymerase and clamp loading
activities. The Pol III-type enzyme is added preferably at a
concentrations of about 0.0002-200 units per milliliter, about
0.002-100 units per milliliter, about 0.2-50 units per milliliter,
and most preferably about 2-50 units per milliliter. The reaction
is incubated at elevated temperature, preferably 60.degree. C. or
more, and could include other proteins to enhance activity such as
a single strand DNA binding protein.
[0248] In another preferred embodiment, the invention provides
methods of extending primers on linear templates in the absence of
the clamp loader. In this reaction, the primers are annealled to
the linear DNA, preferably at the ends such as in standard PCR
applications. The reaction is performed in a suitable buffer,
preferably Tris, at a pH of between 5.5-9.5, preferably 7.5. The
reaction also contains MgCl.sub.2 in the range of 1 mM to 10 mM,
preferably 8 mM; and may contain a suitable salt such as NaCl, KCl
or sodium or potassium acetate. The reaction also contains a
sufficient concentration of deoxynucleoside triphosphates in the
range of 50 .mu.M to 0.5 mM, preferably 60 .mu.M for chain
extension. Tne reaction contains a sliding clamp, such as the
.beta. subunit, in the range of 20 ng to 20 .mu.g, preferably about
2 .mu.g, for ability to slide on the end of the DNA and associate
with the polymerase for action as a clamp to stimulate the DNA
polymerase. The chain extension reaction also contains a Pol
III-type polymerase subunit such as .alpha., core, or a Pol
III*-like particle. The Pol III-type enzyme is added preferably at
a concentrations of about 0.0002-200 units per milliliter, about
0.002-100 units per milliliter, about 0.2-50 units per milliliter,
and most preferably about 2-50 units per milliliter. The reaction
is incubated at elevated temperature, preferably 60.degree. C. or
more, and could include other proteins to enhance activity such as
a single strand DNA binding protein.
[0249] The methods of the present invention thus will provide
high-fidelity amplified copies of a nucleic acid molecule in a more
rapid fashion than traditional amplification methods using the
repair-type enzymes.
[0250] These amplified nucleic acid molecules may then be
manipulated according to standard recombinant DNA techniques. For
example, a nucleic acid molecule amplified according to the present
methods may be inserted into a vector, which is preferably an
expression vector, to produce a recombinant vector comprising the
amplified nucleic acid molecule. This vector may then be inserted
into a host cell, where it may, for example, direct the host cell
to produce a recombinant polypeptide encoded by the amplified
nucleic acid molecule. Methods for inserting nucleic acid molecules
into vectors, and inserting these vectors into host cells, are
well-known to one of ordinary skill in the art (see, e.g.,
Maniatis, 1992).
[0251] Alternatively, the amplified nucleic acid molecules may be
directly inserted into a host cell, where it may be incorporated
into the host cell genome or may exist as an extrachromosomal
nucleic acid molecule, thereby producing a recombinant host cell.
Methods for introduction of a nucleic acid molecule into a host
cell, including calcium phosphate transfection, DEAE-dextran
mediated transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods, are
described in many standard laboratory manuals (see, e.g., Davis,
1986).
[0252] For each of the above techniques wherein an amplified
nucleic acid molecule is introduced into a host cell via a vector
or via direct introduction, preferred host cells include but are
not limited to a bacterial cell, a yeast cell, or an animal cell.
Bacterial host cells preferred in the present invention are E.
coli, Bacillus spp., Streptomyces spp., Erwinia spp., Klebsiella
spp. and Salmonella typhimurium. Preferred as a host cell is E.
coli, and particularly preferred are E. coli strains DH10B and
Stbl2, which are available commercially (Life. Technologies, Inc.
Gaithersburg, Md.). Preferred animal host cells are insect cells,
nematode cells and mammalian cells. Insect host cells preferred in
the present invention are Drosophila spp. cells, Spodoptera Sf9 and
Sf21 cells, and Trichoplusa High-Five cells, each of which is
available commercially (e.g., from Invitrogen; San Diego, Calif.).
Preferred nematode host cells are those derived from C. elegans,
and preferred mammalian host cells are those derived from rodents,
particularly rats, mice or hamsters, and primates, particularly
monkeys and humans. Particularly preferred as mammalian host cells
are CHO cells, COS cells and VERO cells.
[0253] By the present invention, nucleic acid molecules may be
sequenced according to any of the literature-described manual or
automated sequencing methods. Such methods include, but are not
limited to, dideoxy sequencing methods such as "Sanger sequencing"
(Sanger and Coulson, 1975; Sanger et al., 1977; U.S. Pat. No.
4,962,022 to Fleming et al.; and U.S. Pat. No. 5,498,523 to Tabor
et al.), as well as more complex PCR-based nucleic acid
fingerprinting techniques such as Random Amplified Polymorphic DNA
(RAPD) analysis (Williams et al., 1990). Arbitrarily Primed PCR
(AP-PCR) (Welsh and McClelland, 1990), DNA Amplification
Fingerprinting (DAF) (Caetano-Anolls, 1991), microsatellite PCR or
Directed Amplification of Minisatellite-region DNA (DAMD) (Heath et
al., 1993), and Amplification Fragment Length Polymorphism (AFLP)
analysis (EP 534,858 to Vos et al.; Vos et al., 1995; Lin and Kuo,
1995).
[0254] As described above for amplification methods, the nucleic
acid molecule to be sequenced by these methods is typically
contacted with a composition comprising a type A or type B DNA
polymerase. By contrast, in sequencing a nucleic acid molecule
according to the methods of the present invention, the nucleic acid
molecule is contacted with a composition comprising a thermostable
DNA pol III-type enzyme complex instead of necessarily using a DNA
polymerase of the family A or B classes. As for amplification
methods, the DNA pol III-type complexes used in the nucleic acid
sequencing methods of the present invention are preferably
substantially reduced in 3'-5' exonuclease activity; most
preferable for use in the present methods is a DNA polymerase
III-type complex which lacks the .epsilon. subunit. DNA pol
III-type complexes used for nucleic acid sequencing according to
the present methods are used at the same preferred concentration
ranges described above for long chain extension of primers.
[0255] Once the nucleic acid molecule to be sequenced is contacted
with the DNA pol III complex, the sequencing reactions may proceed
according to the protocols disclosed in the above-referenced
techniques.
[0256] As discussed above, the invention extends to kits for use in
nucleic acid amplification or sequencing utilizing DNA polymerase
III-type enzymes according to the present methods. A DNA
amplification kit according to the present invention may comprise a
carrier means, such as vials, tubes, bottles and the like. A first
such container means may contain a DNA polymerase III-type enzyme
complex, and a second such container means may contain a
deoxynucleoside triphosphate. The amplification kit encompassed by
this aspect of the present invention may further comprise
additional reagents and compounds necessary for carrying out
standard nucleic amplification protocols (See U.S. Pat. No.
4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis,
which are directed to methods of DNA amplification by PCR).
[0257] Similarly, a DNA sequencing kit according to the present
invention comprises a carrier means having in close confinement
therein two or mores container means, such as vials, tubes, bottles
and the like. A first such container means may contain a DNA
polymerase III-type enzyme complex, and a second such container
means may contain a dideoxynucleoside triphosphate. The sequencing
kit may further comprise additional reagents and compounds
necessary for carrying out standard nucleic sequencing protocols,
such as pyrophosphatase, agarose or polyacrylamide media for
formulating sequencing gels, and other components necessary for
detection of sequenced nucleic acids (See U.S. Pat. No. 4,962,020
to Fleming et al. and U.S. Pat. No. 5,498,523 to Tabor et al.,
which are directed to methods of DNA sequencing).
[0258] The DNA polymerase III-type complex contained in the first
container means of the amplification and sequencing kits provided
by the invention is preferably a thermostable DNA polymerase
III-type enzyme complex and more preferably a DNA polymerase
III-type enzyme complex that is reduced in 3-5' exonuclease
activity. Naturally, the foregoing methods and kits are presented
as illustrative and not restrictive of the use and application of
the enzymes of the invention for DNA molecule amplification and
sequencing. Likewise, the applications of specific embodiments of
the enzymes, including conserved variants and active fragments
thereof are considered to be disclosed and included within the
scope of the invention.
[0259] As discussed earlier, individual subunits could be modified
to customize enzyme construction and corresponding use and
activity. For example, the region of a that interacts with .beta.
could be subcloned onto another DNA polymerase, thereby causing
.beta. to enhance the activity of the recombinant polymerase.
Alternatively, the .beta. clamp could be modified to function with
another protein or enzyme thereby enhancing its activity or acting
to localize its action to a particular targeted DNA. Finally, the
polymerase active site could be modified to enhance its action, for
example changing Tyrosine enabling more equal site stoppage with
the four ddNTPs (Tabor et al., 1995). This represents a particular
non-limiting illustration of the scope and practice of the present
invention with reference to the utility of individual subunits
hereof.
[0260] Accordingly and as stated above, the present invention also
relates to a recombinant DNA molecule or cloned gene, or a
degenerate variant thereof, which encodes any one or all of the
subunits of the DNA Polymerase III-type enzymes of the present
invention, or active fragments thereof. In the instance of the
.tau. subunit, a predicted molecular weight of about 58 kD and an
amino acid sequence set forth in SEQ ID Nos. 4 or 5 is
comprehended; preferably a nucleic acid molecule, in particular a
recombinant DNA molecule or cloned gene, encoding the 58 kD subunit
of the Polymerase III of the invention, that has a nucleotide
sequence or is complementary to a DNA sequence shown in FIGS. 4A
and 4B (SEQ ID No. 1), and the coding region for dnaX set forth in
FIG. 4C (SEQ ID No. 3). The .gamma. subunit is smaller, and is
approximately 50 kD, depending upon the extent of the frameshift
that occurs. More particularly, and as set forth in FIG. 4E (SEQ ID
No. 4), the .gamma. subunit defined by a -1 frameshift possesses a
molecular weight of 50.8 kD, while the .gamma. subunit defined by a
-2 frameshift, set forth in FIG. 4F (SEQ ID No. 5), possesses a
molecular weight of 49.8 kD.
[0261] As discussed above, the invention also extends to the genes
including holA, holB, dnaX, dnaQ, dnaE, and dnaN from thermophilic
eubacteria (i.e., T.th. and A.ae.) that have been isolated and/or
purified, to corresponding vectors for the genes, and particularly,
to the vectors disclosed herein, and to host cells including such
vectors. In this connection, probes have been prepared which
hybridize to the DNA polymerase III type enzymes of the present
invention, and which are selected from the various oligonucleotide
probes or primers set forth in the present application. These
include, without limitation, the oligonucleotide defined in SEQ ID
No. 6 the oligonucleotide defined in SEQ ID No. 8 the
oligonucleotide defined in SEQ ID No. 10 the oligonucleotide
defined in SEQ ID No. 11 the oligonucleotide defined in SEQ ID No.
12 the oligonucleotide defined in SEQ ID No. 13 the
oligonucleotide-defined in SEQ ID No. 14 the oligonucleotide
defined in SEQ ID No. 15, and the oligonucleotide defined in SEQ ID
No. 16.
[0262] The methods of the invention include a method for producing
a recombinant thermostable DNA polymerase III-type enzyme from a
thermophilic bacterium, such as T.th., A.ae., Th.ma., or B.st.
which comprises culturing a host cell transformed with a vector of
the invention under conditions suitable for the expression of the
present DNA polymerase III. Another method includes a method for
isolating a target DNA fragment consisting essentially of a DNA
coding for a thermostable DNA polymerase III-type enzyme from a
thermophilic bacterium comprising the steps of:
[0263] (a) forming a genomic library from the bacterium;
[0264] (b) transforming or transfecting an appropriate host cell
with the library of step (a);
[0265] (c) contacting DNA from the transformed or transfected host
cell with a DNA probe which hybridizes to a DNA fragment selected
from the group consisting of the DNA fragments defined in SEQ ID
No. 6 and the DNA fragments defined in SEQ ID No. 8 or the
oligonucleotides set forth above; wherein hybridization is
conducted under the following conditions:
[0266] i) hybridization: 1% crystalline BSA (fraction V) (Sigma), 1
mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS at 65.degree. C. for 12
hours and;
[0267] ii) wash: 5.times.20 minutes with wash buffer consisting of
0.5% BSA, fraction V), 1 mM Na2EDTA, 40 mM NaHPO4 (pH 7.2), and 5%
SDS;
[0268] (d) assaying the transformed or transfected cell of step (c)
which hybridizes to the DNA probe for DNA polymerase III-type
activity; and
[0269] (e) isolating a target DNA fragment which codes for the
thermostable DNA polymerase III-type enzyme.
[0270] Also, antibodies including both polyclonal and monoclonal
antibodies, and the DNA Polymerase III-like enzyme complex and/or
their .gamma. and .tau. subunits, .alpha. subunits(s), .delta.
subunit, .delta.' subunit, .beta. subunit, .epsilon. subunit may be
used in the preparation of the enzymes of the present invention as
well as other enzymes of similar thermophilic origin. For example,
the DNA Polymerase III-type complex or its subunits may be used to
produce both polyclonal and monoclonal antibodies to themselves in
a variety of cellular media, by known techniques such as the
hybridoma technique utilizing, for example, fused mouse spleen
lymphocytes and myeloma cells.
[0271] The general methodology for making monoclonal antibodies by
hybridomas is well known. Immortal, antibody-producing cell lines
can also be created by techniques other than fusion, such as direct
transformation of B lymphocytes with oncogenic DNA, or transfection
with Epstein-Barr virus. See, e.g., Schreier et al., 1980;
Hammerling et al., 1981; Kennett et al., 1980; see also U.S. Pat.
No. 4,341,761 to Ganfield et al.; U.S. Pat. No. 4,399,121 to
Albarella et al.; U.S. Pat. No. 4,427,783 to Newman et al.; U.S.
Pat. No. 4,444,887 to Hoffman; U.S. Pat. No. 4,451,570 to Royston
et al.; U.S. Pat. No. 4,466,917 to Nussenzweig et al.; U.S. Pat.
No. 4,472,500 to Milstein et al.; U.S. Pat. No. 4,491,632 to Wands
et al.; and U.S. Pat. No. 4,493,890 to Morris.
[0272] Methods for producing polyclonal anti-polypeptide antibodies
are well-known in the art. See U.S. Pat. No. 4,493,795 to Nestor et
al. A monoclonal antibody, typically containing Fab and/or
F(ab').sub.2 portions of useful antibody molecules, can be prepared
using the hybridoma technology described in Antibodies--A
Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor
Laboratory, New York (1988), which is incorporated herein by
reference. Briefly, to form the hybridoma from which the monoclonal
antibody composition is produced, a myeloma or other
self-perpetuating cell line is fused with lymphocytes obtained from
the spleen of a mammal hyperimmunized with an elastin-binding
portion thereof.
[0273] A monoclonal antibody useful in practicing the present
invention can be produced by initiating a monoclonal hybridoma
culture comprising a nutrient medium containing a hybridoma that
secretes antibody molecules of the appropriate antigen specificity.
The culture is maintained under conditions and for a time period
sufficient for the hybridoma to secrete the antibody molecules into
the medium. The antibody-containing medium is then collected. The
antibody molecules can then be further isolated by well-known
techniques.
[0274] Media useful for the preparation of these compositions are
both well-known in the art and commercially available and include
synthetic culture media, inbred mice and the like. An exemplary
synthetic medium is Dulbecco's minimal essential medium (DMEM)
(Dulbecco et al., 1959) supplemented with 4.5 gm/l glucose, 20 mm
glutamine, and 20% fetal calf serum. An exemplary inbred mouse
strain is the Balb/c.
[0275] Another feature of this invention is the expression of the
DNA sequences disclosed herein. As is well known in the art, DNA
sequences may be expressed by operatively linking them to an
expression control sequence in an appropriate expression vector and
employing that expression vector to transform an appropriate
unicellular host.
[0276] Such operative linking of a DNA sequence of this invention
to an expression control sequence, of course, includes, if not
already part of the DNA sequence, the provision of an initiation
codon, ATG, in the correct reading frame upstream of the DNA
sequence.
[0277] A wide variety of host/expression vector combinations may be
employed in expressing the DNA sequences of this invention. Useful
expression vectors, for example, may consist of segments of
chromosomal, non-chromosomal and synthetic DNA sequences. Suitable
vectors include derivatives of SV40 and known bacterial plasmids,
e.g., E. coli plasmids col E1, pCR1, pBR322, pMB9 and their
derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous
derivatives of phage .lambda., e.g., NM989, and other phage DNA,
e.g., M13 and filamentous single stranded phage DNA; yeast plasmids
such as the 2.mu. plasmid or derivatives-thereof; vectors useful in
eukaryotic cells, such as vectors useful in insect or mammalian
cells; vectors derived from combinations of plasmids and phage
DNAs, such as plasmids that have been modified to employ phage DNA
or other expression control sequences; and the like.
[0278] Any of a wide variety of expression control
sequences--sequences that control the expression of a DNA sequence
operatively linked to it--may be used in these vectors to express
the DNA sequences of this invention. Such useful expression control
sequences include, for example, the early or late promoters of
SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp
system, the TAC system, the TRC system, the LTR system, the major
operator and promoter regions of phage .lambda., the control
regions of fd coat protein, the promoter for 3-phosphoglycerate
kinase or other glycolytic enzymes, the promoters of acid
phosphatase (e.g., Pho5), the promoters of the yeast .alpha.-mating
factors, and other sequences known to control the expression of
genes of prokaryotic or eukaryotic cells or their viruses, and
various combinations thereof.
[0279] A wide variety of unicellular host cells are also useful in
expressing the DNA sequences of this invention. These hosts may
include well known eukaryotic and prokaryotic hosts, such as
strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such
as yeasts, and animal cells, such as CHO, R1.1, B-W and L-M cells,
African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40,
and BMT10), insect cells (e.g., Sf9), and human cells and plant
cells in tissue culture.
[0280] It will be understood that not all vectors, expression
control sequences and hosts will function equally well to express
the DNA sequences of this invention. Neither will all hosts
function equally well with the same expression system; However, one
skilled in the art will be able to select the proper vectors,
expression control sequences, and hosts without undue
experimentation to accomplish the desired, expression without
departing from the scope of this invention. For example, in
selecting a vector, the, host must be considered because the vector
must function in it. The vector's copy number, the ability to
control that copy number, and the expression of any other proteins
encoded by the vector, such as antibiotic markers, will also be
considered.
[0281] In selecting an expression control sequence, a variety of
factors will normally be considered. These include, for example,
the relative strength of the system, its controllability, and its
compatibility with the particular DNA sequence or gene to be
expressed, particularly with regard to potential secondary
structures. Suitable unicellular hosts will be selected by
consideration of, e.g., their compatibility with the chosen vector,
their secretion characteristics, their ability to fold proteins
correctly, and their fermentation requirements, as well as the
toxicity to the host of the product encoded by the DNA sequences to
be expressed, and the ease of purification of the expression
products.
[0282] Considering these and other factors a person skilled in the
art will be able to construct a variety of vector/expression
control sequence/host combinations that will express the DNA
sequences of this invention on fermentation or in large scale
animal culture.
[0283] It is further intended that analogs may be prepared from
nucleotide sequences of the protein complex/subunit derived within
the scope of the present invention. Analogs, such as fragments, may
be produced, for example, by pepsin digestion of bacterial
material. Other analogs, such as muteins, can be produced by
standard site-directed mutagenesis of dnaX, dnaE, dnaQ, dnaN, holA,
or holB coding sequences. Especially useful may be a mutation in
dnaE that provides the polymerase with the ability to incorporate
all four ddNTPs with equal efficiency thereby producing an even
binding pattern in sequencing gels, as discussed above and with
reference to Tabor et al., 1995.
[0284] As mentioned above, a DNA sequence corresponding to dnaX,
dnaQ, holA, holB, dnaE, or dnaN, or encoding the subunits of the
DNA Polymerase M of the invention can be prepared synthetically
rather than cloned. The DNA sequence can be designed with the
appropriate codons for the amino acid sequence of the subunit(s) of
interest. In general, one will select preferred codons for the
intended host if the sequence will be used for expression. The
complete sequence is assembled from overlapping oligonucleotides
prepared by standard methods and assembled into a complete coding
sequence (Edge, 1981; Nambair et al., 1984; Jay et al., 1984).
[0285] Synthetic DNA sequences allow convenient construction of
genes which will express DNA Polymerase III analogs or "muteins".
Alternatively, DNA encoding muteins can be made by site-directed
mutagenesis of native dnaX, dnaQ, holA, holB, dnaE or dnaN genes or
their corresponding cDNAs, and muteins can be made directly using
conventional polypeptide synthesis.
[0286] A general method for site-specific incorporation of
unnatural amino acids into proteins is described in Noren et al.,
1989. This method may be used to create analogs with unnatural
amino acids.
GENERAL DESCRIPTION OF THE INVENTION
[0287] As discussed above, the present invention has as one of its
characterizing features, that a Polymerase III-type enzyme as
defined hereinabove, has been discovered in a thermophile, that has
the structure and function of a chromosomal replicase. This
structure and function confers significant benefit when the enzyme
is employed in procedures such as PCR where speed and accuracy of
DNA reconstruction is crucial.
[0288] Chromosomal replicases are composed of several subunits in
all organisms (Kornberg and Baker, 1992). In keeping with the need
to replicate long chromosomes, replicases are rapid and highly
processive multiprotein machines. All cellular replicases examined
to date derive their processivity from one subunit that is shaped
like a ring and completely encircles DNA (Kuriyan and O'Donnell,
1993; Kelman and O'Donnell, 1994). This "sliding clamp" subunit
acts as a mobile tether for the polymerase machine (Stukenberg et
al., 1991). The sliding clamp does not assemble onto the DNA by
itself, but requires a complex of several proteins, called a "clamp
loader" which couples ATP hydrolysis to the assembly of sliding
clamps onto DNA (O'Donnell et al., 1992). Hence, Pol III-type
cellular replicases are comprised of three components: a clamp, a
clamp loader, and the DNA polymerase.
[0289] An overall goal is to identify and isolate all of the genes
encoding the replicase subunits from a thermophile for expression
and purification in large quantity. Following this, the replication
apparatus can be reassembled from individual subunit components for
use in kits, PCR, sequencing and diagnostic applications (Onrust et
al., 1995).
[0290] As a beginning to identify and characterize the replicase of
a thermophile, we started: by looking for a homologue to the
prokaryotic dnaX gene which encode subunits (.gamma. and .tau.) of
the replicase. The dnaX gene has another homologue, holB, which
encodes yet another subunit (.delta.') of the replicase. The amino
acid sequence of .delta.' (encoded by holA) and .tau./.gamma.
subunits (encoded by dnaX) are particularly highly conserved in
evolution from prokaryotes to eukaryotes (Chen et. al., 1992;
O'Donnell et al., 1993; Onrust et al., 1993; Carter et al., 1993;
Cullman et al., 1995).
[0291] One organism chosen for study and exposition herein is the
exemplary extreme thermophile Thermus thermophilus (T.th.). It is
understood that other members of the class such as the eubacterium
Thermatoga are expected to be analogous in both structure and
function. Thus, the investigation of T.th. proceeded and initially,
a T.th. homologue of dnaX was identified. The gene encodes a full
length protein of 529 amino acids. The amino terminal third of the
sequence shares over 50% homology to dnaX genes as divergent as E.
coli (gram negative) and B. subtilis (gram positive). The T.th.
dnaX gene contains a DNA sequence that provides a translational
frameshift signal for production of two proteins from the same
gene. Such frameshifting has been documented only in the case of E.
coli (Tsuchihashi and Kornberg, 1990; Flower and McHenry, 1990;
Blinkowa and Walker, 1990). No frameshifting has been documented to
occur in the dnaX homologues (RFC subunit genes) of yeast and
humans (Eukaryotic kingdom).
[0292] The presence of a dnaX gene that produces two, subunits
implies that T.th. has a clamp loader (.gamma.) and may be
organized by .tau. into a PolIII*-type replicase like the
replicative DNA polymerase of Escherichia coli, DNA polymerase III
holoenzyme. The E. coli DNA polymerase III holoenzyme contains 10
different subunits, some in copies of two or more for a total
composition of 18 polypeptide chains (Kornberg and Baker, 1992;
Onrust et al., 1995). The holoenzyme is composed of three major
activities: the 3-subunit DNA polymerase core
(.alpha..epsilon..theta.), the .beta. subunit DNA sliding clamp,
and the 5-subunit .gamma. complex clamp loader
(.gamma..delta..delta.'.chi..psi.)- . This 3 component strategy
generalizes to eukaryotes which utilize a clamp (PCNA) and a
5-subunit RFC clamp loader (RFC) which provide processivity to DNA
polymerase .delta. (reviewed in Kelman and O'Donnell, 1994).
[0293] In E. coli, the polymerase and clamp loader components are
organized into one PolIII* particle by the .tau. subunit, that acts
as a "glue" protein (Onrust et al., 1995). One dimer of .tau. holds
together two core polymerases in the particle which are utilized
for the coordinated and simultaneous replication of both strands of
duplex DNA (McHenry, 1982; Maki et al., 1988; Yuzhakov et al.,
1996). The "glue" protein .tau. subunit also binds one clamp loader
(called .gamma. complex) thereby acting as a scaffold for a large
superstructure assembly called DNA polymerase III*. The gene
encoding .tau., called dnaX, also encodes the .gamma. subunit of
DNA polymerase III. The .beta. subunit then associates with Pol
III* to form the DNA polymerase III holoenzyme. The .gamma. subunit
is approximately {fraction (2/3)} the length of .tau.. .gamma.
shares the N-terminus of .tau., but is truncated by a translational
frameshifting mechanism that, after the shift, encounters a stop
codon within two amino acids (Tsuchihashi and Kornberg, 1990;
Flower and McHenry, 1990; Blinkowa and Walker, 1990). Hence,
.gamma. is the N-terminal 453 amino acids of .tau., but contains
one unique residue at the C-terminus (the penultimate codon encodes
a Lys residue which is the same sequence as if the frameshift did
not take place). This frameshift is highly efficient and occurs
approximately 50% of the time.
[0294] The sequence of the .gamma. and .tau. subunits, encoded by
the dnaX gene are homologous to the clamp loading subunits in all
other organisms extending from gram negative bacteria through grain
positive bacteria, the Archeae Kingdom and the Eukaryotic Kingdom
from yeast to humans (O'Donnell et al., 1993). All of these
organisms utilize a three component replicase (DNA polymerase,
clamp and clamp loader) and in these cases the 3 components appear
to behave as independent units in solution rather than forming a
large holoenzyme superstructure. For example, in eukaryotes from
yeast to humans, the clamp loader is the five subunit RFC, the
clamp is PCNA, and the polymerases .delta. and .epsilon. are all
stimulated by the PCNA clamp assembled onto primed DNA by RFC
(reviewed in Kelman and O'Donnell 1994).
[0295] The discovery of a dnaX gene in T.th. provided confidence
that thermophilic bacteria would contain a three component Pol
III-type enzyme. Hence, we proceeded to identify the dnaQ and dnaN
genes encoding, respectively, the proofreading 3-5' exonuclease,
and the .beta. DNA sliding clamp subunits of a Pol III-type enzyme.
Following this, we purified from extracts of T.th. cells, a Pol
III-type enzyme. This enzyme preparation had the unique property of
extending a single primer around a long 7.2 kb single strand DNA
genome of M13 mp18 bacteriophage. Such a primer extension assay
serves as a tool to detect and identify the Pol III-type of enzyme
in cell extracts. The enzyme was confirmed to be a Pol III-type
enzyme based on its reactivity with antibody directed against the
E. coli .alpha. subunit (the DNA polymerase subunit) and antibody
directed against E. coli .gamma. subunit. Proteins corresponding to
.alpha., .tau., .gamma., .delta. and .delta.' were easily visible
and lend themselves to identification of the genes through use of
peptide microsequencing followed by primer design for PCR
amplification. For example, from this DNA pol III-type preparation,
the peptide sequence of the .alpha. subunit was obtained, which
then allowed the dnaE gene encoding the .alpha. subunit (DNA
polymerase) of the Pol III-type enzyme to be obtain.
[0296] These methods should be widely applicable to other
thermophilic bacteria. Additional antibody reagents against other
Pol III-type enzyme components, such as RFC subunits, DNA
polymerase delta, epsilon or beta, and the PCNA clamp from known
organisms can be made quite easily as polyclonal or monoclonal
antibody preparations using as antigen either naturally purified
sequence, recombinant sequence, or synthetic peptide sequence.
Examples of known sequences of these Pol III-type enzymes are to be
found in: DNA polymerases (Braithwaite and Ito, 1993), RFC clamp
loaders (Cullman et al., 1995) and PCNA (Kelman and O'Donnell,
1995).
[0297] The remaining genes of T.th. Pol III needed for efficient
extension of primed templates, holA and holB, are now identified.
The holA coding sequence (SEQ. ID. No. 157) encodes the .delta.
subunit (SEQ. ID. No. 158) and the holB coding sequence (SEQ. ID.
No. 155) encodes the .delta.' subunit (SEQ. ID. No. 156). The holA
and holB coding sequences and the .delta. and .delta.' subunits
were identified via BLAST search (Altschul et al., 1997), and
subsequently isolated following circular PCR. These genes will
provide the subunit preparations through use of standard
recombinant techniques and protein purification protocols. The
protein subunits can then be used to reconstitute the enzyme
complexes as they exist in the cell. This type of reconstitution of
Pol III has been demonstrated using the protein subunits of DNA
polymerase III holoenzyme from E. coli to assemble the entire
particle. See, e.g., U.S. Pat. Nos. 5,583,026 and 5,668,004 to
O'Donnell; and Onrust et al., 1995. The disclosures of these
references are incorporated herein in their entireties.
[0298] Another organism chosen for study and exposition herein is
the extreme thermophile Aquifex aeolicus. Thus, the present
invention also relates to various isolated DNA molecules from
Aquifex aeolicus, in particular the DNA molecules encoding various
replication proteins. These include dnaE, dnaX, dnaE, holA, holB,
ssb DNA molecules from A. aeolicus. These DNA molecules can be
inserted into an expression system or used to transform host cells
from which isolated proteins can be obtained. The isolated proteins
encoded by these DNA molecules are also disclosed.
[0299] Unless otherwise indicated below, the Aquifex aeolicus
sequences were obtained by sequence comparisons using the Thermus
thermophilus counterparts as query against the genome of Aquifex
aeolicus. (Deckert et al., 1998).
[0300] The A. aeolicus dnaE gene has a nucleotide coding sequence
according to SEQ. ID. No. 117 and encodes the .alpha. subunit of
the of DNA Polymerase III, which has an amino acid sequence
according to SEQ. ID. No. 118. The A.ae. .alpha. subunit has
approximately 41% aa identity to the T.th. .alpha. subunit.
[0301] The A. aeolicus dnaX gene has a nucleotide coding sequence
according to SEQ. ID. No. 119 and encodes the .tau. subunit of the
of DNA Polymerase III, which has an amino acid sequence according
to SEQ. ID. No. 120. The A.ae. .tau. subunit has approximately 51%
aa identity to the T.th. .tau. subunit.
[0302] The A. aeolicus dnaN gene has a nucleotide coding sequence
according to SEQ. ID. No. 121 and encodes the .beta. subunit of DNA
Polymerase III, which has an amino acid sequence according to SEQ.
ID. No. 122. The A.ae. .beta. subunit has approximately 27% aa
identity to the T.th. .beta. subunit.
[0303] The A. aeolicus dnaQ gene has a nucleotide coding sequence
according to SEQ. ID. No. 127 and encodes the .epsilon. subunit of
the of DNA Polymerase III, which has an amino acid sequence
according to SEQ. ID. No. 128. The A.ae. .epsilon. subunit has
approximately 26% aa identity to the T.th. .epsilon. subunit.
[0304] The A. aeolicus ssb gene has a nucleotide coding sequence
according to SEQ. ID. No. 129 and encodes the SSB protein, which
has an amino acid sequence according to SEQ. ID. No. 130. The A.ae
SSB protein has approximately 22% aa identity to the T.th. SSB
protein.
[0305] Further, the coding sequences of A. aeolicus genes encoding
the helicase (dnaB), helicase loader (dnaC); and primase (dnaG) are
also disclosed. The A. aeolicus dnaB gene has a nucleotide coding
sequence according to SEQ. ID. No. 131 and encodes the DnaB
protein, which functions as a helicase and has an amino acid
sequence according to SEQ. ID. No. 132. The A. aeolicus dnaG gene
has a nucleotide coding sequence according to SEQ. ID. No. 133 and
encodes the DnaG protein, which functions as a primase and has an
amino acid sequence according to SEQ. ID. No. 134. The A. aeolicus
dnaC gene has a nucleotide coding sequence according to SEQ. ID.
No. 135 and encodes the DnaC protein, which functions as a helicase
loader and has an amino acid sequence according to SEQ. ID. No.
136.
[0306] The A. aeolicus holA and holB genes were previously
unidentified by Deckert et al., 1998. Using Thermus thermophilus
.delta.' subunit amino acid sequence and the Thermatoga maritima
.delta. subunit amino acid sequence (SEQ. ID. No. 146 which itself
was obtained using the T.th. .delta. subunit amino acid sequence of
SEQ. ID. No. 158) in separate BLAST searches (Altschul et al.,
1997), corresponding polypeptide products in Aquifex aeolicus were
identified. The A. aeolicus holA gene has a nucleotide coding
sequence according to SEQ. ID. No. 123 and encodes the .delta.
subunit of the of DNA Polymerase III, which has an amino acid
sequence according to SEQ. ID. No. 124. The A.ae. .delta. subunit
has approximately 21% aa identity to the T.m. .delta. subunit. The
A. aeolicus holB gene has a nucleotide coding sequence according to
SEQ. ID. No. 125 and encodes the .delta.' subunit of the of DNA
Polymerase III, which has an amino acid sequence according to SEQ.
ID. No. 126. The A.ae. .delta.' subunit has approximately 24% aa
identity to the T.th. .delta.' subunit.
[0307] This invention also clones at least the coding regions of a
set of A. aeolicus genes which encode proteins that assemble into
an A. aeolicus DNA polymerase III replication enzyme. These genes
(dnaE, dnaN, dnaX, dnaQ, holA, holB, ssb) were cloned into
expression vectors, the proteins were expressed in E. coli, and the
corresponding protein subunits were purified (alpha, beta, tau,
delta, delta prime, SSB). This invention identifies the major
protein-protein contacts among these subunits, shows how these
proteins can be assembled into higher order multiprotein complexes,
and how to form a rapid and processive DNA polymerase III
holoenzyme.
[0308] In contrast to the E. coli and T. thermophilus dnaX genes
which encode both .tau. and .gamma. subunits, the A. aeolicus dnaX
gene produces only the full length .tau. subunit when expressed in
E. coli. The A. aeolicus .tau. is intermediate in length between
the .gamma. and .tau. subunits of E. coli DNA polymerase III
holoenzyme. The E. coli .tau. binds .alpha.; the .gamma. subunit
does not bind .alpha.. Due to the intermediate size of A. aeolicus
.tau., it was not known whether the A. aeolicus .tau. would bind
the .alpha. subunit. This invention shows that indeed, the A.
aeolicus .tau. binds to .alpha., as well as .delta. and .delta.',
thereby forming an A. aeolicus .alpha..tau..delta..delta.' complex.
Until the identification of the .delta. and .delta.' subunits by
the present invention, their existence, let alone their interaction
with .tau. and .alpha., was not even known.
[0309] The A. aeolicus .alpha..tau..delta..delta.'/.beta. Pol III
can be applied in several useful DNA handling techniques. For
example, the thermophilic Pol III will be useful in DNA sequencing,
especially at high temperature. Also, use of a thermal resistant
rapid and processive Pol III is an important improvement to
polymerase chain reaction technology. The ability of the A.
aeolicus Pol III to extend primers for multiple kilobases makes
possible the amplification of very long segments of DNA (long chain
PCR).
[0310] Another organism chosen for study and exposition herein is
the extreme thermophile Thermotoga maritima. Thus, the present
invention also relates to various isolated DNA molecules from
Thermotoga maritima, in particular the DNA molecules encoding
various replication proteins. These include dnaE, dnaX, dnaN, dnaQ,
holA, holB, ssb DNA molecules from Thermotoga maritima. These DNA
molecules can be inserted into an expression system or used to
transform host cells from which isolated proteins can be obtained.
The isolated proteins encoded by these DNA molecules are also
disclosed.
[0311] Unless otherwise indicated below, the Thermotoga maritima
sequences were obtained by sequence comparisons using the Thermus
thermophilus counterparts as query against the genome of Thermotoga
maritima (Nelson et al., 1999).
[0312] The T. maritima dnaE gene has a nucleotide coding sequence
according to SEQ. ID. No. 137 and encodes the .alpha. subunit of
the of DNA Polymerase III, which has an amino acid sequence
according to SEQ. ID. No. 138. The T.m. .alpha. subunit has
approximately 33% aa identity to the T.th. .alpha. subunit.
[0313] The T. maritima dnaQ gene has a nucleotide coding sequence
according to SEQ. ID. No. 139 and encodes the .epsilon. subunit of
the of DNA Polymerase III, which has an amino acid sequence
according to SEQ; ID. No. 140. The T.m. .epsilon. subunit has
approximately 34% aa identity to the T.th. .epsilon. subunit.
[0314] The T. maritima dnaX gene has a nucleotide coding sequence
according to SEQ. ID. No. 141 and encodes the .tau. subunit of the
of DNA Polymerase III, which has an amino acid sequence according
to SEQ. ID. No. 142. The T.m. .tau. subunit has approximately 48%
aa identity to the T.th. .tau. subunit.
[0315] The T. maritima dnaN gene has a nucleotide coding sequence
according to SEQ. ID. No. 143 and encodes the .beta. subunit of DNA
Polymerase III, which has an amino acid sequence according to SEQ.
ID. No. 144. The T.m. .beta. subunit has approximately 28% aa
identity to the T.th. .beta. subunit.
[0316] The T. maritima ssb gene has a nucleotide coding sequence
according a to SEQ. ID. No. 149 and encodes the SSB protein, which
has an amino acid sequence according to SEQ. ID. No. 150. The T.m.
SSB protein has approximately 18% aa identity to the T.th. SSB
protein.
[0317] Further, the coding sequences of T. maritima a genes
encoding the helicase (dnaB) and primase (dnaG) are also disclosed.
The T. maritima dnaB gene has a nucleotide coding sequence
according to SEQ. ID. No. 151 and encodes the DnaB protein, which
factions as a helicase and has an amino acid sequence according to
SEQ. ID. No. 152. The T. maritima dnaG gene has a nucleotide coding
sequence according to SEQ. ID. No. 153 and encodes the DnaG
protein, which functions as a primase and has an amino acid
sequence according to SEQ. ID. No. 154.
[0318] The T. maritima holA and holB genes were previously
unidentified by Nelson et al., 1999); Using the Thermus
thermophilus .delta. and .delta.' subunit amino acid sequences
(SEQ. ID. Nos. 158 and 156, respectively) in separate BLAST
searches (Altschul et al., 1997), corresponding polypeptide
products in T. maritima were identified. The T. maritima holA gene
has a nucleotide coding sequence according to SEQ. ID. No. 145 and
encodes the .delta. subunit of the of DNA Polymerase III, which has
an amino acid sequence according to SEQ. ID. No. 146. The T.m.
.delta. subunit has approximately 37% aa identity to the T.th.
.delta. subunit. The T.m. holB gene has a nucleotide coding
sequence according to SEQ. ID. No. 147 and encodes the .delta.'
subunit which has an amino acid sequence according to SEQ. ID. No.
148. The T.m. .delta.' subunit has approximately 25% aa identity to
the T.th. .delta.' subunit.
[0319] Yet another organism chosen for study and exposition herein
is the extreme thermophile Bacillus stearothermophilus. Thus, the
present invention also relates to various isolated DNA molecules
from Bacillus stearothermophilus, in particular the DNA molecules
encoding various replication proteins. These include dnaE, dnaX,
dnaN, dnaQ, holA, holB, ssb DNA molecules from Bacillus
stearothermophilus. These DNA molecules can be inserted into an
expression system or used to transform host cells from which
isolated proteins can be obtained. The isolated proteins encoded by
these DNA molecules are also disclosed.
[0320] Unless otherwise indicated below, the Bacillus
stearothermophilus sequences were obtained by searching the
database of this organism (at http://www.genome.ou.edu).
[0321] The B. stearothermophilus polC gene has a nucleotide coding
sequence according to SEQ. ID. No. 183 and encodes the PolC or
.alpha.-large subunit of the DNA Polymerase III, which has an amino
acid sequence according to SEQ. ID. No. 184. The B.st. PolC
subunit, like the PolC subunits of other Gram positive organisms,
contains both polymerase and 3'-5' exonuclease activity. This
subunit, therefore, is essentially a fusion of .alpha. and
.epsilon..
[0322] The B. stearothermophilus dnaX gene has a partial nucleotide
coding sequence according to SEQ. ID. No. 181 and encodes the .tau.
subunit of the of DNA Polymerase III, which has a partial amino
acid sequence according to SEQ. ID. No. 182. The B.st. .tau.
subunit has approximately 31% aa identity to the T.th. .tau.
subunit.
[0323] The B. stearothermophilus dnaN gene has a partial nucleotide
coding sequence according to SEQ. ID. No. 173 and encodes the
.beta. subunit of DNA Polymerase III, which has a partial amino
acid sequence according to SEQ. ID. No. 174. The B.st. .beta.
subunit has approximately 21% aa identity to the T.th. .beta.
subunit.
[0324] The B. stearothermophilus ssb gene has a nucleotide coding
sequence according to SEQ. ID. No. 175 and encodes the SSB protein,
which has an amino acid sequence according to SEQ. ID. No. 176. The
B.st. SSB protein has approximately 23% aa identity to the T.th.
SSB protein.
[0325] The B. stearothermophilus holA gene has a nucleotide coding
sequence according to SEQ. ID. No. 177 and encodes the .delta.
subunit of DNA Polymerase III, which has an amino acid sequence
according to SEQ. ID. No. 178. The B.st. .delta. subunit has
approximately 26% aa identity to the T.th. .delta. subunit.
[0326] The B. stearothermophilus holB gene has a nucleotide coding
sequence according to SEQ. D. No. 179 and encodes the .delta.'
subunit of DNA Polymerase III, which has an amino acid sequence
according to SEQ. ID. No. 180. The B.st. .delta.' subunit has
approximately 25% aa identity to the T.th. .delta.' subunit.
[0327] By conducting BLAST searches of unidentified genomic. DNA
from other thermophilic eubacteria, it is possible to identify
coding regions which encode various functional subunits of other
Pol III replicative machinery.
[0328] Although it is generally appreciated that proteins isolated
from a thermophile should retain activity at high temperature,
there is no guarantee that they will retain temperature resistance
when isolated in pure form. This invention shows that the A.
aeolicus Pol III, like the T. thermophilus Pol III, is resistant to
high temperature. It is expected that the Th. maritima and B.
stearothermophilus Pol III enzymes will similarly be resistant to
high temperature.
[0329] The following experiments illustrate the identification and
characterization of the enzymes and constructs of the present
invention. Accordingly, in Examples 1-8 below, the identification
and expression of the .gamma. and .tau. is presented, as the first
step in the elucidation of the Thermus thermophilus Polymerase III
reflective of the present invention. Examples 9-12 which follow set
forth the protocol for the purification of the remainder of the
sub-units of the enzyme that represent substantial entirety of the
functional replicative machinery of the enzyme. Examples 18-30
demonstrate the preparation of isolated A. aeolicus sequences Pol
III subunits and their thermostable use.
EXAMPLE 1
Experimental Procedures
[0330] Materials
[0331] DNA modification enzymes were from New England Biolabs.
Labelled nucleotides were from Amersham, and unlabeled nucleotides
were from New England Biolabs The Alter-1 vector was from Promega.
pET plasmids and E. coli strains, BL21(DE3) and BL21 (DE3)pLysS
were from Novagen. Oligonucleotides were from Operon. Buffer A is
20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, and 10%
glycerol.
[0332] Genomic DNA
[0333] Thermus thermophilus (strain HB8) was obtained from the
American Type Tissue Collection. Genomic DNA was prepared from
cells grown in 0.1 l of Thermus medium N697 (ATCC: 4 g yeast
extract, 8.0 g polypeptone (BBL 11910), 2.0 g NaCl, 30.0 g agar,
1.0 L distilled water) at 75.degree. C. overnight. Cells were
collected by centrifugation at 4.degree. C. and the cell pellet was
resuspended in 25 ml of 100 mM Tris-HCl (pH 8.0), 0.05 M EDTA, 2
mg/ml lysozyme and incubated at room temperature for 10 min. Then
25 ml 6.10 M EDTA (pH 8.0), 6% SDS was added and mixed followed by
60 ml of phenol. The mixture was shaken for 40 min. followed by
centrifugation at 10,000.times.G for 10 min. at room temperature.
The upper phase (50 ml) was removed and mixed with 50 ml of
phenol:chloroform (50:50 v/v) for 30 min. followed by
centrifugation for 10 min. at room temperature. The upper phase was
decanted and the DNA was precipitated upon addition of 1/10th
volume 3 M sodium acetate (pH 6.5) and 1 volume ethanol. The
precipitate was collected by centrifugation and washed twice with 2
ml of 80% ethanol, dried and resuspended in 1 ml T.E. buffer (10 mM
Tris Hcl (pH 7.5), 1 mM EDTA).
[0334] Cloning of dnaX
[0335] DNA oligonucleotides for amplification of T.th. genomic DNA
were as follows. The upstream 32mer
(5'-CGCAAGCTTCACGCSTACCTSTTCTCCGGSAC-3', S indicating a mixture of
G and C) (SEQ. ID. No. 6) consists of a Hind III site within the
first 9 nucleotides (underlined) followed by codons (SEQ. ID. No.
29) encoding the following amino acid sequence (HAYLFSGT) (SEQ. ID.
No. 7). The downstream 34 mer
(5'-CGCGAATTCGTGCTCSGGSGGCTCCTCSAGSGTC-- 3') (SEQ. ID. No. 8)
consists of an EcoRI site (underlined) followed by codons (SEQ. ID.
No. 30) encoding the sequence KTLEEPPEH (SEQ. ID. No. 9) on the
complementary strand. The amplification reactions contained 10 ng
T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 .mu.l
of Vent polymerase reaction mixture according to the manufacturers
instructions (10 .mu.l ThermoPol Buffer, 0.5 mM each dNTP and 0.5
mM MgSO.sub.4). Amplification was performed using the following
cycling scheme: 5 cycles of: 30 sec. at 95.5.degree. C., 30 sec. at
40.degree. C., 2 min. at 72.degree. C.; 5 cycles of: 30 sec. at
95.5.degree. C., 30 sec. at 45.degree. C., and 2 min. at 72.degree.
C.; and 30 cycles of: 30 sec. at 95.5.degree. C., 30 sec. at
50.degree. C., and 30 sec. at 72.degree. C. Products were
visualized in a 1.5% native agarose gel.
[0336] Genomic DNA was digested with either XhoI, XbaI, StuI, PstI,
NcoI, MluI, KpnI, HindIII, EcoRI, EagI, BglI, or BamHI, followed by
Southern analysis in a native agarose gel (Maniatis et al., 1982).
Approximately 0.5 .mu.g of digest was analyzed in each lane of a
0.8% native agarose gel followed by transfer to an MSI filter
(Micron Separations Inc.). The transfer included the following
steps:
[0337] 1. The agarose gel was soaked in 500 ml of 1% HCl with
gentle shaking for 10 min.
[0338] 2. Then the gel was soaked in 500 ml of 0.5 M NaOH+1.5 M
NaCl for 40 min.
[0339] 3. After that the gel was soaked in 500 ml of 1M ammonium
acetate for 1 h.
[0340] 4. The DNA was transferred to the MSI filter with the use of
blotting paper for 4 h.
[0341] 5. The filter was kept at 80.degree. C. for 15 min. in the
oven.
[0342] 6. The pre-hybridization step was run in 10 ml of
Hybridization solution (1% crystalline BSA (fraction V) (Sigma), 1
mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS) at 65.degree. C. for 30
min.
[0343] 7. The probe, radiolabelled by the random priming method
(see below), was added to the pre-hybridization solution and kept
at 65.degree. C. for 12 h.
[0344] 8. The filter was washed with low stringency with 200 ml of
the wash buffer (0.5% BSA, fraction V), 1 mM Na2EDTA, 40 mM NaHPO4
(pH 7.2), 5% SDS with gentle shaking for 20 min. This step was
repeated 5 times, followed by exposure to X-ray film (XAR-5,
Kodak).
[0345] As a probe, the PCR product was radiolabelled by random as
follows.
[0346] 1. 14 ml of the mixture containing 0.2 .mu.g of PCR product
DNA, 1 .mu.g of the pd(N6) (Promega) and 2.5 ml of the 10.times.
Klenow reaction buffer (100 mM Tris-HCl (pH 7.5), 50 mM MgCl.sub.2,
75 mM dithiothreitol) were boiled for 10 min. and then kept at
4.degree. C.
[0347] 2. The reaction volume was increased up to 25 .mu.l,
containing in addition 33 .mu.M of each dNTP, except dATP, 10
.mu.Ci [.alpha.-.sup.32P] dATP (800 Ci/mM), and 2 units of Klenow
enzyme. The reaction mixture was incubated 1.5 h.
[0348] 3. 2 mg of sonicated herring sperm DNA (GibcoBRL) was added
to the reaction and the volume was increased to 2 ml using
hybridization solution. The sample was then boiled for 10 min.
[0349] A genomic library of XbaI digested DNA was prepared upon
treating 1 .mu.g genomic T.th. DNA with 10 units of XbaI in 100
.mu.l of NEBuffer N2 (50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM
MgCl2, 1 mM DTT for 2 h at 37.degree. C. The digested DNA was
purified by phenol chloroform extraction and ethanol precipitation.
The Alter-1 vector (0.5 .mu.g) (Promega) was digested with 1 unit
of XbaI in NEBuffer N2 and then purified by phenol/chloroform
extraction and ethanol precipitation. One microgram of genomic
digest was incubated with 0.05 .mu.g of digested Alter-1 and 20 U
of T4 ligase in 30 .mu.l of ligase buffer (50 mM Tris-HCl (pH 7.8),
10 mM MgCl.sub.2, 10 mM DTT and 1 mM ATP) at 15.degree. C. for 12
h. The ligation reaction was transformed into the DH5.alpha. strain
of E. coli and transformants were plated on LB plates containing
ampicillin and screened for the dnaX insert using the radiolabelled
PCR probe as follows:
[0350] 1. The colonies tested were lifted onto MSI filters,
approximately 100 colonies to each filter.
[0351] 2. The filters, removed from the LB/Tc plates, were placed
side up on a sheet of Whatman 3 MM paper soaked with 0.5 M NaOH for
5 min.
[0352] 3. The filters were transferred to a sheet of paper soaked
with 1 M Tris-HCl (pH 7.5) for 5 min.
[0353] 4. The filters were placed on a sheet of paper soaked in 0.5
M Tris-HCl (pH 7.5), 1.25 M NaCl for 5 min.
[0354] 5. After drying by air, the filters were heated in the oven
80.degree. C. for 15 min. and then were analyzed by Southern
hybridization.
[0355] Plasmid DNA was prepared from 20 positive colonies; of these
6 contained the expected 4 kb insert when digested with XbaI.
Sequencing of the insert was performed by the Sanger method using
the Vent polymerase sequencing kit according to the manufacturers
instructions (New England Biolabs).
[0356] Identification of the dnaX Gene
[0357] The dnaX genes of the gram negative E. coli and the gram
positive B. subtilis share more than 50% identity in amino acid
sequence within the N-terminal 180 residues containing the
ATP-binding domain (FIG. 2). Two highly conserved regions (shown in
bold in FIG. 2) were used to design oligonucleotide primers for
application of the polymerase chain reaction to T.th. genomic DNA.
The expected PCR product, including the restriction sites (i.e.
before cutting) is 345 nucleotides. Use of these primers with
genomic T.th. DNA resulted in a product of the expected size. The
PCR product was then radiolabelled and used to probe genomic DNA in
a Southern analysis (FIG. 3). Genomic DNA was digested with several
different restriction endonucleases, electrophoresed in a native
agarose gel and then probed with the PCR fragment. The Southern
analysis showed an XbaI fragment of approximately 4 kb, more than
sufficient length to encode the dnaX gene. Other restriction
nucleases produced fragments that were significantly longer, or
produced two or more fragments indicating presence of a site within
the coding sequence of dnaX.
[0358] To obtain full length dnaX, genomic DNA was digested with
XbaI and ligated into XbaI digested Alter-1 vector. Ligated DNA was
transformed into DH5 alpha cells, and colonies were screened with
the labeled PCR probe. Plasmid DNA was prepared from 20 positive
colonies and analyzed for the appropriate sized insert using XbaI.
Six of the twenty clones contained the expected 4 kb XbaI fragment
as an insert, the sequence of which is shown in FIGS. 4A and
4B.
[0359] The Frameshift Site
[0360] The dnaX gene of E. coli produces two proteins, the .gamma.
and .tau. subunits, by a -1 frameshift (Tsuchihashi and Kornberg,
1990; Flower and McHenry, 1990; Blinkowa and Walker, 1990). The
full length product yields .tau., and the frameshift results in
addition of one amino acid before encountering a stop codon to
produce .gamma.. The -1 frameshift site in the E. coli dnaX gene
contains the sequence, A AAA AAG, which follows the X XXY YYZ rule
found in retroviral genes (Jacks et al., 1988). This "slippery
sequence" preserves the initial two residues of the tRNAs in the
aminoacyl and peptidyl sites both before and after the frameshift.
Mutagenesis of the E. coli dnaX frameshifting site has shown that
the first three residues can be nucleotides other than A, but that
A's in the second set of three nucleotides is important to
frameshifting (Tsuchihashi and Brown, 1992).
[0361] Immediately downstream of the stop codon is a potential
stem-loop, structure which enhances frameshifting, presumably by
causing the ribosome to pause. Further, the AAG codon lacks a
cognate tRNA in E. coli and thus the G residue may facilitate the
pause, and has been shown to aid the vigorous frameshifting
observed in the E. coli dnaX gene (Tsuchihashi and Brown, 1992). A
fourth component of frameshifting in the E. coli dnaX gene is
presence of an upstream Shine-Dalgarno sequence which is thought to
pair with the 16S rRNA to increase the frequency of frameshifting
still further (Larsen et al., 1994).
[0362] Examination of the T.th. dnaX sequence reveals a single site
that fulfills the X XXY YYZ rule in which positions 4-7 are A
residues. The site is unique from that in E. coli as all seven
residues are A, and the heptanucleotide sequence is flanked by
another A residue on each side (i.e. A9). Surprisingly, the stop
codon immediately downstream of this site is in the -2 frame,
although there is a stop codon in the -1 frame 28 nucleotides
downstream of the -2 stop codon. Indeed, a -2 frameshift would
fulfill the requirement that the first two nucleotides of each
codon in the peptidyl and aminoacyl sites be conserved during
either a -1 or a -2 frameshift. As with the case of E. coli dnaX,
there are secondary structure step loop structures immediately
downstream. Finally, there is a Shine-Dalgarno sequence immediately
adjacent to the frameshift site, as well as another Shine-Dalgarno
sequence 22 nucleotides upstream of the frameshift site.
[0363] Assuming the first stop codon is utilized (i.e. -2
frameshift), the predicted size of the .gamma. subunit in T.th. is
454 amino acids for a mass of 49.8 kDa, over 2 kDa larger than the
431 residue .gamma. subunit (47.5 kDa) of E. coli. This would
result in 2 residues after the -2 frameshift (i.e. after the
GluLysLys, the residues LysAla would be added) to be compared to
the result of the -1 frameshift in E. coli which also results in 2
residues (LysGlu). In the event that a -1 frameshift were utilized
in the T.th. dnaX gene, then an additional 12 residues would be
added following the frameshift for a molecular mass of 50.8 kDa
(i.e. after the GluLysLys, the residues
LysProAspProLysAlaProProGlyProThrSer would be added at aa 453-464
of SEQ. ID. No. 4). As explained later, this nucleotide sequence
was found to promote both -1 and -2 frameshifting in E. coli (FIG.
8). But first, we examined T.th. cells by Western analysis for the
presence of two subunits homologous to E. coli .gamma. and
.tau..
EXAMPLE 2
[0364] Frameshifting Analysis of the T.th. dnaX Gene
[0365] Frameshifting was analyzed by inserting the frameshift site
into lacZ in the three different reading frames, followed by
plating on X-gal and scoring for blue or white colony formation
(Weiss et al., 1987). The frameshifting region within T.th dnaX was
subcloned into the EcoRI/BamHI sites of pUC19. These sites are
within the polylinker inside of the .beta.-galactosidase gene.
Three constructs were produced such that the insert was either in
frame with the downstream coding sequence of .beta.-galactosidase,
or were out of frame (either -1 or -2). An additional three
constructs were designed by mutating the frameshift sequence and
then placing this insert into the three reading frames of the
.beta.-galactosidase gene. These six plasmids were constructed as
described below.
[0366] The upstream primer for the shifty sequences was 5'-gcg cgg
atc cgg agg gag aaa aaa aaa gcc tca gcc ca-3' (SEQ. ID. No. 10).
The BamHI site for cloning into pUC is underlined. Also, the stop
codon, tga, has been mutated to tca (also underlined). The upstream
primer for the mutant shifty sequence was: 5'-gcg cgg atc cgg agg
gag aga aga aaa gcc tca gcc ca-3' (SEQ. ID. No. 11). The mutant
sequence contains two substitutions of a G for an A residue in the
polyA stretch (underlined). Three downstream primers were utilized
with each upstream primer to create two sets of three inserts in
the 0 frame, -1 frame and -2 frame. The sequence of these primers,
and the length of insert (after cutting with EcoRI and BanHI and
inserting into pUC19) are as follows: 5'-gaa tta aat tcg cgc ttc
ggg agg tgg g-3' (0 frameshift total 58 nucleotide insert) (SEQ.
ID. No. 12); 5'-gcg cga att cgc gct tcg gga ggt ggg-3' (-1 frame,
54mer insert) (SEQ. ID. No. 13); and 5'-gcg cga att cgg gcg ctt cag
gag gtg gg-3' (-2 frame, 56mer insert) (SEQ. ID. No. 14). The
downstream primers have an EcoRI site (underlined); the EcoRI site
of the 0 frame insert was blunt ended to produce the greater length
insert (converting the EcoRI site to an aattaatt sequence). Also,
the tcg sequence, which produces the tga stop codon (underlined)
was mutated to tca in the -2 downstream primer so that readthrough
would be allowed after the frameshift occurred.
[0367] In summary, a region surrounding the frameshift site and
ending at least 5 nucleotides past the -1 frameshift stop codon was
inserted into the .beta.-galactosidase gene of pUC19 in the three
different reading frames (stop codons were mutated to prevent
stoppage following a frameshift). These three plasmids were
introduced into E. coli and plated with X-gal. The results, in FIG.
8, show that blue colonies were observed after 24 h incubation with
all three plasmids and therefore both -1 and -2 frameshifting had
occurred.
[0368] To further these results, two .gamma. residues were
introduced into the polyA tract which should disrupt the ability of
this sequence to direct frameshifts. The mutated slippery sequence
was inserted into pUC19 followed by transformation into E. coli and
plating on X-gal. The results showed that both -1 and -2
frameshifting was prevented, further supporting the fact that
frameshifting requires the polyA tract as expected (FIG. 8).
EXAMPLE 3
[0369] Expression Vector for T.th. .gamma. and .tau.
[0370] The dnaX gene was cloned into the pET16 expression vector in
the steps shown in FIG. 9. First, the bulk of the gene was cloned
into pET16 by removing the PmlI/XbaI fragment from pAlterdnaX, and
placing it into SmaI/XbaI digested Puc19 to yield Puc19dnaXCterm.
The N-terminal sequence of the dnaX gene was then reconstructed to
position an NdeI site at the N-terminus. This was performed by
amplifying the 5' region encoding the N-terminal section of
.gamma./.tau. using an upstream primer containing an NdeI site that
hybridizes to the dnaX gene at the initiating gtg codon (i.e. to
encode Met where the Met is created by the PCR primer, and the Val
is the initiating gtg start codon of dnaX). The primer sequence for
this 5' end was 5'-gtggtgcatatg gtg agc gcc ctc tac cgc c-3' (SEQ.
ID. No. 15) (where the NdeI site is underlined, and the coding
sequence of dnaX follows). The downstream primer hybridizes past
the PmlI site at nucleotide positions 987-1004 downstream of the
initiating gtg (primer sequence: 5'-gtggtggtcgac cca gga ggg cca
cct cca g-3' (SEQ. ID. No. 16) where the initial 12 nucleotides
contain a SalGI restriction site, followed by the sequence from the
region downstream the stop codon). The 1.1 kb nucleotide PCR
product was digested with PmlI/NdeI and the PmlI/NdeI fragment, was
ligated into NdeI/PmlI digested Puc19dnaXCterm to form Puc19dnaX.
The Puc19dnaX plasmid was then digested with NdeI and SalI and the
1.9 kb fragment containing the dnaX gene was purified using the
Sephaglas BandPrep Kit (Pharmacia-LKB). pET16b was digested with
NdeI and XhoI. Then the full length dnaX gene was ligated into the
digested pET16b to form pETdnaX.
EXAMPLE 4
[0371] Expression of T.th. .gamma. and .tau.
[0372] As discussed in the previous example, the dnaX gene was
engineered into the T7 based IPTG inducible pET16 vector such that
the initiation codon was placed precisely following the Met residue
N-terminal leader sequence (FIG. 9). This should produce a protein
containing the entire sequence of .gamma. and .tau., along with a
21 residue leader containing 10 contiguous His residues
(tagged-.tau., =60.6 kDa; tagged-.gamma.=52.4 kDa for -2
frameshift). The pETdnaX plasmid was introduced into. BL21
(DE3)pLysS cells harboring the gene encoding T7 RNA polymerase
under control of the lac repressor. Log phase cells were induced
with IPTG and analyzed before and after induction in an. SDS
polyacrylamide gel (FIG. 10, lanes 1 and 2). The result shows that
upon induction, two new proteins are expressed with the approximate
sizes expected of the T.th. .gamma. and .tau. subunits (larger than
E. coli .gamma., and smaller than E. coli.tau.). The two proteins
are produced in nearly equal amounts, similar to the case of the E.
coli .gamma. and .tau. subunits. Western analysis using antibodies
against the E. coli .gamma. and .tau. subunits cross-reacted with
the induced proteins further supporting their identity as T.th.
.gamma. and .tau. (data not shown, but repeated with the pure
subunits shown in FIG. 10, lane 6).
EXAMPLE 5
[0373] Purification of T.th. .gamma. and .tau.
[0374] The His-tagged T.th. .gamma. and .tau. proteins were
purified from 6 L of induced E. coli cells containing the pETdnaX
plasmid. Cells were lysed, clarified from cell debris by
centrifugation and the supernatant was applied to a HiTrap chelate
affinity column. Elution of the chelate affinity column yielded
approximately 35 mg of protein in which the two predominant bands
migrated in a region consistent with the molecular weight predicted
from the dnaX gene (FIG. 10, lane 3), and produced a positive
signal by Western analysis using polyclonal antibody directed
against the E. coli .gamma. and .tau. subunits (lane 4). The
.gamma. and .tau. subunits; are present in nearly equal amounts
consistent with the nearly equal expression of these proteins in E.
coli cells harboring the pETdnaX plasmid.
[0375] The .gamma. and .tau. subunits were further purified by gel
filtration on a Superose 12 column (FIG. 10, lane 4; FIG. 11).
Recovery of T.th. .gamma. and .tau. subunits through gel filtration
was 81%. The E. coli .gamma. and .tau. subunits, when separated
from one another, elute during gel filtration as tetramers. A
mixture of E. coli .gamma./.tau. results in a mixed tetramer of
.gamma.2.tau.2 along with .gamma.4 and .tau.4 tetramers (Onrust et
al., 1995). The mixture of T.th. .gamma./.tau. elutes ahead of the
150 kDa marker, and thus is consistent with the expected mass of a
.gamma.2.tau.2 tetramer (225 kDa) and .gamma.4 and .tau.4
tetramers.
[0376] As described earlier, the dnaX frameshifting sequence could
produce either a -1 or -2 frameshift to yield a His-tagged .gamma.
subunit of mass either 53.3 kDa or 52.4 kDa, respectively. The
difference in these two possible products is too close to determine
from migration in SDS gels. It also remains possible that two
.gamma. products are present and do not resolve under the
conditions used. The exact protocol for this purification is
described below.
[0377] Six liters of BL21(DE3)pLysSpETdnaX cells were grown in LB
media containing 50 .mu.g/ml ampicillin and 25 .mu.g/ml
chloramphenicol at 37.degree. C. to an O.D. of 0.8 and then IPTG
was added to a concentration of 2 mM. After a further 2 h at
37.degree. C., cells were harvested by centrifugation and stored at
-70.degree. C. The following steps were performed at 4.degree. C.
Cells (115 g wet weight) were thawed and resuspended in 45 ml
1.times. binding buffer (5 mM imidizole, 0.5 M NaCl, 20 mM Tris HCl
(final pH 7.5)) using a dounce homogenizer to complete cell lysis
and 450 ml of 5% polyamine P (Sigma) was added. Cell debris was
removed by centrifugation at 18,000 rpm for 30 min. in a Sorvall
SS24 rotor at 4.degree. C. The supernatant (Fraction I, 40 ml, 376
mg protein) was applied to a 5 ml HiTrap Chelating Separose column
(Pharmacia-LKB). The column was washed with 25 ml of binding
buffer, then with 30 ml of binding buffer containing 60 mM
imidizole, and then eluted with 30 ml of 0.5 M imidizole, 0.5 M
NaCl, 20 mM Tris-HCl (pH 7.5). Fractions of 1 ml were collected and
analyzed on an 8% Coomassie Blue stained SDS polyacrylamide gel.
Fractions containing subunits migrating at the T.th .gamma. and
.tau. positions, and exhibiting cross reactivity with antibody to
E. coli .gamma. and .tau. in a Western analysis, were pooled and
dialyzed against buffer A (20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5
mM DTT and 10% glycerol) containing 0.5 M NaCl (Fraction II, 36 mg
in 7 ml). Fraction II was diluted 2-fold with buffer A and passed
through a 2 ml ATP agarose column equilibrated in buffer A
containing 0.2 M NaCl to remove any E. coli .gamma. complex
contaminant. Then 0.18 mg (300 ml) Fraction II was gel filtered on
a 24 ml Superose 12 column (Pharmacia-LKB) in buffer A containing
0.5 M NaCl. After the first 216 drops, fractions of 200 .mu.l were
collected (Fraction III) and analyzed by Western analysis (by
procedures similar to those described in Example 6), by ATPase
assays and by Coomassie Blue staining of an 8% Coomassie Blue
stained SDS polyacrylamide gel. The Coomassie stained gels and
Western analysis of recombinant T.th. gamma and tau for these
purification steps are summarized in FIG. 10.
EXAMPLE 6
[0378] Western Analysis of T.th. Cells for Presence of .gamma. and
.tau. Subunits
[0379] Polyclonal antibody to E. coli .gamma./.tau.-E. coli .gamma.
subunit was prepared as described (Studwell-Vaughan and O'Donnell,
1991). Pure .gamma. subunit (100 .mu.g) was brought up in Freund's
adjuvant and injected subcutaneously into a New Zealand Rabbit
(Poccono Rabbit Farms). After two weeks, a booster consisting of 50
.mu.g .gamma. in Freund's adjuvant was administered, followed after
two weeks by a third injection (50 .mu.g).
[0380] The homology between the amino terminal regions of T.th. and
E. coli .gamma./.tau. subunits suggested that there may be some
epitopes in common between them. Hence, polyclonal antibody
directed against the E. coli .gamma./.tau. subunits was raised in
rabbits for use in probing T.th. cells by Western analysis. FIG. 7
shows the results of a Western analysis of whole T.th. cells lysed
in SDS. The results show that in T.th. cells, the antibody is
rather specific for two high molecular proteins which migrate in
the vicinity of the molecular masses of E. coli .gamma. and .tau.
subunits.
[0381] Procedure for Western Analysis
[0382] Samples were analyzed in duplicate 10% SDS polyacrylamide
gels by the Western method (Towbin et al. 1979). One gel was
Coomassie stained to evaluate the pattern of proteins present, and
the other gel was then electroblotted onto a nitrocellulose
membrane (Schleicher and Schuell). For molecular size markers, the
kaliedoscope molecular weight markers (Bio-Rad) were used to verify
by visualization that transfer of proteins onto the blotted
membrane had occurred. The gel used in electroblotting was also
stained after electroblotting to confirm that efficient transfer of
protein had occured. Membranes were blocked using 5% non-fat milk,
washed with 0.05% Tween in TBS (TBS-T) and then incubated for over
1 h with a 1/5000 dilution of rabbit polyclonal antibody directed
against E. coli .gamma. and .tau. in 1% gelatin in TBS-T at room
temperature. Membranes were washed using TBS-T buffer and then
antibody was detected on X-ray film (Kodak) by using the, ECL kit
from (Amersham) and the manufactures reccommended procedures.
[0383] Samples included: 1) a mixture of E. coli .gamma. (15 ng)
and .tau. (15 ng) subunits; 2) T.th. whole cells (100 .mu.l)
suspended in cracking buffer; and 3) purified T.th. .gamma. and
.tau. fraction II (0.6 .mu.g as a mixture).
EXAMPLE 7
[0384] Characterization of the ATPase Activity of .gamma./.tau.
[0385] The E. coli .tau. subunit is a DNA dependent ATPase (Lee and
Walker, 1987; Tsuchihashi and Kornberg, 1989). The .gamma. subunit
binds ATP but does not hydrolyze it even in the presence of DNA
unless other subunits of the DNA polymerase III holoenzyme are also
present (Onrust et al., 1991). Next we examined the T.th.
.gamma./.tau. subunits for DNA dependent ATPase activity. The
.gamma./.tau. preparation was, in fact, a DNA stimulated ATPase
(FIG. 11, top panel). The specific activity of the T.th.
.gamma./.tau. was 11.5 mol ATP hydrolyzed/mol .gamma./.tau. (as
monomer and assuming an equal mixture of the two). Furthermore,
analysis of the gel filtration column fractions shows that the
ATPase activity coelutes with the T.th. .gamma./.tau. subunits,
supporting evidence that the weak ATPase activity is intrinsic to
the .gamma./.tau. subunits (FIG. 11). The specific activity of the
.gamma./.tau. preparation before gel filtration was the same as
after gel filtration (within 10%), further indicating that the DNA
stimulated ATPase is an inherent activity of the .gamma./.tau.
subunits. Presumably, only the .tau. subunit contains ATPase
activity, as in the case of E. coli. Assuming only T.th. .tau.
contains ATPase activity, its specific activity is twice the
observed rate (after factoring out the weight of .gamma.). This
rate is still only one-fifth that of E. coli .tau..
[0386] The T.th. .alpha./.tau. ATPase activity is lower at
37.degree. C. than at 65.degree. C. (middle panel), consistent with
the expected-behavior of protein activity from a thermophilic
source. However, there is no apparent increase in activity in
proceeding from 50.degree. C. to 65.degree. C. (the rapid breakdown
of ATP above 65.degree. C. precluded measurement of ATPase activity
at temperatures above 65.degree. C.). In contrast, the E. coli
.tau. subunit lost most of its ATPase activity upon elevating the
temperature to 50.degree. C. (middle panel). These reactions
contain no stabilizers such as a nonionic detergent or gelatin, nor
did they include substrates such as ATP, DNA or magnesium.
[0387] Last, the relative stability of T.th. .gamma./.tau. and E.
coli .gamma./.tau. to addition of NaCl (FIG. 12, bottom panel) was
examined. Whereas the E. coli .tau. subunit rapidly lost activity
at even 0.2 M NaCl, the T.th. .gamma./.tau. retained full activity
in 1.0 M NaCl and was still 80% active in 1.5 M NaCl. The detailed
procedure for the ATPase activity assay is described below.
[0388] ATPase Assays
[0389] ATPase assays were performed in 20 .mu.l of 20 mM Tris-HCl
(pH 7.5), 8 mM MgCl.sub.2 containing 0.72 .mu.g of M13 mp18 ssDNA
(where indicated), 100 mM [.gamma.-.sup.32P]-ATP (specific activity
of 2000-4000 cpm/pmol), and the indicated protein. Some reactions
contained additional NaCl where indicated. Reactions were incubated
at the temperatures indicated in the figure legends for 30 min. and
then were quenched with an equal volume of 25 mM EDTA (final). The
aliquots were analyzed by spotting them (1 .mu.l each) onto thin
layer chromatography (TLC) sheets coated with Cel-300
polyethyleneimine (Brinkmann Instruments Co.). TLC sheets were
developed in 0.5 M lithium chloride, 1 M formic acid. An
autoradiogram of the TLC chromatogram was used to visualize Pi at
the solvent front and ATP near the origin which were then cut from
the TLC sheet and quantitated by liquid scintillation. The extent
of ATP hydrolyzed was used to calculate the mol of Pi released per
mol of protein per min. One mol of E. coli .tau. was calculated
assuming a mass of 71 kDa per monomer. The T.th. .gamma. and .tau.
preparation was treated as an equal mixture and thus one mole of
protein as monomer was the average of the predicted masses of the
.gamma. and .tau. subunits (54 kDa).
EXAMPLE 8
[0390] Homolog of T.th. .gamma./.tau. to dnaX Gene Products of
Other Organism
[0391] The XbaI insert encoded an open reading frame, starting with
a GTG codon, of 529 amino acids in length (58.0 kDa), closer to the
predicted length of the B. subtilis .tau. subunit (563 amino acids,
62.7 kDa mass) (Alonso et al., 1990) than the E. coli .tau. subunit
(71.1 kDa) (Yin et al., 1986). The dnaX gene encoding the
.gamma./.tau. subunits of E. coli DNA polymerase III holoenzyme is
homologous to the holB gene encoding the .delta.' subunit of the
.gamma. complex clamp loader, and this homology extends to all 5
subunits of the eukaryotic RFC clamp loader as well as the
bacteriophage gene protein 44 of the gp44/62 clamp loading complex
(O'Donnell et al., 1993). These gene products show greatest
homology over the N-terminal 166 amino acid residues (of E. coli
dnaX); the C-terminal regions are more divergent. FIG. 4 shows an
alignment of the amino acid sequence of the N-terminal regions of
the T.th. dnaX gene product to those of several other bacteria. The
consensus GXXGXGKT (SEQ. ID. No. 17) motif for nucleotide binding
is conserved in all these protein products. Further, the E. coli
.delta.' crystal structure reveals one atom of zinc coordinated to
four Cys residues (Guenther, 1996). These four Cys residues are
conserved in the E. coli dnaX gene, and the .gamma. and .tau.
subunits encoded by E. coli dnaX bind one atom of zinc. These Cys
residues are also conserved in T.th. dnaX (shown in FIG. 4).
Overall, the level of amino acid identity relative to E. coli dnaX
in the N-terminal 165 residues of T.th. dnaX is 53%. The T.th. dnaX
gene is just as homologous to the B. subtilis dnaX (53% identity)
gene relative to E. coli dnaX. After this region of homology, the
C-terminal region of T.th. dnaX shares 26% and 20% identity to E.
coli and B. subtilis dnaX, respectively. A proline rich region,
downstream of the conserved region, is also present in T.th. dnaX
(residues 346-375), but not in the B. subtilis dnaX (see FIGS. 3A
and 3B). The overall identity between E. coli dnaX and T.th. dnaX
over the entire gene is 34%. Identity of T.th. dnaX to B. subtilis
dnaX over the entire gene is 28%.
[0392] Comparison of dnaX Genes from T.th. and E. coli
[0393] The above identifies a homologue of the dnaX gene of E. coli
in Thermus thermophilus. Like the E. coli gene, T.th. dnaX encodes
two related proteins through use of a highly efficient
translational frameshift. The T.th. .gamma./.tau. subunits are
tetramers, or mixed tetramers, similar to the .gamma. and .tau.
subunits of E. coli. Further, the .gamma./.tau. subunits a DNA
stimulated ATPase like its E. coli counterpart. As expected for
proteins from a thermophile, the T.th. .gamma./.tau. ATPase
activity is thermostabile and resistant to added salt.
[0394] In E. coli, .gamma. is a component of the clamp loader, and
the .tau. subunit serves the function of holding the clamp loading
apparatus together with two DNA polymerases for coordinated
replication of duplex DNA. The presence of .gamma. in T.th.
suggests it has a clamp loading apparatus and thus a clamp as well.
The presence of the .tau. subunit of T.th. implies that T.th.
contains a replicative polymerase with a structure similar to that
of E. coli DNA polymerase III holoenzyme.
[0395] A significant difference between E. coli and T.th. dnaX
genes is in the translational frameshift sequence. In E. coli, the
heptamer frameshift site contains six A residues followed by a G
residue in the context A AAA AAG. This sequence satisfies the X XXY
YYZ rule for -1 frameshifting. The frameshift is made more
efficient by the absence of the AAG tRNA for Lys which presumably
leads to stalling of the ribosome at the frameshift site and
increases the efficiency of frameshifting (Tsuchihashi and Brown,
1992). Two additional aids to frameshifting include a downstream
hairpin and an upstream Shine-Dalgarno sequence. (Tsuchihashi and
Kornberg, 1990; Larsen et al., 1994). The -1 frameshift leads to
incorporation of one unique residue at the C-terminus of E. coli
.gamma. before encounter with a stop codon.
[0396] In T.th., the dnaX frameshifting heptamer is A AAA AAA, and
it is flanked by two other A residues, one on each side. There is
also a downstream region of secondary structure. The nearest
downstream stop codon is positioned such that gamma would contain
only one unique amino acid, as in E. coli. However, the T.th. stop
codon is in the -2 reading frame thus requires a -2 frameshift. No
precedent exists in nature for -2 frameshifting, although -2
frameshifting has been shown to occur in test cases (Weiss et al.,
1987). In vivo analysis of the T.th. frameshift sequence shows that
this natural sequence promotes both -1 and -2 frameshifting in E.
coli. Whereas the -2 frameshift results in only one unique.
C-terminal residue; a -1 frameshift would result in an extension of
12 C-terminal residues. At present, the results do not discriminate
which path occurs in T.th., a -1 or -2 frameshift, or a combination
of the two.
[0397] There are two Shine-Dalgarno sequences just upstream of the
frameshift site in T.th. dnaX. In two cases of frameshifting in E.
coli, an upstream Shine-Dalgarno sequence has been shown to
stimulate frameshifting (reviewed in Weiss et al., 1897). In
release factor 2 (RF2), the Shine-Dalgarno is 3 nucleotides
upstream of the shift site, and it stimulates a +1 frameshift
event. In the case of E. coli dnaX; a Shine-Dalgarno sequence 10
nucleotides upstream of the shift sequence stimulates the -1
frameshift. One of the T.th. dnaX Shine-Dalgarno sequences is
immediately adjacent to the frameshift sequence with no extra
space, the other is 22 residues upstream of the frameshift site.
Which of these Shine-Dalgarno sequences plays a role in T.th. dnaX
frameshifting, if any, will-require future study.
[0398] In E. coli, efficient separation of the two polypeptides,
.gamma. and .tau., is achieved by mutation of the frameshift site
such that only one polypeptide is produced from the gene
(Tsuchihashi and Kornberg, 1990). Substitution of G-to-A in two
positions of the heptamer of T.th. dnaX eliminates frameshifting
and thus should be a source to obtain .tau. subunit free of
.gamma.. To produce pure .gamma. subunit free of .tau., the
frameshifting site and sequence immediately downstream of it can be
substituted for an in-frame sequence with a stop codon.
[0399] Examination of the B. subtilis dnaX gene shows no frameshift
sequence that satisfies the X XXY. YYZ rule. Hence, it would appear
that dnaX does not make two proteins in this gram positive
organism.
[0400] Rapid thermal motions associated, with high temperature may
make coordination of complicated processes more difficult. It seems
possible that organizing the components of the replication
apparatus may become yet more important at higher temperature.
Hence, production of a .tau. subunit that could be used to
crosslink two polymerases and a clamp loader into one organized
particle may be most useful at elevated temperature.
[0401] As stated above, the following examples describe the
continued isolation and purification of the substantial entirety of
the Polymerase III from the extreme thermophile Thermus
thermophilus. It is to be understood that the following exposition
is reflective of the protocol and characteristics, both
morphological and functional, of the Polymerase III-type enzymes
that are the focus of the present invention, and that the invention
is hereby illustrated and comprehends the entire class of enzymes
of thermophilic origin.
EXAMPLE 9
[0402] Purification of the Thermus thermophilus DNA Polymerase
III
[0403] All steps in the purification assay were performed at
4.degree. C. The following assay was used in the purification of
DNA polymerase from T.th. cell extracts. Assays contained 2.5 mg
activated calf thymus DNA (Sigma Chemical Company) in a final
volume of 25 ml of 20 mM Tris-Cl (pH 7.5), 8 mM MgCl.sub.2, 5 mM
DTT, 0.5 mM EDTA, 40 mg/ml BSA, 4% glycerol, 0.5 mM ATP, 3 mM each
dCTP, dGTP, dATP, and 20 mM [.alpha.-.sup.32P]dTTP. An aliquot of
the fraction to be assayed was added to the assay mixture on ice
followed by incubation at 60.degree. C. for 5 min. DNA synthesis
was quantitated using DE81 paper followed by washing off
unincorporated nucleotide. Incorporated nucleotide was determined
by scintillation counting of the filters.
[0404] Thermus thermophilus cell extracts were prepared by
suspending 35 grams of cell paste in 200 ml of 50 mM TRIS-HCl,
pH=7.5, 30 mM spermidine, 100 mM NaCl 0.5 mM EDTA, 5 mM DTT, 5%
glycerol, followed by disruption by passage through a French
pressure cell (15,000 PSI). Cell debris was removed by
centrifugation (12,000 RPM, 60 min). DNA polymerase III in the
clarified supernatant was precipitated by treatment with ammonium
sulphate (0.226 gm/liter) and recovered by centrifugation. This
fraction was then backwashed with the same buffer (but lacking
spermidine) containing 0.20 gm/l ammonium sulfate. The pellet was
then resuspended in buffer A and dialyzed overnight against 2
liters of buffer A; a precipitate which formed during dialysis was
removed by centrifugation (17,000 RPM, 20 min).
[0405] The clarified dialysis supernatant, containing approximately
336 mg of protein, was applied onto a 60 ml heparin agarose column
equilibrated in buffer A which was washed with the same buffer
until A280 reached baseline. The column was developed with a 500 ml
linear gradient of buffer A from 0 to 500 mM NaCl. More tightly
adhered proteins were washed off the column by treatment with
buffer A (20 mM Tris Hcl, pH=7.5, 0.1 nM EDTA, 5 mM DTT, and 10%
glycerol) and 1M NaCl. Some DNA polymerase activity flowed through
the column. Two peaks (HEP.P1 and HEP.P2) of DNA polymerase
activity eluted from the heparin agarose column containing 20 mg
and 2 mg of total protein respectively (FIG. 13A). These were kept
separate throughout the remainder of the purification protocol.
[0406] The Pol III resided in HEP.P1 as indicated by the following
criteria: 1) Western analysis using antibody directed against the
.alpha. subunit of E. coli Pol III indicated presence of Pol III in
HEP.P1; 2). Only the HEP.P1 fraction was capable of extending a
single primer around an M13mp18 7.2 kb ssDNA circle (explained
later in Example 16), such long primer extension being a
characteristic of Pol III type enzymes; and 3) Only the HEP.P1
provided DNA polymerase activity that was retained on an
ATP-agarose affinity column, which is indicative of a Pol III-type
DNA polymerase since the .gamma. and .tau. subunits are ATP
interactive proteins.
[0407] The first peak of the heparin agarose column. (HEP.P1: 20 mg
in 127.5 ml) was dialyzed against buffer A and applied onto a 2 ml
N6-linkage ATP agarose column pre-equilibrated in the same buffer.
Bound protein was eluted by a slow (0.05 ml/min) wash with buffer A
+2M NaCl and collected into 200 .mu.l fractions. Chromatography of
peak HEP.P1 yielded a flow-through (HEP.P1-ATP-FT) and a bound
fraction (HEP.P1-ATP-Bound) (FIG. 13B). Binding of peak HEP.P2 to
the ATP column could not be detected, though DNA polymerase
activity was recovered in the flow-trough.
[0408] The HEP.P1-ATP-Bound fractions from the ATP agarose
chromatographic step were further purified by anion exchange over
monoQ. The HEP.P1-ATP-Bound fractions were diluted with buffer A to
approximately the conductivity of buffer A plus 25 mM NaCl and
applied to a 1 ml monoQ column equilibrated in Buffer A. DNA
polymerase activity eluted in the flow-through and in two resolved
chromatographic peaks (MONOQ peak1 and peak2) (FIG. 13C). Peak 2was
by far the major source of DNA polymerase activity. Western
analysis using rabbit antibody directed against the E. coli .alpha.
subunit confirmed presence of the ax subunit in the second peak
(see the Western analysis in FIG. 14B). Antibody against the E.
coli .tau. subunit also confirmed the presence of the .tau. subunit
in the second peak. Some reaction against .alpha. and .tau. was
also present in the minor peak (first peak). The Coomassie Blue SDS
polyacrylamide gel of the MonoQ fractions (FIG. 14A) showed a band
that co-migrated with E. coli .alpha. and was in the same postion
as the antibody reactive material (antibody against E. coli
.alpha.). Also present are bands corresponding to .tau., .gamma.,
.delta., and .delta.'. These subunits, along with .beta., are all
that is necessary for rapid and processive synthesis and primer
extension over a long (>7 kb) stretch of ssDNA in the case of E.
coli DNA Polymerase III holoenzyme.
[0409] The Pol III-type enzyme purified from T.th. may be a Pol
III*-like enzyme that contains the DNA polymerase and clamp loader
subuits (i.e., like the Pol, III* of E. coli). The evidence for
this is 1) the presence of dnaX and dnaE gene products in the same
column fractions as indicated by Western analysis (see above); 2)
the ability of this enzyme to extend a primer around a 7.2 kb
circular ssDNA upon adding only .beta. (see Example 16); 3)
stimulation of Pol III by adding .beta. on linear DNA, indicating
.beta. subunit is not present in saturating amounts (see Example
15); and 4) the presence of .tau. in T.th. which may glue the
polymerase and clamp loader into a Pol III* as in E. coli; and 5)
the comigration of .alpha. with subunits .tau., .gamma., .delta.
and .delta.' of the clamp loader in the column fractions of the
last chromatographic step (MonoQ, FIG. 14A).
[0410] Micro-Sequencing of T. th DNA Polymerase III .alpha.
Subunit
[0411] The .alpha. subunit from the purified T.th DNA polymerase
III (HEP.P1.ATP-Bound.MONOQ peak2) was blotted onto PVDF membrane
and was cut out of the SDS-PAGE gel and submitted to the
Protein-Nucleic Acid Facility at Rockefeller University for
N-terminal sequencing and proteolytic digestion, purification and
microsequencing of the resultant peptides. Analysis of the .alpha.
candidate band (Mw 130 kD) yielded four peptides, two of which
(TTH1, TTH2) showed sequence similarity to .alpha. subunits from
various bacterial sources (see FIG. 15).
EXAMPLE 10
[0412] Identification of the Thermus thermophilus dnaE Gene
Encoding the .alpha. Subunit of DNA Polymerase III Replication
Enzyme
[0413] Cloning of the dnaE gene was started with the sequence of
the TTH1 peptide from the purified .alpha. subunit (FFIEIQNHGLSEQK)
(SEQ. ID. No. 61). The fragment was aligned to a region at
approximately 180 amino acids downstream of the N-termini of
several other known .alpha. subunits as shown in FIG. 15. The
upstream 33mer (5'-GTGGGATCCGTGGTTCTGGATCTCGATGA- AGAA-3') (SEQ.
ID. No. 31) consists of a BamHI site within the first 9 nucleotides
(underlined) and the sequence coding for the following peptide
HGLSEQK on the complementary strand. The downstream 29 mer
(5'-GTGGGATCCACGGSCTSTCSGAGCAGAAG-3') (SEQ. ID. No. 32) consists of
a BamHI site within the first 9 nucleotides (underlined) and the
following sequence coding for the peptide FFIEIQNH (SEQ. ID. No.
62).
[0414] These two primers were directed away from each other for the
purpose of performing inverse PCR (also called circular PCR). The
amplification reactions contained 10 ng. T.th. genomic DNA (that
had been cut and religated with XmaI), 0.5 mM of each primer, in a
volume of 100 .mu.l of Vent polymerase reaction mixture containing
10 .mu.l ThermoPol Buffer, 0.5 mM of each dNTP and 0.25 mM
MgSO.sub.4. Amplification was performed using the following cycling
scheme:
[0415] 1. 4 cycles of: 95.5.degree. C.--30 sec., 45.degree. C.--30
sec., 75.degree. C.--8 min.
[0416] 2. 6 cycles of: 95.5.degree. C.--30 sec., 50.degree. C.--30
sec., 75.degree. C.--6 min.
[0417] 3. 30 cycles of: 95.5.degree. C.--30 sec., 52.5.degree.
C.--30 sec., 75.degree. C.--5 min.
[0418] A 1.4 kb fragment was obtained and cloned into pBS-SK:BamHI
(i.e. pBS-SK (Stratagene) was cut with BamHI). This sequence was
bracketted by the 29mer primer on both sides and contained the
sequence coding for the N-terminal part of the subunit up to the
peptide used for primer design.
[0419] To obtain further dnaE gene sequence, the TTH2 peptide was
used. It was aligned to a legion about 600 amino acids from the
N-termini of the other known subunits (FIG. 15B).
[0420] The upstream 34mer (5-GCGGGATCCTCAACGAGGACCTCTCCATCTTCAA-3')
(SEQ. ID. No. 33) consists of a BamHI site within the first 9
nucleotides (underlined) and the sequence from the end of the
fragment previously obtained. The downstream 35mer
(5'-GCGGGATCCTTGTCGTCSAGSGTSAGSGCGTCGTA-3'- ) (SEQ. ID. No. 34)
consists of a BamHI site within the first 9 nucleotides
(underlined) and the following sequence coding for the peptide
YDALTLDD (SEQ. ID. No. 63) on the complementary strand. The
amplification reactions contained 10 ng T.th. genomic DNA, 0.5 mM
of each primer, in a volume of 100 .mu.l of Vent polymerase
reaction mixture containing 10 .mu.l ThermoPol Buffer, 0.5 mM of
each dNTP and 0.25 mM MgSO.sub.4. Amplification was performed using
the following cycling scheme:
[0421] 1.4 cycles of 95.5.degree. C. --30 sec., 45.degree. C.--30
sec., 75.degree. C.--8 min.
[0422] 2. 6 cycles of: 95.5.degree. C.--30 sec., 50.degree. C.--30
sec., 75.degree. C.--6 min.
[0423] 3. 30 cycles of: 95.5.degree. C.--30 sec., 55.degree. C.--30
sec., 75.degree. C.--5 min.
[0424] A 1.2 kb PCR fragment was obtained and cloned into
pUC19:BamHI. The fragment was bracketted by the downstream primer
on both sides and contained the region overlapping in 56 bp with
the fragment previously cloned.
[0425] To obtain yet more dnaE sequence, the following primers were
used. The upstream 39mer
(3'-GTGTGGATCCTCGTCCCCCTCATGCGCGACCAGGAAGGG-5') (SEQ. ID. Nos. 35
and 114) consists of a BamHI site within the first 10 nucleotides
(underlined) and the sequence from the end of the fragment
previously obtained. The downstream 27mer
(5'-GTGTGGATCCTTCTTCTTSCCCATSGC- -3') (SEQ. ID. No. 36) consists of
a BamHI site within the first 10 nucleotides (underlined), and the
sequence coding for the peptide AMGKKK (SEQ. ID. No. 64) (at
position approximately 800 residues from the N terminus) on the
complementary strand. The AMGKKK (SEQ. ID. No. 64) sequence was
chosen for primer design as it is highly conserved among the known
gram-negative .alpha. subunits. The amplification reactions
contained 10 ng T.th. genomic DNA, 0.5 mM of each primer, in a
volume of 100 .mu.l of Taq polymerase reaction mixture containing
10 .mu.l PCR Buffer, 0.5 mM of each dNTP and 2.5 mM MgCl.sub.2.
Amplification was performed using the following cycling scheme:
[0426] 1. 3 cycles of: 95.5.degree. C.--30 sec., 45.degree. C.--30
sec., 72.degree. C.--8 min.
[0427] 2. 6 cycles of: 94.5.degree. C.--30 sec., 55.degree. C.--30
sec., 72.degree. C.--6 min.
[0428] 3. 32 cycles of: 94.5.degree. C.--30 sec., 50.degree. C.--30
sec., 72.degree. C.--5 min.
[0429] A 2.3 kb PCR fragment was obtained instead of the expected
0.6 kb fragment. BamHI digestion of the PCR product resulted in
three fragments of 1.1 kb, 0.7 kb and 0.5 kb. The 1.1 kb fragment
was cloned into pUC19:BamHI. It turned out to be the one adjacent
to the fragment previously obtained and contained the dnaE sequence
right up to the region coding for the AMGKKK (SEQ. ID. No. 64)
peptide, but was disrupted by an intron just upstream of this
region. The sequence that follows this was amplified from the 2.3
kb original PCR product using the same conditions and cycling
scheme as for the 2.3 kb fragment. The downstream primer was the
same as in the previous step. The upstream 27mer
(3'-GTGTGGATCCGTGGTGACCTTAGCCAC-5') (SEQ. ID. Nos. 37 and 115)
consisted of a BamHI site within the first 9 nucleotides
(underlined) and the sequence from the end of the 1.1 kb fragment
previously described.
[0430] The expected 1.2 kb PCR fragment was obtained and cloned
into pUC19:SmaI. This fragment coded for the rest of the intein and
the end of it was used to obtain the next sequence of dnaE
downstream of this region. The upstream 30mer
(3'-TTCGTGTCCGAGGACCTTGTGGTCCACAAC-5') (SEQ. ID. Nos. 38 and 116)
was a sequence from the end of the intron. The downstream 23mer
(5'-CCAGAATCGTCTGCTGGTCGTAG-3') (SEQ. ID. No. 39) was the sequence
from the end of the dnaE gene of D.rad. (coding on the
complementary strand for the region slightly homologous in the
distantly related .alpha. subunits and possibly highly homologous
between T.th. and D.rad. .alpha. subunits). The amplification
reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer,
in a volume of 100 .mu.l of Vent polymerase reaction mixture
containing 10 .mu.l ThermoPol Buffer, 0.5 mM of each dNTP and 0.1
mM MgSO.sub.4. Amplification was performed using the following
cycling scheme:
[0431] 1.3 cycles of: 95.5.degree. C.--30 sec., 55.degree. C.--30
sec., 75.degree. C.--8 min.
[0432] 2. 32 cycles of: 94.5.degree. C.--30 sec., 50.degree. C.--30
sec., 75.degree. C.--5 min.
[0433] A 2.5 kb PCR fragment was obtained and cloned into
pUC19:SmaI. This fragment contained the dnaE sequence coding for
the 300 mino acids next to the AMGKKK (SEQ. ID. No. 64) region
disrupted by yet a second intein inside another sequence that is
conserved among the known .alpha. subunits (FNKSHSAAY) (SEQ. ID.
No. 65).
[0434] To obtain the rest of the dnaE gene the upstream 19mer
(5'-AGCACCCTGGAGGAGCTTC-3') (SEQ. ID. No. 49) from the end of the
known dnaE sequence was used. The downstream primer was:
5'-CATGTCGTACTGGGTGTAC-3' (SEQ. ID. No. 41). The amplification
reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer,
in a volume of 100 .mu.l of Vent polymerase reaction mixture
containing 10 .mu.l ThermoPol Buffer, 0.5 mM of each dNTP and 0.1
mM MgSO.sub.4. Amplification was performed using the following
cycling scheme:
[0435] 1. 3 cycles of: 95.5.degree. C.--30 sec., 55.degree. C.--30
sec., 75.degree. C.--8 min.
[0436] 2. 32 cycles of: 94.5.degree. C.--30 sec., 50.degree. C.--30
sec., 75.degree. C.--5 min.
[0437] A 1.0 kb fragment bracketed by this upstream primer was
obtained. It contained the 3' end of the dnaE gene.
EXAMPLE 11
[0438] Cloning and Expression of the Thermus thermophilus dnaQ Gene
Encoding the .epsilon. Subunit of DNA Polymerase III Replication
Enzyme
[0439] Cloning of dnaQ
[0440] The dnaQ gene of E. coli and the corresponding region of
PolC of B. subtilis, evolutionary divergent organisms, share
approximately 30% identity. Comparison of the predicted amino acid
sequences for DnaQ (.epsilon.) of E. coli and PolC of B. subtilis
revealed two highly conserved regions (FIG. 17). Within each of
these regions, a nine amino acid sequence was used to design two
oligonucleotide primers for use in the polymerase chain
reaction.
[0441] The regions highly conservative among Pol III exonucleases
were chosen to design the degenerate primers for the amplification
of a T.th. dnaQ internal fragment (see FIG. 17). DNA
oligonucleotides for amplification of T.th. genomic DNA were as
follows. The upstream 27mer (5'-GTSGTSNNSGACNNSGAGACSACSGGG-3'
(SEQ. ID. No. 42)) encodes the following sequence (VVXDXETTG) (SEQ.
ID. No. 66). The downstream 27mer
(5'-GAASCCSNNGTCGAASNNGGCGTTGTG-3') (SEQ. ID. No. 43) encodes the
sequence HNAXFDXGF (SEQ. ID. No. 67) on the complementary strand.
The amplification reactions contained 10 ng T.th. genomic DNA, 0.5,
mM of each primer, in a volume of 100 .mu.l of Vent polymerase
reaction mixture containing 10 .mu.l ThermoPol Buffer, 0.5 mM of
each dNTP and 0.5 mM MgSO.sub.4. Amplification was performed using
the following cycling scheme:
[0442] 1. 5 cycles of: 95.5.degree. C.--30 sec., 40.degree. C.--30
sec., 72.degree. C.--2 min.
[0443] 2. 5 cycles of: 95.5.degree. C.--30 sec., 45.degree. C.--30
sec., 72.degree. C.--2 min.
[0444] 3. 30 cycles of: 95.5.degree. C.--30 sec., 50.degree. C.--30
sec., 72.degree. C.--30 min.
[0445] Products were visualized in a 1.5% native agarose gel. A
fragment of the expected size of 270 bp was cloned into the SmaI
site of pUC19 and sequenced with the CircumVent Thermal Cycle DNA
sequencing kit according to the manufacturer's instructions (New
England Biolabs).
[0446] To obtain further sequence of the dnaQ gene, genomic DNA was
digested with either mhoI, BamHI KpnI or NcoI. These restriction
enzymes were chosen because they cut T.th. genomic DNA frequently.
Approximately 0.1 .mu.g of DNA for each digest was ligated by T4
DNA ligase in 50 .mu.l of ligation buffer (50 mM Tris-HCl (pH 7.8),
10 mM MgCl.sub.2, 10 mM dithiothreitol, 1 mM ATP, 25 mg/ml bovine
serum albumin) overnight at 20.degree. C. The ligation mixtures
were used for cicular PCR.
[0447] DNA oligonucleotides for amplification of T.th. genomic DNA
were the following. The upstream 27mer
(5'-CGGGGATCCACCTCAATCACCTCGTGG-3') (SEQ. ID. No. 44) consists of a
BamHI site within the first 9 nucleotides (underlined) and the
sequence complementary to 42-61 bp region of the previously cloned
dnaQ fragment. The downstream 30mer
(5'-CGGGGATCCGCCACCTTGCGGCTCCGGGTG-3') (SEQ. ID. No. 45) consists
of a BamHI site within the first 9 nucleotides (underlined) and the
sequence corresponding to 240-261 bp region of the dnaQ fragment
(see FIG. 17).
[0448] The amplification reactions contained 1 ng T.th. genomic DNA
(that had been cut with NcoI and religated into circular DNA for
circular PCR), 0.4 mM of each primer, in a volume of 100 .mu.l of
Vent polymerase reaction mixture containing 10 .mu.l ThermoPol
Buffer, 0.5 nM of each dNTP, 0.5 mM MgSO.sub.4, and 10% DMSO.
Circular amplification was performed using the following cycling
scheme:
[0449] 1. 5 cycles of: 95.5.degree. C.--30 sec., 50.degree. C.--30
sec., 72.degree. C.--8 min.
[0450] 2. 35 cycles of: 95.5.degree. C.--30 sec., 55.degree. C.--30
sec., 72.degree. C.--6 min.
[0451] 3. 72.degree. C.--10 min.
[0452] A 1.5 kb fragment was obtained and cloned into the BamHI
site of the pUC19 vector. Partial sequencing of the fragment
reveiled that it contained the dnaQ regions adjacent to sequences
corresponding to the PCR primers and hence contained the sequences
both upstream and downstream of the previously cloned dnaQ
fragment. One of NcoI sites turned out to be approximatly 300 bp
downstream of the end of the first cloned dnaQ sequence and hence
did not include the 3' end of dnaQ. To obtain the 3' end, another
inverse PCR reaction was performed. Since an ApaI restiction site
was recognized within this newly sequenced dnaQ fragment, the
circular PCR procedure was performed using as template an ApaI
digest of T.th. genomic DNA that was ligated (circularized) under
the same conditions as described above.
[0453] DNA oligonucleotides for amplification of the ApaI/religated
T.th. genomic DNA were as follows. The upstream 31 mer
(5'-GCGCTCTAGACGAGTTCCCA- AAGCGTGCGGT-3') (SEQ. ID. No. 46)
consists of a mbaI site within the first 10 nucleotides
(underlined) and the sequence complementary to the region
downstream of the ApaI restriction site in the newly sequenced dnaQ
fragment. The downstream 25 mer (5'-CGCGTCTAGATCACCTGTATCCAGA-3')
(SEQ. ID. No. 47) consists of a XbaI site within the first 10
nucleotides (underlined) and the sequence corresponding to another
region downstream of the ApaI restriction site in the newly
sequenced dnaQ fragment. The 1.7 kb PCR fragment was cloned into
the XbaI site of the pUC19 vector and partially sequenced. The
sequence of dnaQ, and the protein sequence of the .epsilon. subunit
encoded by it, is shown in FIG. 18.
[0454] The dnaQ gene is encoded by an open reading frame of 209 (or
190 depending on which Val is used as the initiating residue) amino
acids in length (23598.5 kDa--or 21383.8 kDa for shorter version),
similar to the length of the E. coli subunit (243 amino acids,
27099.1 kDa mass) (see FIG. 17).
[0455] The entire amino acid sequence of the .epsilon. subunit
predicted from the T.th. dnaQ gene aligns with the predicted amino
acid sequence of the dnaQ genes of other organisms with only a few
gaps and insertions (the first two amino acids, and four positions
downstream) (FIG. 17). The consensus motifs VVXDXETTG (SEQ. ID.
Nos. 66 and 68), HNAXFDXGF (SEQ. ID. No. 67), and HRALYD (SEQ. ID.
No. 70), characteristic for exonucleases, are conserved. Overall,
the level of amino acid identity relative to most of the known
.epsilon. subunits, or corresponding proofreading exonuclease
domains of gram positive PolC genes is approximately 30%. Upstream
of start 1 (FIG. 17) there were stop codons in all three reading
frames.
[0456] Expression of dnaQ
[0457] The dnaQ gene was cloned gene into the pET24-a expression
vector in two steps. First, the PCR fragment encoding the
N-terminal part of the gene was cloned into the pUC19 plasmid,
containing the ApaI inverse PCR fragment into NdeI/ApaI sites. DNA
oligonucleotides for amplification of T.th. genomic DNA were as
follows. The upstream 33mer
(5'-GCGGCGCATATGGTGGTGGTCCTGGACCTGGAG-3') (SEQ. ID. No. 48)
consists of an NdeI site within the first 12 nucleotides
(underlined) and the begining of the dnaQ gene. The downstream 25
mer (5'-CGCGTCTAGATCACCTGTAT- CCAGA-3') (SEQ. ID. No. 49), already
used for ApaI circular PCR, consists of an XbaI site within the
first 10 nucleotides (underlined) and the sequence corresponding to
the region downstream of the ApaI restriction site. The 2.2 kb
NdeI/SalI fragment was then cloned into the NdeI/XhoI sites of the
pET16 vector to produce pET24-a:dnaQ. The .epsilon. subunit was
expressed in the BL21/LysS strain transformed by the pET24-a:dnaQ
plasmid.
EXAMPLE 12
[0458] The Thermus thermophilus dnaN Gene Encoding the .beta.
Subunit of DNA Polymerase III Replication Enzyme
[0459] Strategy of Cloning dnaN by Use of dnaA
[0460] DnaN proteins are highly divergent in bacteria making it
difficult to clone them by homology. The level of identity between
DnaN representatives from E. coli and B. subtilis is as low as 18%.
These 18% of identical amino acid residues are dispersed through
the proteins rather then clustering together in conservative
regions, further complicating use of homology to design PCR
primers. However, one feature of dnaN genes among widely different
bacteria is their location in the chromosome. They appear to be
near the origin, and immediately adjacent to the dnaA gene. The
dnaA genes show good homology among different bacteria and, thus,
dnaA was first cloned in order to obtain a DNA probe that is likely
near dnaN.
[0461] Identification of dnaA and dnaN
[0462] The dnaA genes of E. coli and B. subtilis share 58% identity
at the amino acid sequence level within the ATP-binding domain (or
among the representatives of gram-positive and gram-negative
bacteria, evolutionary divergent organisms). Comparison of the
predicted amino acid sequences encoded by dnaA of E. coli and B.
subtilis revealed two highly conserved regions (FIG. 19). Within
each of these regions, a seven amino acid sequence was used to
design two oligonucleotide primers for use in the polymerase chain
reaction. The DNA oligonucleotides for amplification of T.th.
genomic DNA were as follows. The upstream 20mer
(5'-GTSCTSGTSAAGACSCACTT-3') (SEQ. ID. No. 50) encodes the
following sequence: VLVKTHL (SEQ. ID. No. 69). The downstream 21mer
(5'-SAGSAGSGCGTTGAASGTGTG-3', where S is G or C) (SEQ. ID. No. 51)
encodes the sequence: HTFNALL (SEQ. ID. No. 71), on the
complementary strand. The amplification reactions contained 10 ng
T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 .mu.l
of Vent polymerase reaction mixture containing 10 .mu.l ThermoPol
Buffer, 0.5 mM of each dNTP and 0.5 mM MgSO.sub.4. Amplification
was performed using the following cycling scheme:
[0463] 1. 5 cycles of: 95.5.degree. C.--30 sec., 45.degree. C.--30
sec., 75.degree. C.--2 min.
[0464] 2. 5 cycles of: 95.5.degree. C.--30 sec., 50.degree. C.--30
sec., 75.degree. C.--2 min.
[0465] 3. 30 cycles of: 95.5.degree. C.--30 sec., 52.degree. C.--30
sec., 75.degree. C.--30 min.
[0466] Products were visualized in a 1.5% native agarose gel. A
fragment of the expected size of 300 bp was cloned into the SmaI
site of pUC19 and sequenced with the CircumVent Thermal Cycle DNA
sequencing kit (New England Biolabs).
[0467] To obtain a larger section of the T.th. dnaA gene, genomic
DNA was digested with either HaeII, HindIII, KasI, KpnI, MluI,
NcoI, NgoMI, NheI, NsiI, PaeR7I, PstI, SacI, SalI, SpeI, SphI,
StuI, or XhoI, followed by Southern analysis in a native agarose
gel. The filter was probed with the 300 bp PCR product radiolabeled
by random priming. Four different restriction digests showed a
single fragment of reasonable size for further cloning. These were,
KasI, NgoMI, and StuI, all of which produced fragments of about 3
kb, and NcoI that produced a 2 kb fragment. Also, a KpnI digest
resulted in two fragments of about 1.5 kb and 10 kb.
[0468] Genomic DNA digests using either NgoMI and StuI were used to
obtain the dnaA gene by inverse PCR (also referred to as circular
PCR). In this procedure, 0.1 .mu.g of DNA from each digest was
treated separately with T4 DNA ligase in 50 .mu.l of ligation
buffer (50 mM Tris-HCl (pH 7.8), 10 mM MgCl.sub.2, 10 mM
dithiothreitol, 1 mM ATP, 25 mg/ml bovine serum albumin) overnight
at 20.degree. C. This results in circularizing the genomic DNA
fragments. The ligation mixtures were used as substrate in inverse
PCR.
[0469] DNA oligonucleotides for amplification of recircularized
T.th. genomic DNA were as follows. The upstream 22mer was
(5'-CTCGTTGGTGAAAGTTTCCGTG-3') (SEQ. ID. No. 52), and the
downstream 24mer was (5'-CGTCCAGTTCATCGCCGGAAAGGA-3') (SEQ. ID. No.
53). The amplification reactions contained 5 ng T.th. genomic DNA,
0.5 .mu.M of each primer, in a volume of 100 .mu.l of Taq
polymerase reaction mixture containing 10 .mu.l PCR Buffer, 0.5 mM
of each dNTP and 2.5 mM MgCl.sub.2. Amplification was performed
using the following cycling scheme:
[0470] 1. 5 cycles of: 95.0.degree. C.--30 sec., 55.degree. C.--30
sec., 72.degree. C.--10 min.
[0471] 2. 35 cycles of: 95.5.degree. C.--30 sec., 50.degree. C.--30
sec., 72.degree. C.--8 min.
[0472] The PCR fragments of the expected length for NgoMI and StuI
treated and then ligated chromosomal DNA were digested with either
BamHI or Sau3a and cloned into pUC19:BamHI and pUC19:(BamHI+SmaI)
and sequenced with CircumVent Thermal Cycle DNA, sequencing kit.
The 1.6 kb (BamHI+BamH) fragment from the NgoMI PCR product
contained a sequence coding for the N-terminal part of dnaN,
followed by the gene for enolase. The 1 kb (Sau3a+Sau3a) fragment
from the same PCR product included the start of dnaN gene and
sequence characteristic of the origin of replication (i.e., 9mer
DnaA-binding site sequences). The 0.6 kb (BamHI+BamHI) fragment
from the StuI PCR reaction contained starts for dnaA and gidA genes
in inverse orientation to each other. The 0.4 kb (Sau3a+Sau3a)
fragment from the same PCR product contained the 3' end of the dnaA
gene and DNA sequence characteristic for the origin of
replication.
[0473] This sequence information provided the beginning and end of
both the dnaA and the dnaN genes. Hence, these genes were easily
cloned from this information. Further, the dnaN gene was readily
cloned and expressed in a pET24-a vector. These steps are described
below.
[0474] Cloning and Sequence of the dnaA Gene
[0475] The dnaA gene was cloned for sequencing in two parts: from
the potential start of the gene up to its middle and from the
middle up to the end. For the N-terminal part, the upstream 27mer
(5'-TCTGGCAACACGTTCTGGAGCACATCC-3') (SEQ. ID. No. 54) was 20 bp
downsteam of the potential start codon of the gene. The downstream
23mer (5'-TGCTGGCGTTCATCTTCAGGATG-3') (SEQ. ID. No. 55) was
approximately from the middle of the dnaA gene. For the C-terminal
part, the upstream 23mer (5'-CATCCTGAAGATGAACGCCAGCA-3') (SEQ. ID.
No. 56) was complementary to the previous primer. The downstream
25mer (5'-AGGITATCCACAGGGGTCATGTGCA-3- ') (SEQ. ID. No. 57) was 20
bp upstream the potential stop codon for the dnaA gene. The
amplification reactions contained 10 ng T.th. genomic DNA, 0.5
.mu.M of each primer, in a volume of 100 .mu.l of Vent polymerase
reaction mixture containing 10 .mu.l ThermoPol Buffer, 0.5 mM of
each dNTP and 0.5 mM MgSO.sub.4. Amplification was performed using
the following cycling scheme:
[0476] 1. 5 cycles of: 95.5.degree. C.--30 sec., 55.degree. C.--30
sec., 75.degree. C.--3 min.
[0477] 2. 30 cycles of: 95.5.degree. C.--30 sec., 50.degree. C.--30
sec., 75.degree. C.--2 min.
[0478] Products were visualized in a 1.0% native agarose gel.
Fragments of the expected sizes of 750 bp and 650 bp were produced,
and were sequenced using CircumVent Thermal Cycle DNA sequencing
method (New England Biolabs). The nucleotide and amino acid
sequences of dnaA and its protein product are shown in FIG. 20. The
DnaA protein is homologous to the DnaA proteins of several other
bacteria as shown in FIG. 19.
[0479] Cloning and Expression of dnaN
[0480] The full length dnaN gene was obtained by PCR from T.th.
total. DNA. DNA oligonucleotides for amplification of T.th. dnaN
were the following: the upstream 29mer
(5'-GTGTGTCATATGAACATAACGGTTCCCAA-3') (SEQ. ID. No. 58) consists of
an NdeI site within first 11 nucleotides (underlined), followed by
the sequence for the start of the dnaN gene; the downstream 29mer
(5'-GCGCGAATTCTCCCTTGTGGAAGGCTTAG-3') (SEQ. ID. No. 59) consists of
an EcoRI site within the first 10 nucleotides (underlined),
followed by the sequence complementary to a section just downstream
of the dnaN stop codon. The amplification reactions contained 10 ng
T.th. genomic DNA, 0.5 .mu.M of each primer, in a volume of 100
.mu.l of Vent polymerase reaction mixture containing 10 .mu.l
Thermopol Buffer, 0.5 mM of each dNTP and 0.2 mM MgSO.sub.4.
Amplification was performed using the following cycling scheme:
[0481] 1. 5 cycles of: 95.0.degree. C.--30 sec., 55.degree. C.--30
sec., 75.degree. C.--5 min.
[0482] 2. 35 cycles of: 95.5.degree. C.--30 sec., 50.degree. C.--30
sec., 75.degree. C.--4 min.
[0483] The nucleotide and amino acid sequences of dnaN and the
.beta. subunit, respectively, are shown in FIG. 21. The T.th.
.beta. subunit shows limited homology to the .beta. subunit
sequences of several other bacteria over its entire length (FIG.
22).
[0484] The approximately 1 kb dnaN gene was cloned into the pET24-a
expression vector using the NdeI and EcoRI restriction sites both
in the dnaN containing PCR product and in pEt24-a (FIG. 23).
Expression of T.th. .beta. subunit was obtained under the following
conditions: a fresh colony of Bl21(DE3) E. coli strain was
transformed by the pET24-a:dnaN plasmid, and then was grown in LB
broth containing 50 mg/ml kanamycin at 37.degree. C. until the cell
density reached 0.4 OD.sub.600. The cell culture was then induced
for dnaN expression upon addition of 2 mM IPTG. Cells were
harvested after 4 additional hours of growth under 37.degree. C.
The induction of the T.th. .beta. subunit is shown in FIG. 24.
[0485] Two liters of BL21(DE3)pETdnaN cells were grown in LB media
containing 50 mg/ml ampicillin at 37.degree. C. to an O.D. of 0.8
and then IPTG was added to a concentration of 2 mM. After a further
2 h at 37.degree. C., cells were harvested by centrifugation and
stored at -70.degree. C. The following steps were performed at
4.degree. C. Cells were thawed and resuspended in 40 ml of 5 mM
Tris-HCl (pH 8.0), 1% sucrose, 1M NaCl, 5 mM DTT; and 30 mM
spermidine. Cells were lysed using a French Pressure cell at 20,000
psi. The lysate was allowed to sit at 4.degree. C. for 30 min. and
then cell debris was removed by centrifugation (Sorvall SS-34
rotor, 45 min. 18,000 rpm). The supernatant was incubated at
65.degree. C. for 20 minutes with occasional stirring. The
resulting protein precipitate was removed by centrifugation as
described above. The supernatant was dialyzed against 4 liters of
buffer A containing 50 mM NaCl overnight. The dialyzed supernatant
was clarified by centrifugation (35 ml, 150 mg total) and then
loaded onto an 8 ml MonoQ column equilibrated in buffer A
containing 50 mM NaCl. The column was washed with 5 column volumes
of the same buffer and then eluted with a 120 ml gradient of buffer
A plus 50 mM NaCl to buffer A plus 500 mM NaCl. Fractions of 2 ml
were collected. Over 50 mg of T.th. .beta. was recovered in
fractions 5-21.
EXAMPLE 13
[0486] Identification and Cloning of T. thermophilus holA
[0487] A search of the incomplete T.th. genome database
(www.g21.bio.uni-goettingen.de) showed a match to E. coli .delta.
encoded by holA. The sequence obtained from the database was as
follows (SEQ. ID. No. 185):
4 TPKGKDLVRHLENRAKRLGLRLPGGVAQYLA-SLEGDLEALERELEKLAL
LSP-PLTLEKVEKVVALRPPLTGFDLVRSVLEKDPKEALLRLGRLKEEGE
EPLRLLGALSWQFALLARAFFLLREMPRPKEEDLARLEAHPYAAKKALL-
EAARRLTEEALKEALDALMEAEKRAKG-GKDPWLALEAAVLRLAR-PAGQ PRVD
[0488] Next, the following PCR primers were designed from the codon
usage of T.th.: upstream 27mer (5'- GCC CAG TAC CTC GCC TCC CTC GAG
GGG -3') (SEQ. ID. No. 186) and downstream 27mer (5'- GGC CCC CTT
GGC CTT CTC GGC CTC CAT -3' (SEQ. ID. No. 187) to obtain a partial
holA nucleotide sequence (SEQ. ID. No. 188):
5 AGACTCGAGG CCCTGGAGCG GGAGCTGGAG AAGCTTGCCC TCCTCTCCCC ACCCCTCACC
60 CTGGAGAAGG TGGAGAAGGT GGTGGCCCTG AGGCCCCCCC TCACGGGCTT
TGACCTGGTG 120 CGCTCCGTCC TGGAGAAGGA CCCCAAGGAG GCCCTCCTGC
GCCTCAGGCG CCTCAGGGAG 180 GAGGGGGAGG AGCCCCTCAG GCTCCTCGGG
GCCCTCTCCT GGCAGTTCGC CCTCCTCGCC 240 CGGGCCTTCT TCCTCCTCCG
GGAAAACCCC AGGCCCAAGG AGGAGGACCT CGCCCGCCTC 300 GAGGCCCACC
CCTACGCCGC CAAGAAGGCC A 331
[0489] This sequence codes for a partial amino acid sequence of the
T.th. .delta. subunit (SEQ. ID. No. 189):
6 RLEALERELEKLALLSPPLTLEKVEKVVALRPPLTGFDLVRSVLEKDPKE
ALLRLRRLREEGEEPLRLLGALSWQFALLARAFFLLRENPRPKEEDLARL EAHPYAAKKA
[0490] The DNA sequence obtained by PCR (SEQ. ID. No. 188) was used
to design internal primers for inverted PCR. The upstream 31 mer
(5'-GTGGTGTCTAGACATCATAACGGTTCTGGCA-3') (SEQ. ID. NO. 190)
introduced an XbaI site for cloning holA into a pGEX vector. The
downstream 27mer (5'-GAGGGCCACCACCTTCTCCACCTTCTC-3') (SEQ. ID. No.
191) encodes holA sequence EKVEKVVAL (aa residues 159-167 of SEQ.
ID. No. 158) on the complementary strand. The amplification
reactions contained 50 ng T.th. genomic DNA and 0.1 uM of each
primer in a volume of 100 .mu.l of Vent polymerase reaction mixture
containing 10 .mu.l ThermoPol Buffer, 2.5 mM of each dNTP, 2 mM
MgSO.sub.4, and 10 .mu.l of formamide. Amplification was performed
using the following cycling scheme:
[0491] 1. 5 cycles of: 95.degree. C.--30 sec., 65.degree. C.--20
sec., 75.degree. C.--5 min.
[0492] 2. 5 cycles of: 95.degree. C.--20 sec., 58.degree. C.--10
sec., 75.degree. C.--5 min.
[0493] 3. 35 cycles of: 95.degree. C.--20 sec., 50.degree. C.--5
sec., 75.degree. C.--4 min.
[0494] Products were visualized in a 1.0% native agarose gel. A
fragment of 1.5 Kb was gel purified and partially sequenced.
[0495] A different set of primers were used to obtain the 3'-end of
T.th. holA, including an upstream 25mer
(5'-CTCCGTCCTGGAGAAGGACCCCAAG-3') (SEQ. ID. No. 192) which encoded
the amino acid sequence SVLEKDPK from T.th. holA (aa residues
179-186 of SEQ. ID. No. 158), and a downstream 29mer
(5'-CGCGAATTCAACGCSCTCCTCAAGACSCT-3' where S=C or G) (SEQ. ID. No.
193) was not related to the holA sequence. The amplification
reactions contained 50 ng T.th. genomic DNA and 0.1 .mu.M of each
primer in a volume of 100 .mu.l of Vent polymerase reaction mixture
containing 10 .mu.l ThermoPol Buffer, 2.5 mM of each dNTP, and 1-2
mM MgSO.sub.4, and 10 .mu.l of formamide. Amplification was
performed using the following cycling scheme:
[0496] 1. 5 cycles of: 95.degree. C.--30 sec., 65.degree. C.--20
sec., 75.degree. C.--5 min.
[0497] 2. 5 cycles of: 95.degree. C.--20 sec., 55.degree. C.--10
sec., 75.degree. C.--5 min.
[0498] 3. 35 cycles of: 95.degree. C.--20 sec., 50.degree. C.--5
sec., 75.degree. C.--4 min.
[0499] Products were visualized in a 1.0% native agarose gel. A
fragment of 1.2 Kb was gel purified and partially sequenced to
obtain the remainder of the T.th. holA gene.
[0500] The T.th. holA gene was cloned into the NdeI/EcoRI sites in
the pET24 vector using a pair of primers. The upstream 31 mer
(5'-GACACTTAACATATGGTCATCGCCTTCACCG-3') (SEQ. ID. No. 194) contains
a NdeI site within the first 15 nucleotides (underlined) and has a
sequence corresponding to 5' region of T.th. holA. The downstream
38 mer (5'-GTGTGTGAATTCGGGTCAACGGGCGAGGCGGAGGACCG-3'). (SEQ. ID.
No. 195) contains a EcoRI site within the first 12 nucleotides
(underlined) and has a sequence complementary to the 3' end of holA
gene.
EXAMPLE 14
[0501] Identification of T.th. holB Encoding .delta.40 Subunit
[0502] To clone the ends of T.th. holB gene, it was assumed that
the order of genes in Thermus thermophilis could be the same as in
related Deinococcus radiodurance. Multiple alignment of the
upstream neighbor (probable phosphoesterase, DNA repair Rad24c
related protein) revealed a conservative region close to the
C-terminus of the protein sequence:
7 (SEQ. ID. No. 196) Deinococcus radiodurance VILNPGSVGQ (SEQ. ID.
No. 197) Methanococcus janaschii YLINPGSVGQ (SEQ. ID. No. 198)
Thermotoga maritima LVLNPGSAGR
[0503] The D.rad. sequence was used to design an upstream 28mer
primer (5'-CTGGTGAACCCGGGCTCCGTGGGCCAGC-3') (SEQ. ID. No. 199) that
encodes the amino acid sequence LLVNPGSVGQ (SEQ. ID. No. 200) and a
downstream 27mer (5'-CTCGAGGAGCTTGAGGAGGGTGTTGGC-3') (SEQ. ID. No.
201) encodes the sequence ANTLLKLLE (SEQ. ID. No. 202) on the
complementary strand. The amplification reactions contained 50 ng
T.th. genomic DNA and 0.1 .mu.M of each primer in a volume of 100
.mu.l of Deep Vent polymerase reaction mixture containing 10 .mu.l
ThermoPol Buffer, 2.5 nM of each dNTP, 1.5 mM MgSO.sub.4 and 10
.mu.l formamide. Amplification was performed using the following
cycling scheme:
[0504] 1. 5 cycles of: 95.degree. C.--30 sec., 68.degree. C.--20
sec., 75.degree. C.--3 min.
[0505] 2. 5 cycles of: 95.degree. C.--20 sec., 63.degree. C.--20
sec., 75.degree. C.--3 min.
[0506] 3. 35 cycles of: 95.degree. C.--20 sec., 55.degree. C.--10
sec., 75.degree. C.--3 min.
[0507] Product was visualized in a 1.0% native agarose gel as a
single band of 0.7 Kb. The fragment was purified and partially
sequenced.
[0508] Multiple alignment of the gene downstream of D.rad.
identified the following conservative region:
8 Deinococcus radiodurans GFGGVQLHAAHGYLLSQFLSPRHNVREDEYGG (SEQ.
ID. No. 203) Caenorhabditis elegans
GFDGIQLHGAHGYLLSQFTSPTTNKRVDKYGG (SEQ. ID. No. 204) Pseudomonas
aeruginosa GFSGVEIHAAHGYLLSQFLSPLSNRRSDAWGG (SEQ. ID. No. 205)
Archaeoglobus fulgidus GFDAVQLHAAHGYLLSEFISPHVNRRKDEYGG (SEQ. ID.
No. 206)
[0509] The fragment in bold was used to design primers,
specifically the downstream primer, for cloning of the 3' region of
the T.th. holB gene. The upstream 30mer
(5'-CATCCTGGACTCGGCCCACCTCCTCACCGA-3') (SEQ. ID. No. 207) encodes
the amino acid sequence ILDSAHLLT (SEQ. ID. No. 208). The
downstream 33mer (5'- GAGGAGGTAGCCGTGGGCCGCGTGGAGCTCCAC-3') (SEQ.
ID. No. 209) encodes the sequence VELHAAHGYLL (SEQ. ID. No. 210) on
the complementary strand. The amplification reactions contained 50
ng T.th. genomic DNA and 0.1 .mu.M of each primer in a volume of
100 .mu.l of Deep Vent polymerase reaction mixture containing 10
.mu.l ThermoPol Buffer, 2.5 mM of each dNTP, 2 mM MgSO.sub.4, and
10 .mu.l DMSO. Amplification was performed using the following
cycling scheme:
[0510] 1. 5 cycles of: 95.degree. C.--30 sec., 70.degree. C.--20
sec., 75.degree. C.--4 min.
[0511] 2. 5 cycles of: 95.degree. C.--20 sec., 66.degree. C.--20
sec., 75.degree. C.--4 min.
[0512] 3. 30 cycles of: 95.degree. C.--20 sec., 60.degree. C.--10
sec., 77.degree. C.--4 min.
[0513] Products were visualized in a 1.0% native agarose gel as a
single band of 1.1 kb. The Kb fragment was gel purified and
sequenced to provide the remainder of the holB gene encoding T.th.
.delta.'.
[0514] For protein expression, the T.th. holB gene was cloned into
the pET124 vector at the Nde:EcoR sites using a pair of primers.
The upstream 32mer (5'-GGCTTTCCCATATGGCTCTACACCCGGCTCAC-3') (SEQ.
ID. No. 211) contains a NdeI site within the first 15 nucleotides
(underlined) and the sequence corresponding to the 5' region of
T.th. holB. The downstream 29 mer
(5'-GCGTGGATCCACGGTCATGTCTCTAAGTC-3') (SEQ. ID. No. 212) contains a
BamHI site within the first 10 nucleotides (underlined) and a
sequence complementary to the 3' end of the holB gene.
EXAMPLE 15
[0515] Alternate Synthetic Path in Absence of Clamp Loader
Activity
[0516] As discussed earlier, the Pol III-type enzyme of the present
invention is capable of application and use in a variety of
contexts, including a method wherein the clamp loader component
that is traditionally involved in the initiation of enzyme
activity, is not required. The clamp loader generally functions to
increase the efficiency of ring assembly onto circular primed DNA,
because both the ring and the DNA are circles and one must be
broken transiently for them to become interlocked rings. In such a
reaction, the clamp loader increases the efficiency of opening the
ring.
[0517] The procedure described below illustrates the instance where
the clamp loader need not be present. For example, the .beta. clamp
can be assembled onto DNA in the absence of the clamp loader.
Particularly, the bulk of primed templates in PCR reactions are
linear ssDNA fragments that are primed at the ends. On linear
primed DNA, the ring need not open at all. Instead, the ring can
simply thread onto the end of the linear primed template (Bauer and
Burgers, 1988; Tan et al, 1986; O'Day et al., 1992; Burgers and
Yoder, 1993). Hence, on linear primed templates, such as those
generated in PCR, the beta clamp can simply slide over the DNA end.
After the ring slides onto the end, the DNA polymerase can
associate with the ring for enhanced DNA synthesis.
[0518] Such "end assembly" is common among Pol III-type enzymes and
has been demonstrated in yeast and human systems. Rings assembling
onto linear DNA for use by their respective DNA polymerases are
shown in the following example demonstrated in the E. coli
bacterial system, in the human system, and in the T.th. system.
[0519] The bulk of the primed templates in PCR reactions are linear
ssDNA fragments that are primed at their ends. However, these end
primed linear fragments are not generated until after the first
step of PCR has already been performed. In the very first step, PCR
primers generally anneal at internal sites in a heat denatured
ssDNA template. Primed linear templates are then generated in
subsequent steps enabling use of this alternate path. For this
first step, the clamp may be assembled onto an internal site in the
absence of the clamp loader using special conditions that allow
clamp assembly in the absence of a clamp loader.
[0520] For example, a set of conditions that lead to assembly of
the clamp onto circular DNA (i.e., internal primed sites) have been
described in the protocol for the use of the bacteriophage T4 ring
shaped clamp (gene 45 protein) without the clamp loader (Reddy et
al., 1993). In this case, polyethylene glycol leads to
"macromolecular crowding" such that the clamp and DNA are pushed
together in close proximity, leading to the ring self assembling
onto internal primed sites on circular DNA. Other possible
conditions that may lead to assembly of rings onto internal sites
include use of a high concentration of beta such that use of heat
or denaturant to break the dimeric ring into two half rings
(crescents) followed by lowering the heat (or dilution or removal
of denaturant) leading to rings assembling around the DNA.
[0521] The ring shaped sliding clamps of E. coli and human slide
over the end of linear DNA to activate their respective DNA
polymerase in the absence of the clamp loader. This clamp loader
independent assay is performed in the bacterial system in FIG. 25A.
For this assay, the linear template is polydA primed with oligodT.
The polydA is of average length 4500 nucleotides and was purchased
from SuperTecs. OligodT35 was synthesized by Oligos etc. The
template was prepared using 145 .mu.l of 5.2 mM (as nucleotide)
polydA and 22 .mu.l of 1.75 mM (as nucleotide) oligodT. The mixture
was incubated in a final volume of 2100 .mu.l T.E. buffer (ratio as
nucleotide was 21:1 polydA to oligodT). The mixture was heated to
boiling in a 1 ml Eppendorf tube, then removed and allowed to cool
to room temperature. Assays were performed in a final volume of 25
.mu.l 20 mM Tris-Cl (pH 7.5), 8 mM MgCl.sub.2, 5 mM DTT, 0.5 mM
EDTA, 40 mg/ml BSA, 4% glycerol, containing 20 .mu.M
[.alpha.-.sup.32P]dTTP, 0.1 .mu.g polydA-oligodT, 25 ng Pol III
and, where present, 5 .mu.g of .beta. subunit. Proteins were added
to the reaction on ice, then shifted to 37.degree. C. for 5 min.
DNA synthesis was quantitated using DE81 paper as described (Rowen
and Kornberg, 1978).
[0522] In the linear template assay, no ATP or dATP is provided and
therefore, a clamp loader, even if present, is not active. Thus,
the clamp (e.g., .beta.) can only stimulate the DNA polymerase
provided the clamp threads onto the DNA (see diagram in FIG. 25).
Hence, threading of the clamp is shown by a stimulation of the DNA
polymerase. In lane 1 of FIG. 25A, the DNA polymerase is incubated
with the the linear DNA in the absence of the clamp, and lane 2
shows the result of adding the clamp. The results show that the
clamp is able to thread onto the DNA ends and stimulate the DNA
polymerase in the absence of ATP and thus, in the absence of clamp
loading as well.
[0523] This clamp loader independent assay is performed in the
human system in FIG. 25B. The assay reaction (25 .mu.l) contains 50
mM Tris-HCl (pH=7.8), 8 mM MgCl2, 1 mM DTT, 1 mM creatine
phosphate, 40 .mu.g/ml bovine serum albumin, 0.55 Fg human SSB, 100
ng PCNA (where present), 7 units DNA polymerase delta (1 unit
incorporates 1 pmol dTMP in 60 min.), 40 mM [.alpha.-.sup.32P]dTTP
and 0.1 .mu.g polydA-oligodT. Proteins were added to the reaction
on ice, then shifted to 37.degree. C. for 60 min. DNA synthesis was
quantitated using DE81 paper as described (Rowen and Kornberg,
1978). In lane 3, (FIG. 25) the DNA polymerase .delta. is incubated
with the linear DNA in the absence of the clamp, and lane 4 showes
the result of adding the PCNA clamp. The results demonstrate that
the clamp is able to thread onto the DNA ends and stimulate the DNA
polymerase in the absence of ATP and thus, the absence of clamp
loading.
[0524] This clamp loader independent assay is performed in the
T.th. system in FIG. 25C. The assay reaction is exactly as
described above for use of the E. coli Pol III and beta system
except the temperature is 60.degree. C. and here the Pol III is
HEP.P1 T.th. Pol III (0.5 .mu.l, providing 0.1 units where one unit
is equal to 1 pmol of dTMP incorporated in 1 minute under these
conditions and in the absence of beta), and the beta subunit is 7
.mu.g T.th. .beta. (from the MonoQ column). Proteins were added to
the reaction on ice, then shifted to 37.degree. C. for 60 min. DNA
synthesis was quantitated using DE81 paper as described (Rowen and
Kornberg, 1978). In lane 3 (FIG. 25C), the T.Th. Pol III is
incubated with the linear DNA in the absence of the clamp, and lane
4 shows the result of adding the T.th. .beta. clamp. The results
demonstrate that the clamp is able to thread onto the DNA ends and
stimulate the DNA polymerase in the absence of clamp loader
activity.
EXAMPLE 16
[0525] Use of T.th. Pol III in Long Chain Primer Extension
[0526] A characteristic of Pol III-type enzymes is their ability to
extend a single primer for several kilobases around a long (e.g. 7
kb) circular single stranded DNA genome of a bacteriophage. This
reaction uses the circular .beta. clamp protein. For the circular
.beta. to be assembled onto, a circular DNA genome, the circular
.beta. must be opened, positioned around the DNA, and then closed.
This assembly of the circular beta around DNA requires the action
of the clamp loader, which uses ATP to open and close the ring
around DNA. In this example, the 7.2 kb circular single strand DNA
genome of bacteriophage M13 mp18 was used as a template. This
template was primed with a single DNA 57mer oligonucleotide and the
Pol. III enzyme was tested for conversion of this template to a
double strand circular form (RFII). The reaction was supplemented
with recombinant T.th. .beta. produced in E. coli. This assay is
summarized in the scheme at the top of FIG. 26. M13 mp18 ssDNA was
phenol extracted from phage purified as described (Turner and
O'Donnell, 1995). M13 mp18 ssDNA was primed with a 57mer DNA
oligomer synthesized by Oligos etc. The replication assays
contained 73 ng singly primed M13mp18 ssDNA and 100 ng T.th. .beta.
subunit in a 25 .mu.l reaction containing 20 mM Tris-HCl (pH 7.5),
8 mM MgCl.sub.2, 40 .mu.g/ml BSA, 0.1 mM EDTA, 4% glycerol, 0.5 mM
ATP, 60 .mu.M each of dCTP, dGTP, dATP and 20 .mu.M
.alpha.-.sup.32P-TTP (specific activity 2,000-4,000 cpm/pmol).
Either T.th. Pol III from the Heparin, peak 1 (HEP.P1; 5 .mu.l,
0.21 units where 1 unit equals 1 pmol nucleotide incorporated in 1
min.) or a non-Pol III from the Heparin peak 2 (HEP.P2; 5 .mu.l,
2.6 units) were added to the reaction. Reactions were shifted to
60.degree. C. for 5 min., and then DNA synthesis was quenched upon
adding 25 .mu.l of 1% SDS, 40 mM EDTA. One half of the reaction was
analyzed in a 0.8% native agarose gel, and the other half was
quantitated using DE81 paper as described (Studwell and O'Donnell,
1990).
[0527] The results of the assay are shown in FIG. 26. Lane 1 is the
result obtained using the T.th. Pol III (HEP.P1) which was capable
of extending the primer around the ssDNA circle to form RFII. Lane
2 shows the result of using the non-Pol III (HEP.P2) which was not
capable of this extension and produced only incomplete DNA products
(the result shown included 0.8 .mu.g E. coli SSB which did not
increase the chain length of the product). In the absence of SSB,
the same product was observed, although the band contained more
counts. The greater amount of total synthesis observed in lane 2 is
due to the build up of immature products in a small region of the
gel. The presence of immature products in lane 1 is likely due to a
contaminating polymerase in the preparation that can not convert
the single primer to the full length RFII form. Alternatively, the
presence of incomplete products in lane 1 (Pol III type enzyme) is
due to secondary structure in the DNA which causes the Pol III to
pause. In this case it may be presumed that performing the reaction
at higher temperature could remove the secondary structure barrier.
Alternatively, SSB could be added to the assay (although T.th. SSB
would be needed, because addition of E. coli SSB was tried and did
not alter the quality of the product profile). Generally, SSB is
needed to remove secondary structure elements from ssDNA at
37.degree. C. for complete extension of primers by mesophilic Pol
III-type enzymes.
[0528] The assay described above was performed at 60.degree. C. The
T.th. Pol III HEP.P1 gained activity as the temperature was
increased from 37.degree. C. to 60.degree. C., as expected for an
enzyme from a thermophilic source. The E. coli Pol III lost
activity at 60.degree. C. compared to 37.degree. C., as expected
for an enzyme from a mesophilic source.
EXAMPLE 17
[0529] Materials Used in Examples 18-29
[0530] Radioactive nucleotide were from Dupont NEN; unlabeled
nucleotides were from Pharmacia Upjohn. DNA oligonucleotides were
synthesized by Gibco BRL. M13mp18 ssDNA was purified from phage
that was isolated by two successive bandings in cesium chloride
gradients. M13 mp18 ssDNA was primed with a 30-mer (map position
6817-6846) as described. The pET protein expression vectors and
BL21 (DE3) protein expression strain of E. coli were purchased from
Novagen. DNA modification enzymes were from New England Biolabs.
Aquifex aeolicus genomic DNA was a gift of Dr. Robert Huber and Dr.
Karl Stetter (Regensburg University, Germany). Protein
concentrations were determined by absorbance at 280 nm using
extention coefficients calculated from their known Trp and Tyr
content using the equation .epsilon..sub.280=Trp.sub.m (5690
M.sup.-1 cm.sup.-1)+Tyr.sub.n (1280 M.sup.-1 cm.sup.-1).
EXAMPLE 18
[0531] Purification of a Encoded by dnaE
[0532] The Aquifex aeolicus dnaE gene was previously identified
(Deckert et al., 1998). The dnaE was obtained by searching the
Aquifex aeolicus genome, with the amino acid sequence of T.th
.alpha. subunit (encoded by dnaE). The dnaE gene was amplified from
Aquifex aeolicus genomic DNA by PCR using the following primers:
the upstream 37mer (5'-GTGTGTCATATGAGTAAG GATTTCGTCCACCTTCACC-3')
(SEQ. ID. No. 157) contains an NdeI site (underlined); the
downstream 34mer (5'-GTGTGTGGATCCGGGGACTACTCGGAAGTAAGGG-3') (SEQ.
ID. No. 158) contains a BamHI site (underlined). The PCR product
was digested with NdeI and BamHI, purifed, and ligated into the
pET24 NdeI and BamHI sites to produce pETAadnaE.
[0533] The pETAadnaE plasmid was transformed into the BL21 (DE3)
strain of E. coli. Cells were grown in 50 L of LB containing 100
.mu.g/ml of kanamycin, 5 mM MgSO.sub.4 at 37.degree. C. to
OD.sub.600=2.0, induced with 2 mM IPTG for 20 h at 20.degree. C.,
then collected by centrifugation. Cells were resuspended in 400 ml
50 mM Tris-HCl (pH 7.5), 10% sucrose, 1M NaCl, 30 mM spermidine, 5
mM DTT and 2 mM EDTA. The following procedures were performed at
4.degree. C. Cells were lysed by passing them twice through a
French Press (15,000 psi) followed by centrifugation at 13,000 rpm
for 90 min at 4.degree. C. In this protein preparation, as well as
each of those that follow, the induced Aquifex aeolicus protein was
easily discernible as a large band in an SDS polyacrylamide gel
stained with Coomassie Blue. Hence, column fractions were assayed
for the presence of the Aquifex aeolicus protein by SDS PAGE
analysis, which forms the basis for pooling column fractions.
[0534] The clarified cell lysate was heated to, 65.degree. C. for
30 min and the precipitate was removed by centrifugation at 13,000
rpm in a GSA rotor for 1 h. The supernatant (1.4 gm, 280 ml) was
dialyzed against buffer A (20 mM Tris-HCl (pH 7.5)), 10%0 glycerol,
0.5 nM EDTA, 5 mM DTT) overnight, then diluted to 320 ml with
buffer A to a conductivity equal to 100 mM NaCl. The dialysate was
applied to a 150 ml Fast Flow Q (FFQ) Sepharose column (Pharmacia)
equilibrated in buffer A, and eluted with a 1.5 L linear gradient
of 0-500 mM NaCl in buffer A. Eighty fractions were collected.
Fractions 38-58 (1 g, 390 ml) were pooled, dialyzed versus buffer A
overnight, and applied to a 250 ml Heparin Agarose column (Bio-Rad)
equilibrated with buffer A. Protein was eluted with a 1 L linear
0-5 mM NaCl gradient in buffer A. One hundred fractions were
collected. Fractions 69-79 (320 mg in 200 ml) were pooled and
dialyzed against buffer A containing 100 mM NaCl. The .alpha.
preparation was aliquoted and stored frozen at -80.degree. C. (see
FIG. 27).
EXAMPLE 19
[0535] Purification of .delta. Encoded by holA
[0536] The Aquifex aeolicus holA gene was not previously identified
by the genome sequencing group at Diversa (Deckert et al., 1998).
Aquifex aeolicus holA was identified by searching the Aquifex
aeolicus genome with the amino acid sequence of the T.th. .delta.
subunit (encoded by holA). The Aquifex aeolicus holA was amplified
by PCR using the following primers: the upstream 36mer
(5'-GTGTGTCATATGGAAACCACAATATTCCAGTTCCAG-3') (SEQ. ID. No. 159)
contains an NdeI site (underlined); the downstream 39mer
(5'-GTGTGTGGATCCTTATCCACCATGAGAAGTATTTTTCAC-3') (SEQ. ID. No. 160)
contains a BamHI site (underlined). The PCR product was digested
with NdeI and BamHI, purified, and ligated into the pET24 NdeI and
BamHI sites to produce pETAaholA.
[0537] The pETAaholA plasmid was transformed into E. coli strain
BL21 (DE3). Cells were grown in 50 L of LB media containing 100
.mu.g/ml kanamycin. Cells were grown at 37.degree. C. to
OD.sub.600=2.0, induced for 20 h upon addition of 2 mM IPTG, then
collected by centrifugation. Cells from 25 L of culture were lysed
as described in Example 18.
[0538] The cell lysate was heated to 65.degree. C. for 30 min and
the precipatate was removed by centrifugation. The supernatant (650
mg, 240 ml) was dialyzed against buffer A, adjusted to a
conductivity equal to 160 mM NaCl by addition of 40 ml of buffer A,
and applied to a 220 ml Heparin Agarose column equilibrated in
buffer A containing 100 mM NaCl. The column was eluted with 1.0 L
linear gradient of 150-700 mM NaCl in buffer A. One hundred and
four fractions were collected. Fractions 45-56 were pooled (250 mg,
210 ml), diluted with 230 ml buffer A to a conductivity equal to
230 mM NaCl, then loaded onto a 100 ml FFQ Sepharose column
equilbrated in buffer A containing 150 mM NaCl. The column was
eluted with 200 ml linear gradient of 150-750 mM NaCl in buffer A;
seventy-three fractions were collected. Fractions 16-38 were pooled
(95 mg, 40 ml), aliquoted, and stored at -80.degree. C. (see FIG.
27).
EXAMPLE 20
[0539] Purification of .delta.' Encoded by holB
[0540] The Aquifex aeolicus holB gene was previously identified by
the genome sequencing facility at Diversa (Deckert et al., 1998).
The Aquifex aeolicus holB sequence was obtained by searching the
Aquifex aeolicus genome with the sequence of the T.th. .delta.'
(encoded by holB). The Aquifex aeolicus holB gene was amplified by
PCR using the following primers: the upstream 39mer
(5'-GTGTGTCATATGGAAAAAGTTTTTTTTGGAAA AAACTCCAG-3') (SEQ. ID. No.
161) contains an NdeI site (underlined); the downstream 35mer
(5'-GTGTGTGGATCCTTAATCCGCCTGAACGGCTAACG-3') (SEQ. ID. No. 162)
contains a BamHI site (underlined). The PCR product was digested
with NdeI and BamHI, purified, and ligated into the pET24 NdeI and
BamHI site to produce pETAaholB.
[0541] The pETAaholB plasmid was transformed into E. coli strain
BL21 (DE3). Cells were grown at 37.degree. C. in 50 L media
containing 100 .mu.g/ml kanamycin to OD.sub.600 2.0, then induced
for 3 h upon addition of 0.2 mM IPTG. Cells were collected by
centrifugation and were lysed using lysozyme by the heat lysis
procedure (Wickner and Kornberg, 1974). The cell lystate was heated
to 65.degree. C. for 30 min and precipatate was removed by
centrifugation. The supernatant (2.4 g, 400 ml) was dialyzed versus
buffer A, then applied to a 220 ml FFQ Sepharose column
equilibrated in buffer A. Protein was eluted with a 1 L linear
gradient of 0-500 mM NaCl in buffer A; eighty fractions were
collected. Fractions 23-30 were pooled and diluted 2-fold with
buffer A to a conductivity equal to 100 mM NaCl, then loaded onto a
200 ml Heparin Agarose column equilibrated in buffer A. Protein was
eluted with a 1 L linear gradient of 0-1.0M NaCl in buffer A;
eighty-four fractions were collected. Fractions 46-66 were pooled
(1.3 g, 395 ml), dialyzed versus buffer A containing 100 mM NaCl,
then aliquoted and stored frozen at -80.degree. C. (see FIG.
27).
EXAMPLE 21
[0542] Purification of .tau. Encoded by dnaX
[0543] The Aquifex aeolicus dnaX gene was previously identified.
(Deckert et al., 1998). The dnaX gene sequence was obtained by
searching the Aquifex aeolicus genome with the sequence of T.th.
.tau. subunit (encoded by dnaX). The Aquifex aeolicus dnaX was
amplified by PCR using the following primers: the upstream 41mer
(5'-GTGTGTCATATGAACTACGTTCCCTTCGCGA- GAAAGTACAG-3') (SEQ. ID. No.
163) contains an NdeI site (underlined); the downstream 36mer
(5'-GTGTGTGGATCCTTAAAACAGCCTCGTCCCGCTGGA-3') (SEQ. ID. No. 164)
contains a BamHI site (underlined). The PCR product was digested
with NdeI and BamHI, purified, and ligated into the pET24 NdeI and
BamHI sites to produce pETAadnaX.
[0544] The pETAadnaX plasmid was transformed into E. coli strain
BL21 (DE3). Cells were grown in 50 L LB containing 100 .mu.g/ml
kanamycin at 37.degree. C. to OD.sub.600=0.6, then induced for 20 h
at 20.degree. C. upon addition of IPTG to 0.2 mM. Cells were
collected by centrifugation and lysed as described in Example 18.
The clarified cell lysate was heated to 65.degree. C. for 30 min
and the protein precipitate was removed by centrifugation. The
supernatant (1.1 g in 340 ml) was treated with 0.228 g/ml ammonium
sulfate followed by centrifugation. The .tau. subunit remained in
the pellet which was dissolved in buffer B (20 mM Hepes (pH 7.5),
0.5 mM EDTA, 2 mM DTT, 10% glycerol) and dialyzed versus buffer B
to a conductivity equal to 87 mM NaCl. The dialysate (1073 mg, 570
ml) was applied to a 200 ml FFQ Sepharose column equilibrated in
buffer A. The column was eluted with a 1.5 L linear gradient of
0-500 mM NaCl in buffer A; eighty fractions were collected.
Fractions 28-37 were pooled (289 mg, 138 ml), dialyzed against
buffer A to a conductivity equal to 82 mM NaCl, then loaded onto a
150 ml column of Heparin Agarose equilibrated in buffer A. The
column was eluted with a 900 ml linear gradient of 0-500 mM NaCl in
buffer A; thirty-two fractions were collected. Fractions 15-18 (187
mg, 110 ml) were dialyzed versus buffer A, then aliquoted and
stored at -80.degree. C. (see FIG. 27).
EXAMPLE 22
[0545] Purification of .beta. Encoded by dnaN
[0546] The Aquifex aeolicus dnaN gene was previously identified
(Deckert et al., 1998). The dnaN sequence was obtained by searching
the Aquifex aeolicus genome with the sequence of T.th. .beta.
subunit (encoded by dnaN). The Aquifex aeolicus dnaN gene was
amplified by PCR using the following primers: the upstream 33mer
(5'-GTGTGTCATATG CGCGTTAAGGTGGACAGGGAG-3') (SEQ. ID. No. 165)
contains an NdeI site (underlined); the downstream 36mer
(5'-TGTGTCTCGAG TCATGGCTACACCCTCATCGGCAT-3') (SEQ. ID. No. 166)
contains a XhoI site (underlined). The PCR product was digested
with NdeI and BamHI, purified, and ligated into the pET24 NdeI and
BamHI sites to produce pETAadnaN.
[0547] The pETAadnaN plasmid was transformed into E. coli strain
BL21 (DE3). Cells were grown in 1 L LB containing 100 mg/ml
kanamycin at 37.degree. C. to OD.sub.600=1=0.0, then induced for 6
h upon addition of 2 mM IPTG. Cells were collected (7 g) and lysed
as described in Example 18. The cell lysate was heated to
65.degree. C. for 30 min and the protein precipitate was removed by
centrifugation. The supernatant (39 mg, 45 ml) was applied to a 10
ml DEAE Sephacel column (Pharmacia) equilibrated in buffer A. The
column was eluted with a 100 ml linear gradient of 0-500 mM NaCl in
buffer A; seventy-five fractions were collected. Fractions 45-57
were pooled (18.7 mg), dialyzed versus buffer A, and applied to a
30 ml Heparin Agarose column equilibrated in buffer A. The column
was eluted with a 300 ml linear gradient of 0-500 mM NaCl in buffer
A; sixty-five fractions were collected. Fractions 27-33 were pooled
(11 mg, 28 ml) and stored at -80.degree. C. (see FIG. 27).
EXAMPLE 23
[0548] Purification of SSB Encoded by ssb
[0549] The Aquifex aeolicus ssb gene was previously identified
(Deckert et al., 1998g). The ssb gene sequence was obtained by
searching the Aquifex aeolicus genome with the sequence of T.th.
SSB (encoded by ssb). The Aquifex aeolicus ssb gene was amplified
by PCR using the following primers: the upstream 47mer
(5'-GTGTGTCATATGCTCAA TAAGGTTTTTATAATAGGAAGAC- TTACGGG-3') (SEQ.
ID. No. 167) contains an NdeI site (underlined); the downstream
39mer (5'-GTGTGGATCCTTA AAAAGGTATTTCGTCCTCTTCATCGG-3') (SEQ. ID.
No. 0.168) contains a BamHI site (underlined). The PCR product was
digested with NdeI and BamHI, purified, and ligated into the pET16
NdeI and BamHI sites to produce pETAassb.
[0550] The pETAassb plasmid was transformed into E. coli strain
BL21 (DE3). Cells were grown in 6 L of LB media containing 200
.mu.g/ml ampicillin. Cells were grown at 37.degree. C. to
OD.sub.600=0.6, then induced at 15.degree. C. overnight in the
presence of 2 mM IPTG and collected by centrifugation. Cells were
lysed as described above in Example 18, except cells were
resuspended in buffer C (20 mM Tris-HCl (pH 7.9), 500 mM NaCl).
[0551] The cell lysate was heated to 65.degree. C. for 30 min, then
the precipitate was removed by centrifugation. The supernatant (1.4
g, 190 ml) was applied to 25 ml Chelating Sepharose column
(Pharmacia-Biotech) charged with 50 mM Nickel Sulfate and then
equilibrated in buffer C containing 5 mM Imidazole. The column was
eluted with a 300 ml linear gradient of 5-100 mM Imidazole in
buffer C. Fractions of 4 ml were collected. Fractions 81-92 were
pooled (.about.240 mg in 48 ml) and dialyzed overnight against 2 L
of buffer B containing 200 mM NaCl. The dialysate was diluted to a
conductivity equal to 92 mM NaCl using buffer A and then loaded
onto an 8 ml MonoQ column equilibrated in buffer A containing 100
mM NaCl. The column was eluted with a 120 ml linear gradient of
100-500 mM Imidazole in buffer A. Seventy-four fractions were
collected. Fractions 57-70 were pooled (100 mg, 25 ml), aliquoted,
and stored at -80.degree. C. (see FIG. 27).
EXAMPLE 24
[0552] MonoQ Preparation of .tau..delta..delta.'
[0553] The .delta. subunit (0.29 mg) purified in Example 1.9 and
.delta.' subunit (0.31 mg) purified in Example 20 were mixed in a
volume of 2.8 ml of buffer A at 15.degree. C. After 30 min, the
.tau. subunit (0.5 mg in 1.4 ml), purified in Example 21, was added
and the reaction was incubated a further 1 h at 15.degree. C. The
reaction was applied to a 1 ml MonoQ column equilibrated in buffer
A. The .tau..delta..delta.' complex elutes later than either .tau.,
.delta. or .delta.' alone. Protein was eluted with a 32 ml linear
gradient of 100-500 mM NaCl in buffer A; eighty fractions were
collected. Analysis of the MonoQ fractions in a SDS polyacylamide
gel shows a peak of .tau..delta..delta.' complex that elutes in
fractions of 32-38 (see FIG. 28). The peak fractions 850 .mu.g were
stored at -80.degree. C. This procedure can easily be scaled up.
For example, a much larger amount of .tau..delta..delta.' was
constituted by following a similar protocol and using a 8 ml MonoQ
column, which yielded 9.6 mg of .tau..delta..delta.'.
EXAMPLE 25
[0554] Constitution of .alpha..tau..delta..delta.' Complex
[0555] The reaction mixture contained 1.2 mg .alpha. subunit (9
nmol; 133,207 da) purified in Example 18, 0.41 mg .tau. subunit
(7.5 nmol; 54,332 da) purified in Example 21, 0.41 mg .delta.
subunit (10 nmol; 40,693 da) purified in Example 19, and 0.2 mg
.delta.' subunit (9 nmol; 29,000 da) purified in Example 20 in 1.1
ml buffer A. The .alpha. and .tau. subunit solutions were premixed
in 871 .mu.l for 2 h at 15.degree. C. before adding .delta. and
.delta.' subunit solution, then the complete mixture was allowed to
incubate an additional 12 h at 15.degree. C. The reaction may not
require an order of addition, or these extended incubation times.
The reaction mixture was concentrated to 200 .mu.l using a
Centricon 30 at 4.degree. C., then applied to an FPLC Superose 6 HR
10/30 column (25 ml) at 4.degree. C. developed with a continuous
flow of buffer A containing 100 mM NaCl. After the first 216 drops
(6.6 ml), fractions of 7 drops each were collected. Fractions were
analyzed on a SDS polyacrylamide gel stained with Coomassie Blue
(FIG. 29). The analysis was repeated using the .alpha. subunit
alone (FIG. 29). The results show that the peak fractions of
.alpha. shift to a considerably earlier position when .tau.,
.delta. and .delta.' are present and .alpha. comigrates with .tau.,
.delta., and .delta.', when compared to the elution position of
.alpha. alone, indicating that .alpha. assembles with .tau.,
.delta. and .delta.' into a .alpha..tau..delta..delta.'
complex.
EXAMPLE 26
[0556] .alpha..tau..delta..delta.' Functions with the .beta.
Clamp
[0557] Replication reactions were performed using circular M13 mp18
ssDNA primed with a synthetic DNA 90 mer oligonucleotide. Reactions
contained 8.6 kg primed M13 mp18 ssDNA, 9.4 .mu.g SSB purified in
Example 23, 1.0 .mu.g .alpha..tau..delta..delta.' prepared in
Example 25, and 2.0 .mu.g .beta. subunit purified in Example 22
(when present), in 230 .mu.l of 20 mM Tris-HCl (pH 7.5), 5 mM DTT,
4% glycerol, 8 mM MgCl.sub.2, 0.5 mM ATP, 60 .mu.M each dATP and
dGTP (buffer composition is for a final volume of 250 .mu.l).
Reactions were mixed on ice, then aliquoted into separate tubes
containing 25 .mu.l each. For each timed reaction, the mixture was
brought to 65.degree. C. for 2 min before initiating syntheses upon
addition of 2 .mu.l of dCTP and .alpha..sup.2P-dTTP (final
centrations, 60 and 40 .mu.M, respectively). Aliquots were quenched
at the times indicated in FIG. 30 upon adding 4 .mu.l of 0.25M
EDTA, 1% SDS. Quenched reactions were then analyzed in a 0.8%
alkaline agarose gel. The results, illustrated in FIG. 30;
demonstrate that efficient synthesis requires addition of the
.beta. subunit. Comparison with size standards in the same gel
indicates an average speed of .about.125 nucleotides; the leading
edge of the product smear indicates a maximum speed of 375
nucleotides/s.
EXAMPLE 27
[0558] Purification of T.th. .alpha. Subunit
[0559] To obtain T.th. .alpha. subunit, 8 L of E. coli BL21(DE3)
cells harboring pETtthalpha were grown to O.D.=0.3 and induced upon
adding IPTG. Cells were collected by centrifugation and resuspended
in 200 ml 50 mM Tris-HCl (pH 7.5), 10% sucrose, 1M NaCl, 30 mM
spermidine, 5 mM DTT and 2 mM EDTA. The following procedures were
performed at 4.degree. C. Cells were lysed by passing them three
times through a French Press (20,000 psi) followed by incubation at
4.degree. C. for 30 min and then centrifugation at 18,000 rpm in an
SS-34 rotor for 45 min at 4.degree. C. Induced protein was less
that 1% total cell protien but was discernible as a band that
migrated in the appropriate position for its predicted molecular
weight in an SDS polyacrylamide gel stained with Coomassie Blue.
Hence, column fractions were assayed for the presence of the
protein by SDS PAGE analysis, which forms the basis for pooling
column fractions.
[0560] The clarified cell lysate was heated to 65.degree. C. for 30
min and the precipitate was removed by centrifugation. The
supernatant (1.4 gm, 280 ml) was dialyzed against buffer A (20 mM
Tris-HCl (pH 7.5), 10% glycerol, 0.5 mM EDTA, 5 mM DTT) overnight,
then diluted to 320 ml with buffer A to a conductivity equal to 100
mM NaCl. The dialysate (approximately 150 mg) was applied to a 60
ml DEAE Fast Flow Q (FFQ) Sepharose column (Pharmacia) equilibrated
in buffer A, and eluted with a 600 ml linear gradient of 0-500 mM
NaCl in buffer A. Fractions of 8 ml each were collected. The Tth.
.alpha. subunit could be seen as a major band in several fractions,
especially in fractions 26-30. In these peak fractions the Tth.
.alpha. subunit was approximately 20-30 percent pure.
EXAMPLE 28
[0561] Purification of Tth. .epsilon. Subunit
[0562] The dnaQ gene was cloned into the pET16 expression plasmid
using the Val within the context "VGL WEW . . . " and transformed
into E. coli (BL21 (DE3). This pET plasmid places an N-terminal
leader containing six histidines onto the expressed protein to
facilitate purification via use of chelate affinity chromatography.
Twelve liters of cells were grown to an OD of 0.7 and induced with
IPTG. Induced cells were collected by centrifugation and
resuspended in 150 ml of buffer C (20 mM Tris-HCl (pH 7.9), 500 mM
NaCl). Cells were lysed by passing them two times through a French
Press (20,000 psi) followed by incubation at 4.degree. C. for 30
min and then centrifugation at 13,800 rpm in an SLA-1500 rotor for
45 min at 4.degree. C. induced protein appeared greater than 5%
total cell protien and was easily discernible as a band that
migrated in the appropriate position for its predicted molecular
weight in an SDS polyacrylamide gel stained with Coomassie Blue.
Hence, column fractions were assayed for the presence of the
protein by SDS PAGE analysis, which forms the basis for pooling
column fractions.
[0563] Upon analyzing the precipitate from the cell lysis, and the
supernatent, it was determined that the epsilon subunit was
insoluble and appeared in the precipitate. Therefore the cell
pellet was resuspended in 100 ml of binding buffer containing 6M
freshly deionized urea. This resuspension was then placed in
centrifuge bottles and spun at 13,800 rpm for 45 min in the
SLA-1500 rotor. The epsilon was in the supernatent and was applied
to a 25 ml Chelating Sepharose column (Pharmacia-Biotech) charged
with 50 mM Nickel Sulfate and then equilibrated in buffer C
containing 5 mM Imidazole. The column was washed with two column
volumes of buffer C, then washed with 5 column volumes of beffer C
containing 80 mM Imidazole (final). Then the Tth epsilon was eluted
with a 250 ml linear gradient of 60-1000 mM Imidazole in buffer C.
Fractions of 4 ml were collected. Fractions 15-24 were pooled
(.about.131 mg) and dialyzed overnight against 2 L of buffer A
containing 6M urea, but no NaCl or glycerol. The dialysate was then
loaded onto an 8 ml MonoQ column equilibrated in buffer A
containing 6M urea. The column was eluted with a 120 ml linear
gradient of 0-500 mM NaCl in buffer A containing urea Sixty five
fractions were collected. The epsilon is approximately 80-90
percent pure at this stage. Fractions 13-17 were stored at
-80.degree. C. The epsilon is in urea but is at a concentration of
5-10 mg/ml, and thus can be used with other proteins by diluting it
such that the final urea concentration is less than 0.5 M. This
level of urea does not generally denature protein, and should allow
epsilon to renature for catalytic activity.
EXAMPLE 29
[0564] Temperature Optimum of Aquifex and Thermus .alpha. Subunit
DNA Polymerases
[0565] The temperature optimum of the alpha subunits of the Aquifex
and Thermus replicases was tested in the calf thymus DNA
replication assay. In this experiment, the reactions were assembled
on ice in 25 .mu.l containing 2.5 .mu.g calf thymus activated DNA,
and either 0.88 ug Aquifex .alpha., or 0.6 .mu.g of the Thermus
.alpha. DEAE pool of peak fractions (obtained from Examples 18 and
28, respectively) in 20 mM Tris-HCl (pH 8.8), 8 mM MgCl.sub.2, 10
mM KCl, 10 mM (NH.sub.4)SO.sub.4, 2 mM MgSO.sub.4, 0.1% Triton
X-100, 60 .mu.M each dATP, dCTP, dGTP, and 20 .mu.M
.alpha..sup.32P-dTTP. Reactons were shifted to either 30, 40, 50,
60, 70, 80, or 90.degree. C., then stopped after 5 minutes and
spotted onto DE81 filters to quantitate DNA synthesis. The results,
illustrated in FIGS. 31-32, show that these enzymes increase in
activity as the temperature is raised. The Thermus .alpha. has a
broad peak of activity from 70-80.degree. C. (FIG. 31), while the
Aquifex .alpha. is maximal at 80.degree. C. (FIG. 32). The Aquifex
.alpha. retains considerable activity at 90.degree. C., whereas the
Thermus a is nearly inactive at 90.degree. C., a result that is
consistent with the higher temperature at which the Aquifex
aeolicus may live relative to the Thermus bacterium.
EXAMPLE 30
[0566] Temperature Optimum of Aquifex
.alpha..tau..delta..delta.'/.beta.
[0567] Aquifex .alpha., .beta., .tau..delta..delta.', SSB and
.alpha..tau..delta..delta. were tested for stability at different
temperatures by incubating the protein in a solution, followed by
performing a replication assay of the protein. Incubation was
performed in 0.4 ml tubes under mineral oil. The 5 .mu.l reaction
mixture contained: buffer B (20 mM Tris-HCl (pH 7.5), 5 mM DTT, 5
mM EDTA), and either: 0.352 .mu.g of .alpha. (FIG. 33A), 0.2 .mu.g
of .beta. (FIG. 33B), 0.125 .mu.g .tau. complex (FIG. 33C), 0.32
.mu.g SSB and 0.042 .mu.g primed M13 mp18 ssDNA (FIG. 33D), 0.82
.mu.g Pol III* (FIG. 33E). Reactions were incubated for 2 min. at
either 70, 80, 85, or 96.degree. C. in the presence of either 0.1%
Triton X-100 (filled diamonds); 0.05% Tween-20 and 0.01% NP-40
(filled circles); 4 mM CaCl.sub.2 (filled triangles); 40% Glycerol
(inverted filled triangles); 0.01% Triton X-100, 0.05% Tween-20,
0.01% NP-40, 4 mM CaCl.sub.2 (half-filled square); 40% Glycerol,
0.1% Triton X-100 (open diamonds); 40% Glycerol, 0.05% Tween-20,
0.01% NP-40 (open circles); 40% Glycerol, 4 mM CaCl.sub.2 (open
triangles); 40% Glycerol, 0.01% Triton X-100, 0.05% Tween-20, 0.01%
NP-40, 4 mM CaCl.sub.2. (half-filled diamonds). After heating,
reactions were shifted to ice and 20 .mu.l of replication assay
buffer was added followed by incubation for 1.5 min at 70.degree.
C.; 15 .mu.l was then spotted onto a DE81 filter and DNA synthesis
was quantitated. The replication assay buffer contained: 60 mM
Tris-HCl (pH 9.1 at 25.degree. C.), 8 mM MgCl.sub.2, 18 mM
(NH.sub.4).sub.2SO.sub.4, 2 mM ATP, 60 .mu.M each of dATP, dCTP,
dGTP, and 20 .mu.M [.alpha..sup.-32P] TTP (specific activity 10,000
cpm/pmol), and 0.264 .mu.g primed M13 mp18 ssDNA. To assay for
.beta., 0.1 ng .alpha..tau..delta..delta.' was added to the
reaction. To assay .tau..delta..delta.', 0.9 ng .beta. and 0.17 ng
.alpha. were added to the reaction. To assay for SSB, 0.17 ng E.
coli .beta. and 0.1 ng E. coli .alpha..tau..delta..delta.' were
added to the reaction followed by incubation for 1.5 min at
37.degree. C. To assay for .alpha..tau..delta..delta.', 0.9 ng
.beta. was added to the reaction. To assay .alpha., the calf thymus
DNA replication assay was performed in the buffer as described
above but 2.5 .mu.g activated calf thymus DNA was used instead of
primed M13 mp18 ssDNA, no other replication proteins were added,
and incubation was for 8 min at 70.degree. C.
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[0697] This invention may be embodied in other forms or carried out
in other ways without departing from the spirit or essential
characteristics thereof. The present disclosure is therefore to be
considered as in all respects illustrative and not restrictive, the
scope of the invention being indicated by the appended claims, and
all changes which come within the meaning and range of equivalency
are intended to be embraced therein.
Sequence CWU 0
0
* * * * *
References