U.S. patent application number 10/187496 was filed with the patent office on 2003-10-30 for dna polymerase iii holoenzyme.
Invention is credited to O'Donnell, Michael E..
Application Number | 20030203465 10/187496 |
Document ID | / |
Family ID | 34380695 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030203465 |
Kind Code |
A1 |
O'Donnell, Michael E. |
October 30, 2003 |
DNA polymerase III holoenzyme
Abstract
The present invention is directed toward the 5 previously
unknown genes, for subunits .delta., .delta.', .chi., .theta., and
.psi., of the DNA polymerase III holoenzyme, and toward a unique
man-made enzyme containing 5, preferably 6, protein subunits which
shows the same activity as the naturally occurring 10 protein
subunit DNA polymerase III holoenzyme.
Inventors: |
O'Donnell, Michael E.;
(Hastings on Hudson, NY) |
Correspondence
Address: |
Michael L. Goldman
NIXON PEABODY LLP
Clinton Square
P.O. Box 31051
Rochester
NY
14603-1051
US
|
Family ID: |
34380695 |
Appl. No.: |
10/187496 |
Filed: |
July 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10187496 |
Jul 1, 2002 |
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08828323 |
Mar 28, 1997 |
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08828323 |
Mar 28, 1997 |
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08279058 |
Jul 22, 1994 |
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5668004 |
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08279058 |
Jul 22, 1994 |
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07826926 |
Jan 24, 1992 |
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Current U.S.
Class: |
435/199 ;
536/23.2 |
Current CPC
Class: |
C12N 9/1252
20130101 |
Class at
Publication: |
435/199 ;
536/23.2 |
International
Class: |
C12N 009/22; C07H
021/04 |
Goverment Interests
[0002] Research support which led to the making of the present
invention was provided in part by funding from the National
Institutes of Health under Grant No. GM-38839. Accordingly, the
federal government has certain statutory rights to the invention
described herein under 35 U.S.C. 200 et seq.
Claims
I claim:
1. A genetic sequence for an isolated subunit for a DNA polymerase
III gene selected from the subunit group consisting of .delta.,
.delta.', .chi., .theta., and .psi. genes.
2. An isolated and purified enzyme showing the activity of the 10
subunit DNA polymerase III holoenzyme which contains only the
polymerase peptide subunits for .alpha., .epsilon., .beta.,
.delta., and .gamma. subunits of the naturally occurring
holoenzyme.
3. The enzyme according to claim 2 which further contains the
polymerase peptide subunit .delta.' of the naturally occurring
holoenzyme.
4. An isolated and purified peptide selected from the group of the
.delta., .delta.', .chi., .theta., and .psi. DNA polymerase III
holoenzyme peptides.
Description
[0001] The present application for Letters Patent is a
Continuation-in-Part of my earlier U.S. patent application Ser. No.
07/826,926, filed Jan. 24th 1992., said Continuation-in-Part having
been filed as International Patent Application PCT US93/00627 on
Jan. 22nd 1993.
[0003] In 1982, Arthur Kornberg was the first to purify DNA
polymerase III holoenzyme (holoenzyme) and determine that it was
the principal polymerase that replicates the E. coli
chromosome.
[0004] In common with chromosomal replicases of phages T4 and T7,
yeast, Drosophila, mammals and their viruses, the E. coli
holoenzyme contains at least ten subunits in all (.alpha.,
.epsilon., .theta., .tau., .sub..chi., .delta., .delta.',
.sub..chi., .sub..psi., .beta.) [see J. Biol Chem, 257:11468
(1982)]. It has been proposed that chromosomal replicases may
contain a dimeric polymerase in order to replicate both the leading
and lagging strands concurrently. Indeed the 1 MDa size of the
holoenzyme and apparent equal stoichiometry of its subunits (except
.beta. which is twice the abundance of the others) is evidence that
the holoenzyme has the following dimeric composition:
(.alpha..epsilon..theta-
.).sub.2.tau..sub.2(.gamma..delta..delta.'.sub..chi..psi.).sub.2.beta..sub-
.4.
[0005] One of the features of the holoenzyme which distinguish it
as a chromosomal replicase is its use of ATP to form a tight, gel
filterable, "initiation complex" on primed DNA. The holoenzyme
initiation complex completely replicates a uniquely primed
bacteriophage single-strand DNA (ssDNA) genome coated with the
ssDNA binding protein (SSB), at a speed of at least 500 nucleotides
per second (at 30.degree. C.) without dissociating from an 8.6 kb
circular DNA even once. This remarkable processivity (nucleotides
polymerized in one template binding event) and catalytic speed is
in keeping with the rate of replication fork movement in E. coli (1
kb/second at 37.degree. C.). In comparison, DNA polymerase I as
well as the T4 polymerase, Taq polymerase, and T7 polymerase
(sequence) are all very slow (10-20 nucleotides) and lack high
processivity (10-12 nucleotides per binding event). With these
distinctive features the polyIII holoenzyme has commercial
application. However, there is a good reason it has not yet been
applied commercially. Namely, there are only a few (10-20)
molecules of polyIII holoenzyme per cell and thus it Is difficult
to purify; only a few tenths of a milligram ban be obtained from
1000 liters of cells; and it can not be simply overproduced by
genetic engineering because it is composed of 10 different
subunits.
[0006] The subunits of DNA polymerase III holoenzyme are set forth
in the following table:
1 Gene Subunit Mass (kda) Function .alpha. 130 DNA polymerase
.epsilon. 27 Proofreading 3'-5' exonuclease .theta. 10 .tau. 71
Dimerizes core, DNA-dependent ATPase .gamma. 47 Binds ATP holA
.delta. 35 Interact with g to transfer .beta. to DNA holB .delta.'
33 DNA-dependent ATPase with g holC .chi. 15 holD .psi. 12 holE
.beta. 40 Sliding clamp on DNA, binds core
[0007] As discovered in making the present Invention, the .delta.'
is a mixture of two proteins, both encoded by the same holB gene,
and therefore it may be regarded as two subunits of the holoenzyme,
thus bringing the total number of subunits in the holoenzyme to
eleven.
[0008] The genes for 5 of the holoenzyme's subunits have been
identified [see Nucleic Acids Research 14(20): 8091 (1986); Gene
28:159 (1984); PNAS (USA) 80:7137 (1982); J. of Bacteriology
169(12): 5735(1987); and J. of Bacteriology 158:455 (1984)]. These
5 genes have been cloned and overproducing expression plasmids for
these 5 subunits are commercially available. However, prior to the
present invention, the identification for the remaining 5 subunits
which make up the holoenzyme was not known.
[0009] The present invention describes, for the first time, the
genetic and peptide sequences for the remaining five subunits of
the polymerase III holoenzyme. In addition, to sequence these
genes, very efficient overproducing plasmids for each of them have
been constructed, and purification protocols for each have been
devised. Whereas the low amount of holoenzyme in cells has allowed
the subassemblies to be available in microgram quantities prior to
the present invention (milligrams of pure .alpha., .epsilon.,
.tau., .delta. and .beta. subunits are available using molecular
cloned genes in overproducing expression plasmids), utilizing
techniques according to the present invention it has been possible
to obtain approximately 100 mg of homogeneous subunit from 4 L of
cells.
[0010] Prior to the identification of the remaining 5 genes of the
holoenzyme, a few micrograms of each subunit was resolved from the
holoenzyme. The sequence analysis of these resolved subunits
eventually lead to the identification of their genetic sequences,
and then to the genes per se. In addition, reconstitution studies
were carried out to determine which subunits were essential to the
speed and processivity of the holoenzyme. In addition,
overproducing expression plasmids for these 5 subunits were
produced.
[0011] Following these studies, it has now been determined,
according to the present invention, that at least 5 subunits are
required for the action of this enzyme (.alpha., .epsilon., .beta.,
.delta., and .gamma.), and preferably 6 subunits are essential for
the speed and processivity of the holoenzyme. These subunits, the
combination of which are essential for the unique synthetic
capabilities of the holoenzyme, according to the present invention,
are: .alpha., .epsilon., .beta., .delta., .delta.', and
.gamma..
[0012] The 5 subunits according to the present invention which have
been identified, sequenced, cloned, provided in overproducing
expression plasmids, expressed, and purified for the first time are
subunits .delta., .delta.', .sub..chi., .theta., and
.sub..psi..
[0013] The following figures, detailed description and examples are
provided in order to allow the reader to obtain a fuller and more
complete understanding of the present invention. With regard to the
figures,
[0014] FIG. 1 depicts the pET-.delta. expression vector according
to the present invention;
[0015] FIG. 2 depicts the PET-.delta.' expression vector according
to the present invention;
[0016] FIGS. 3A, B, and C depict the replication activity of
.delta., .delta.' and .delta..delta.' with .gamma. and .tau.
according to the present invention;
[0017] FIGS. 4A and B depict the effect of .delta.' and .delta. on
the ATPase activity of .gamma. and .tau. according to the present
invention;
[0018] FIG. 5 depicts the pET-.theta. expression plasmid according
to the present invention;
[0019] FIGS. 6A and B depict the reconstitution assay according to
the present invention indicating that .theta. does not stimulate
DNA synthesis;
[0020] FIG. 7 depicts that .theta., according to the present
invention stimulates .epsilon. in excision of an incorrect 3' TG
base pair;
[0021] FIGS. 8A and B depicts the native molecular weight of ae and
polIII core according to the present invention;
[0022] FIG. 9 depicts the construction of the pET-.sub..psi.
overproducing plasmid according to the present invention;
[0023] FIGS. 10A and B depict the stimulation of the DNA dependent
ATPase of .gamma. and .tau. by .sub..psi. and .sub..chi., according
to the present invention;
[0024] FIG. 11 depicts the construction of the PET-.sub..chi.
expression plasmid according to the present invention; and
[0025] FIGS. 12A and B depicts native molecular mass of .sub..chi.,
.sub..psi. and the .sub..chi..psi. complex, according to the
present invention.
[0026] More specifically with regard to FIG. 1, there is shown the
expression vector for .delta. as prepared and described in the
following examples. The holA gene excised from M13-.delta.-NdeI
using NdeI is shown above the pET3c vector. The open reading frame
encoding .delta. is inserted into the NdeI site of pET3c such that
the initiating ATG is positioned downstream of the Shine-Dalgarno
sequence and a T7 promoter. Downstream of the holA Insert are 492
nucleotides of E. coli DNA and 591 nucleotides of M13mp18 DNA. The
T7 RNA polymerase termination sequence is downstream of the holA
insert.
[0027] More specifically with regard to FIG. 2, the holB fragment
excised from M13-.delta.'-NdeI using NdeI is shown above the
expression vector. The open reading frame encoding .delta.' is
inserted into the NdeI site of pET3c such that the initiating ATG
is positioned downstream of the Shine-Dalgarno sequence and a T7
promoter. The holB insert also contains 158 nucleotides of E. coli
DNA downstream of the the holB stop codon to an NdeI site. The T7
polymerase termination sequence is downstream of the holB
insert.
[0028] With regard to FIG. 3, replication assays were performed as
described below. FIG. 3C summaries the replication assays. Either
.gamma. or .tau. was titrated into assays containing SSB "coated"
primed M13mp18 ssDNA, .intg., ae and either 2 ng .delta., 2 ng
.delta.' or an equal mixture (1 ng each) of .delta. and .delta.'
(.delta..delta.'). The reaction mixture was preincubated for 8
minutes to allow reconstitution of the processive polymerase prior
to initiating a 20 second pulse of DNA synthesis. FIG. 3A depicts
the results of the .gamma. subunit being titrated into the
replication mixture either alone (open squares) or containing
either .delta.' (closed circles), .delta. (open circles), or
.delta..delta.' (closed squares). FIG. 3B depicts the results of
the .gamma. subunit being titrated into the replication mixture
either alone (open triangles), or containing either .delta.'
(closed circles), .delta. (open circles), or .delta..delta.'
(closed squares).
[0029] With regard to FIG. 4, ATPase assays were performed in the
presence of M13mp18 ssDNA as described in detail below. The
subunits in each assay are identified below the plot in the figure.
FIG. 4A refers to the effect of .delta., .delta.' and .beta. on the
.gamma.ATPase; FIG. 4A refers to the effect of .delta., .delta.'
and .beta. on the .tau.ATPase.
[0030] With regard to FIG. 5, the shaded NdeI-BamHI segment
includes the holE gene (arrow). Transcription of the holE is driven
by a T7 promoter. The T7 RNA polymerase termination sequence is
downstream from the E. coli DNA insert. Translation of .delta. is
aided by an upstream Shine-Dalgarno sequence.
[0031] With regard to FIG. 6, the replication reactions were
performed as described below. FIG. 6A outlines the protocol
summarizing the assay. Either the .alpha..epsilon. complex or
reconstituted polIII core (.alpha..epsilon..theta.) were titrated
into the assay which contains .beta., .gamma. complex and primed
phage X 174 ssDNA "coated" with SSB. Proteins and DNA were
preincubated for 6 minutes to allow time for assembly of the
processive polymerase. A 15 second round of synthesis was initiated
upon addition of remaining deoxynucleoside triphosphates. Circles:
titration of .alpha..epsilon. complex; triangles: titration of
.alpha..epsilon..theta.. The .alpha. subunit was limiting in these
assays and therefore the amount of .alpha..epsilon. and
.alpha..epsilon..theta. added to the assay is taken as the amount
of .alpha. added.
[0032] With regard to FIG. 7, there is depicted the results of a
titration of .theta. into the assay containing .epsilon. and a
mismatched 3'32P-end-labelled T residue on a synthetic "hooked"
primer template.
[0033] With regard to FIG. 8, the is shown a comparison of the
migration of .alpha..epsilon. and polIII core relative to protein
standards on gel filtration and in glycerol gradients. The position
of polIII core reconstituted using either excess or
substoichiomtetric .theta. was the same in both types of analysis.
FIG. 8A depicts gel filtration analysis on Superose 12. The Stokes
radius of protein standards was calculated from their known
diffusion coefficients. FIG. 8B depicts glycerol gradient
sedimentation analysis. Sedimentation coefficients of the standards
are Amy, sweet potato .beta.-amylase (152 kDa, 8.9S); Apf, horse
apoferritin (467 kDa, 59.5 .ANG.); IgG; bovine immunoglobulin G
(158 kDa, 52.3 .ANG., 7.4S); BSA, bovine serum albumin (67 kDa,
34.9 .ANG., 4.41S); Ova, chicken ovalbumin (43.5 kDa, 27.5 .ANG.,
3.6S); Myo, horse myoglobin (17.5 kDa, 19.0 .ANG., 2.0S). The
positions of ae and polIII core relative to the protein standards
are indicated in the plots. The Stokes radii and S values of
.alpha..epsilon. and polIII were measured by comparison to the
standards.
[0034] With regard to FIG. 9, the holD gene was amplified from
genomic DNA using primers which form an NdeI site at the Initiating
ATG and downstream BamHI site. Due to an internal NdeI site within
holD, insertion of the complete holD gene into the pET3c expression
plasmid required the two steps shown below. Additional details
appear in the following description.
[0035] With regard to FIG. 10, ATPase assays were performed using a
two-fold molar excess of .sub..chi. and .sub..psi. (as monomers)
over .gamma. and .tau. (as dimers) and using M13mp18 ssDNA as an
effector. FIG. 10A depicts ATPase assays of .sub..psi., .sub..chi.,
.gamma. and combination of these proteins; FIG. 10B depicts the
affect of .sub..psi. and .sub..chi. subunits on the ATPase of
.tau.. Subunits in the assays are indicated below the plots, and
assays performed in the presence of SSB are indicated.
[0036] With regard to FIG. 11, the holC gene was amplified from
genomic DNA using primers which generate an NdeI site at the start
codon of holC and a BamHI site 152 nucleotides downstream of holC
as described below. The 604 bp amplified product was purified,
digested with NdeI and BamHI, and ligated into the NdeI and BamHI
sites of pET3c to yield pET-.sub..chi.. The open reading frame
encoding .sub..chi. was inserted into the NdeI-BamHI sites of pET3c
such that the initiating ATG is positioned downstream of the
Shiner-Dalgarno sequence and a T7 promoter. The T7 RNA polymerase
termination sequence is downstream of the holC insert. The
Amp.sup.r indicates the ampicillin resistance gene; the ori
indicates the pB322 origin of replication.
[0037] With regard to FIG. 12A, the Stokes radius of .sub..chi.,
.sub..psi. and .sub..chi..psi. complex was determined by comparison
with protein standards in gel filtration on Superdex 75. With
regard to FIG. 12B, the S value of .sub..chi., .sub..psi. and
.sub..chi..psi. complex determined by comparison to protein
standards in glycerol gradient analysis are given. The protein
standards were: bovine serum albumin (BSA), 34.9 .ANG., 4.41S;
chicken ovalbumin (Ova) 27.5 .ANG., 3.6S; soybean trypsin inhibitor
(Ti), 23.8 .ANG.; bovine carbonic anhydrase II (Carb), 3.06S; horse
myobglobin (Myo), 19.0 .ANG., 2.0S; and horse kidney metalothionin
(Met), 1.75S.
[0038] In general, the sequence for each of the genes for the five
subunit peptides, according to the present invention, began with
isolating, purifying and sequencing the individual peptides.
[0039] The .delta., .delta.', .sub..chi., .sub..psi. subunits were
purified by a combination of two published procedures. First the
.gamma. complex (.gamma., .delta., .delta.', .sub..psi., was
purified from 1.5 Kg E. coli HB101 (pNT203-pSK100) as described by
Maki [see J. Bio.
[0040] Chem 263:6555(1988)]. Second, the complex was split into two
fractions--"a.gamma..sub..chi..psi." complex and a
".delta..delta.'" complex--as described by O'Donnell [see J. Bio.
Chem 265:1179 (1990)]. The peptide sequences for .delta. and
.delta.' were obtained from the .delta..delta.' fraction, whereas
the peptide sequences of .sub..chi. and .sub..psi. are obtained
from the .gamma..psi..sub..chi. fraction. The .theta. subunit
sequence was obtained from a side fraction off this procedure which
contained nearly pure polymerase III ( .alpha., .epsilon., .theta.)
subunits.
[0041] For all 5 proteins, the amino acid sequences were obtained
in the same manner, by the use of N-terminal analysis and tryptic
analysis. N-terminal analysis was conducted using known techniques
of SDS-PAG electrophoresis [see Nature 227:680(1970)] in a 15% gel,
and subsequent electroelution onto PVDF membrane. The resolved
peptides were removed from the membrane and sequenced. For tryptic
analysis, either .delta..delta.' or .gamma..sub..chi..psi. comples
was chromatographed in a 15% SDS-PAG gel to separate the individual
subunits. However, for this procedure, the 100 pmol was
electroblotted onto nitrocellulose. The nitrocellulose membrane was
then digested with trypsin, and the peptides resolved by microbore
HPLC. The resolved peptides were then sequenced.
[0042] The electroblotting procedure used in the tryptic analysis
protocol is more fully described in the following general
example:
EXAMPLE I
Electroblotting
[0043] SDS (Bio-Rad) was purified by crystallization from
ethanol-water. SDS (100 g) was added to ethanol (450 g), stirred,
and heated to 55.degree. C. Additional hot water was added (50-75
ml) until all of the SDS dissolved. Activated charcoal (10 g) was
added to the solution, and after 10 minutes the slurry was filtered
through a Buchner funnel (Whatman No. 5 paper) to remove all the
charcoal. The filtered solution was chilled, first a 4.degree. C.
for 24 hrs and then at -20.degree. C. for an additional 24 hrs.
Crystalline SDS was collected on a coarse-frit sintered-glass
funnel and washed with 800 ml of ethanol chilled to -20.degree. C.
The partial purified SDS was then recrystallized using the above
procedure but without the charcoal. 0.75 mm SDS-Laemmli gel was
made using ultra-pure reagents. Prior to electrophoresis 10 mM
Glutatnhione (to a final concentration of 0.05 mM) was added to the
upper chamber buffer, and the system preelectrophotesed 2 hr at 3-5
mA (3 mA for mini-gel, 5 mA for normal). After 2 hrs, the upper
chamber was emptied and standard tris-glycine buffer was added. The
samples to be run were denatured using Laemmli denaturaton solution
made from the ultra-pure reagents (in the presence of 5 mM DTT).
The gel was run under conditions such that separation was acheived
in less than 2 hrs. After the gel run, the gel was soaked for 30
min in 10 mM CAPS pH 11, 5% methanol (% of methanol will vary
depending on the size of the protein: in general, high molecular
weight proteins transfer more efficiently in absence of methanol
while low molecular weight proteins require methanol in the
buffer). CAPS buffer was made by titrating a 10 mM solution with 10
N NaOH. For gel transfer, slices of Immobilon were wet in 100%
methanol and equilibrated 10 min in the CAPS transfer buffer, and
the protein transferred using Bio-Rad mini blotter (transfer time
will vary depending on protein size, methanol, etc.; -70 kDa
polypeptide will transfer in 90 min in the presence of 5% methanol
at 15V). After transfer, Immobilon was soaked in distilled water
for 5 min, and the membrane was stained with 0.1% Commassie Blue
R250 in 50% methanol for 1 min, and destained in 50% methanol and
10% aldehyde-free acetic acid. The membranes were soaked in
distilled water for 10 min, and allowed to air dry. Protein bands
of interest were cut from the membrane and stored in eppendorf
tubes at -20.degree. C. until sequenced.
[0044] The identification of the subunit gene of DNA polymerase III
.delta. was accomplished by purifying the .delta..delta.' proteins
to apparent homogeneity through an ATP-agarose column from 1.3 kg
of the .delta./.tau. overproducing strain of E. coli [HB 101 (pNT
203, pSK 100)].
[0045] The .delta..delta.' subunits were separated by
electrophoresis in a 15% SDS-PAG (polyacrylamide gel), then
electroblotted onto PVDF membrane (Whatman) for N-terminal
sequencing (50 pmol each) [see J. Biol. Chem. 262:10035 (1987)] ,
and onto nitrocellulose membrane (Schleicher and Schuell) for
tryptic analysis (140 pmol each) [see PNAs USA 84:6970 (1987)].
Proteins were visualized by Ponceau S (Sigma). Protein sequences
were determined using an Applied Biosystems 470A gas-phase
microsequenator. Sequence results were as follows:
2 N-terminal sequence: NH.sub.2-Met Leu Arg Leu Tyr Pro Glu Gln Leu
Arg Ala Gln Leu Asn 5 10 Glu Gly Leu Arg Ala Ala Tyr Leu Leu Leu
Gly Asn Asp Pro; 15 20 25 tryptic peptide .delta.-1: NH.sub.2-Ala
Ala Tyr Leu Leu Leu Gly Asn Asp Pro Leu Leu Leu Gln 5 10 Glu Ser
Gln A sp Ala Val Arg; 15 20 tryptic peptide .delta.-2: NH.sub.2-Ala
Gln Glu Asn Ala Ala Trp Phe Thr Ala Leu Ala Asn Arg 5 10 tryptic
peptide .delta.-3: NH.sub.2-Val Glu Gln Ala Val Asn Asp Ala Ala His
Phe Thr Pro Phe 5 10 His Trp Val Asp Ala Leu Leu Met (Gly) (Lys).
15 20
[0046] Paranthesis in the above sequence indicate uncertainty in
the amino acid assignment.
[0047] The DNA sequencing, construction of the overproducing
vector, and DNA replication assays for this subunit were conducted
according to the following example:
EXAMPLE II
[0048] DNA Sequencing:
[0049] The 3.2 kb KpnI/Bg1II (restriction enzymes, New England
Biolabs) fragment containing .delta. was excised from .lambda.169
(Kohara) and directionally ligated into pUC18 to yield pUCdelta.
Both strands of DNA were sequenced by the chain termination method
of Sanger using the United States Biochemicals sequenase kit,
[.alpha.-.sup.35S]dCTP (New England Nuclear), and synthetic DNA
17-mers (Oligos etc. inc.). All sequence information presented here
was determined on both strands using both dGTP and dITP in
sequencing reactions.
[0050] Construction of the Overproducing Vector:
[0051] Approximately 1.7 kb of DNA upstream of .delta. was excised
from pUCdelta using KpnI (polylinker site) and BstXI (the BstXI
site is 13 base pairs upstream of the start codon of holA) followed
by self-ligation of the plasmid. A 1.5 kb fragment containing the
holA gene was then excised using EcoRI and XbaI (these sites are in
the pUC polylinker on either side of the .delta. insert) followed
by directional ligation into M13mp18 to yield M13delta. An NdeI
site was generated at the start codon of holA by primer directed
mutagenesis [see Methods Enzymol 154:367 (1987)] using a DNA 33-mer
(5'->3'):
[0052] GTACAACCGA ATCATATGTT ACCCAGCGAG CTC 33
[0053] containing the NdeI site (underlined) at the start codon of
holA to prime replication of M13delta viral ssDNA, and using DNA
polymerase and SSB in place of Klenow polymerase to completely
replicate the circle without strand displacement [see J. Biol.
Chem. 260:12884 (1985)]. The NdeI site was verified by DNA
sequencing. An NdeI fragment (2.1 kb) containing the .delta. gene
was excised from the NdeI mutated M13 delta and ligated into pET-3c
linearized using NdeI to yield pETdelta. The orientation of the
holA gene in pETdelta was determined by sequencing.
[0054] DNA Replication Assays:
[0055] The replication assay contained 72 ng M13mp18 ssDNA (0.03
pmol as circles) uniquely primed with a DNA 30-mer [see J. Biol.
Chem. 266:11328 (1991)], 980 ng SSB (13.6 pmol as tetramer), 22 ng
.beta. (0.29 pmol as dimer), 200 ng .gamma.(2.1 pmol as tetramer),
,55 ng .alpha..epsilon. complex in a final volume (after addition
of proteins) of 25 .mu.l 20 mM Tris-HCL (pH7.5), 8 mM MgCl.sub.2, 5
mM DTT, 4% glycerol, 40 .mu.g/ml BSA, 0.5 mM ATP, 60 .mu.M each
dCTP, dGTP, dATP and 20 .mu.M [.alpha.-.sup.32P]dTTP (New England
Nuclear). Proteins used in the reconstitution assay were purified
to apparent homogeneity and their concentration determined. Delta
protein or column fraction containing .delta., was diluted in
buffer (20 mM Tris-HCL (pH7.5), 2 mM DTT, 0.5 mM EDTA, 20%
glycerol, 60 mM NaCl and 50 .mu.g/ml BSA) such that 1-10 ng of
protein was added to the assay on ice, shifted to 37.degree. C. for
5 minutes, then quenched upon spotting directly onto DE81 filter
paper. DNA synthesis was quantitated as described.
[0056] Gel Filtration:
[0057] Gel filtration of .delta., .delta.' and the .delta..delta.'
complex was performed using an HR 10/30 Superdex 75 column
equilibrated in 20 mM Tris-HCL (pH 7.5), 10% glycerol, 2 mM DTT,
0.5 mM EDTA and 100 mM (buffer B). Either .delta. (30 .mu.g, 0.78
nmol as monomer), .delta.' (30 .mu.g, 0.81 nmol as monomer) or a
mixture of .delta. and .delta.' (30 .mu.g of each) were incubated
for 30 minutes at 15.degree. C. in 100 .mu.l of buffer B then the
entire 100 .mu.l sample was injected onto the column. The column
was developed with buffer B at a flow rate of 0.3 ml/minute and
after the first 6 ml, fractions of 170 .mu.l were collected.
Fractions were analyzed by 13% SDS polyacrylamide gels (100 .mu.l
per lane) stained with Coomassie Brillant Blue. Densitometry of
stained gels was performed using a Pharmacia-LKB Ultrascan XL laser
densitometer.
[0058] Gel filtration of .gamma. or .gamma. mixed with either
.delta. or .delta.' or both .delta. and .delta.' was performed
using an HR 10/30 Superose 12 column equilabrated in buffer B.
Protein mixtures were preincubated 30 minutes at 15.degree. C. in
100 .mu.l buffer B then injected onto the column and the column was
developed and analyzed as described above. Replication activity
assays of these column fractions were performed as described above
with the following modifications. The .gamma. subunit was omitted
from the assay and each fraction was diluted 50-fold with 20 mM
Tris-HCL (pH 7.5), 10% glycerol, 2 mM DTT, 0.5 mM EDTA and 50
.mu.g/ml BSA. Then 2 .mu.l of diluted fraction was withdrawn and
added to the assay.
[0059] Protein standards were a mixture of proteins obtained from
BioRad and from Sigma Chemical Co. and were brought to a
concentration of approximately 50 .mu.g each in 100 .mu.l buffer B
before analysis on either Superdex 75 or Superose 12 columns.
[0060] Glycerol Gradient Sedimentation:
[0061] Sedimentation analysis of .delta., .delta.' and a mixture of
.delta. and .delta.' were performed using 11.6 ml 10%-30% glycerol
gradients in buffer B. Either .delta. (57 .mu.g, 1.5 nmol as
monomer), .delta.' (56 .mu.g, 1.5 nmol as monomer) or a mixture of
.delta. and .delta.' (57 .mu.g and 56 .mu.g, respectively) were
incubated at 15.degree. C. for 30 minutes in a final volume of 100
.mu.l buffer B then each sample was layered onto a separate
gradient. Protein standards (50 .mu.g each in 100 .mu.l buffer B)
were layered onto another gradient and the gradients were
centrifuged at 270,000.times.g for 60 hours at 4.degree. C.
Fractions of 170 .mu.l were collected from the bottom of the tube
and analyzed (100 .mu.l/lane) in a 13% SDS polyacrylamide gel
stained with Coomassie Blue.
[0062] Light Scattering:
[0063] The diffusion coefficient of .delta., .delta.' and the
.delta..delta.' complex was determined by dynamic light scattering
at 780 nm in a fixed angle (90.degree.) Biotage model dp-801 light
scattering instrument (Oros instruments). Samples of .delta. (200
.mu.g, 5.2 nmol as monomer), and .delta.' (200 .mu.g, 5.4 nmol as
monomer) were in 400 .mu.l of 20 mM Tris-HCL (pH 7.5), 100 mM NaCl
and 1.2% glycerol. The mixture of .delta. and .delta.' (100 .mu.g
of each) was in 400 .mu.l of 20 mM Tris-HCl (pH 7.5) and 100 mM
NaCl. The observed diffusion coefficient of .delta.' in the
presence of 1.2% glycerol was 0.6% higher than in the absence of
glycerol. Hence, the 1.2% glycerol in the .delta. and .delta.'
samples had little effect on the observed diffusion
coefficient.
[0064] The purification of .delta. was preformed according to the
following example:
EXAMPLE III
Purification of .delta.
[0065] BL21 (DE3) cells harboring pETdelta were grown at 37.degree.
C. in 12 liters of LB media containing 100 .mu.g/ml of ampicillin.
Upon growth to OD 1.5, the temperature was lowered to 25.degree.
C., and IPTG was added to 0.4 mM. After a further 3 hrs. of growth,
the cells (50 g) were collected by centrifugation. Cells were lysed
using lysozyme as described in prior publications, and the debris
removed by centrifugation. The following purification steps were
performed at 4.degree. C. The assay for .delta. is as described
above.
[0066] The clarified cell lysate (300 ml) was diluted 2-fold with
20 mM Tris-HCl (pH 7.5), 20% glycerol, 0.5 mM EDTA, 2 mM DTT
(buffer A) to a conductivity equal to 112 mM NaCl, and then loaded
(over 3 hrs.) onto a 60 ml Hexylamine Sepharose column equilibrated
with buffer A plus 0,1 M NaCl. The Hexylamine column was washed
with 60 ml buffer A plus 0.1 M NaCl, and then eluted (over 14 hrs)
using a 600 ml linear gradient of 0.1 M NaCl to 0.5 M NaCl in
buffer A. Eighty fractions were collected. Fractions 16-34 (125
mls) were dialyzed against 2 liters of buffer A plus 90 mM NaCl
overnight, and then diluted 2-fold with buffer A to yield a
conductivity equal to 65 mM NaCl just prior to loading (over 45
min) onto a 60 ml column of Heparin Sepharose equilibrated in
buffer A plus 50 mM NaCl. The heparin column was washed with 120 ml
buffer A plus 50 mM NaCl, and then eluted (over 14 hrs) using a 600
ml linear gradient of 0.05 M NaCl to 0.5 M NaCl in buffer A. Eighty
fractions were collected. Fractions 24-34 were pooled and diluted
3-fold (final volume of 250 mls) with buffer A to a conductivity
equal to 85 mM NaCl just prior to loading (over 50 min) onto a 50
ml Hi-Load 26/10 Q Sepharose fast flow FPLC column. The column was
washed with 150 ml buffer A plus 50 mM NaCl, and then eluted using
a 600 ml linear gradient of 0.05 M NaCl to 0.5 mM NaCl in buffer A.
Eighty fractions were collected. Fractions 28-36 which contained
pure .delta. were pooled (74 mls, 1.9 mg/ml); passed over a 1 ml
ATP-agarose column (N-6 linked) to remove any possible
.gamma.complex contaminant, and then dialyzed versus two changes of
2 liters each of buffer A containing 0.1 M NaCl (the DTT was
ommitted for the purpose of determining protein concentration
spectrophotometrically) before storing at -70.degree. C.
[0067] The following table gives the results obtained from
measuring the protein levels obtained from the fractions taken in
Example III.
3 total specific fold protein total activity purifica- % Fractions
(mg) units.sup.1 (units/mg) tion yield I Lysate.sup.2 2070 5.4
.times. 10.sup.9 2.6 .times. 10.sup.6 1.0 100 II Hexylamine 446 2.5
.times. 10.sup.9 5.6 .times. 10.sup.6 2.2 46 III Heparin 197 2.0
.times. 10.sup.9 10.2 .times. 10.sup.6 3.9 37 IV Q Sepharose 141
1.5 .times. 10.sup.9 10.6 .times. 10.sup.6 4.1 28 .sup.1One unit is
defined as pmol nucleotide incorporated per minute .sup.2Ommission
of gamma from the assay of the lysate resulted in a 200-fold
reduction of specific activity (units/mg)
[0068] The .delta. gene was identified using amino acid sequence
information from .delta.. The sequence of the N-terminal 28 amino
acids of .delta. and the sequence of three internal tryptic
peptides were determined. One of the tryptic peptides (tryptic
peptide .delta.-1) overlapped 10 amino acids of the N-terminal
sequence. A search of the GenBank revealed a sequence which
predicted the exact amino acid sequence of the 21 amino acid
tryptic peptide .delta.-1 which overlapped the N-terminal sequence.
The matching sequence occurred just downstream of the rIpB gene at
15.2 minutes of the E. coli chromosome. The match of the published
DNA sequence to the N-terminal sequence of .delta. was imperfect
due to a few errors in the published sequence of this region. The
published sequence of r1pB accounted for approximately 22% of the
.delta. gene and did not encode either of the other two tryptic
fragments. The Kohara lamda phage 169 contains 19 kb of DNA
surrounding the .delta. gene. The 3.2 kb KpnI/Bg1II fragment
containing .delta. was excised from .lambda.169, cloned into pUC18
and the .delta. gene was sequenced. The DNA sequence predicts the
correct N-terminal sequence of .delta. (except the Ile instead of
Leu at position 2) and encodes the other two internal tryptic
peptides of .delta. in the same reading frame, and predicts a 343
amino acid protein of 38.7 kda consistent with the mobility of the
.delta. in SDS-PAGE (35 kDa).
[0069] The full nucleic acid sequence for the .delta. gene
according to the present invention was determined to be:
4 ATG ATT CGG TTG TAC CCG GAA CAA CTC CGC GCG CAG CTC 39 AAT GAA
GGG CTG CGC GCG GCG TAT CTT TTA CTT GGT AAC 78 GAT CCT CTG TTA TTG
CAG GAA AGC CAG GAC GCT GTT CGT 117 CAG GTA GCT GCG GCA CAA GGA TTC
GAA GAA CAC CAC ACT 156 TTT TCC ATT GAT CCC AAC ACT GAC TGG AAT GCG
ATC TTT 195 TCG TTA TGC CAG GCT ATG AGT CTG TTT GCC AGT CGA CAA 234
ACG CTA TTG CTG TTG TTA CCA GAA AAC GGA CCG AAT GCG 273 GCG ATC AAT
GAG CAA CTT CTC ACA CTC ACC GGA CTT CTG 312 CAT GAC GAC CTG CTG TTG
ATC GTC CGC GGT AAT AAA TTA 351 AGC AAA GCG CAA GAA AAT GCC GCC TGG
TTT ACT GCG CTT 390 GCG AAT CGC AGC GTG CAG GTG ACC TGT CAG ACA CCG
GAG 429 CAG CTC AAC TTA GAA CTG GAT GAC GCG GCA AAT CAG GTG 507 CTC
TGC TAC TGT TAT GAA GGT AAC CTG CTG GCG CTG GCT 546 CAG GCA CTG GAG
CGT TTA TCG CTG CTC TGG CCA GAC GGC 585 AAA TTG ACA TTA CCG CGC GTT
GAA CAG GCG GTG AAT GAT 624 GCC GCG CAT TTC ACC CCT TTT CAT TGG GTT
GAT GCT TTG 663 TTG ATG GGA AAA AGT AAG CGC GCA TTG CAT ATT CTT CAG
702 CAA CTG CGT CTG GAA GGC AGC GAA CCG GTT ATT TTG TTG 741 CGC ACA
TTA CAA CGT GAA CTG TTG TTA CTG GTT AAC CTG 780 AAA CGC CAG TCT GCC
CAT ACG CCA CTG CGT GCG TTG TTT 819 GAT AAG CAT CGG GTA TGG CAG AAC
CGC CGG GGC ATG ATG 858 GGC GAG GCG TTA AAT CGC TTA AGT CAG ACG CAG
TTA CGT 897 CAG GCC GTG CAA CTC CTG ACA CGA ACG GAA CTC ACC CTC 936
AAA CAA GAT TAC GGT CAG TCA GTG TGG GCA GAG CTG GAA 975 GGG TTA TCT
CTT CTG TTG TGC CAT AAA CCC CTG GCG GAC 1014 GTA TTT ATC GAC GGT
TGA 1032
[0070] The underlined portions of this sequence refer to, subunits
which are .delta.-1 (55-117), .delta.-2 (358-399), and .delta.-3
(604-672). In addition, the upstream sequence:
5 CCGAACAGCT GATTCGTAAG CTGCCAAGCA TCCGTGCTGC GGATATTCGT 50
TCCGACGAAG AACAGACGTC GACCACAACG GATACTCCGG CAACGCCTGC 100
ACGCGTCTCC ACCACGCTGG GTAACTG 127
[0071] wherein the last underlined TG denotes two-thirds of rIpB
stop codon; in addition, the positive RNA polymerase promoter
signals (TCGCCA and GATATT) and the Shine-Dalgarno sequence (ACGCT)
are underlined.
[0072] In addition, the downstream nucleic acid sequence for holA
begins with a stop codon:
6 TGA ATGAAATCT TTACAGGCTC TGTTTGGCGG CACCTTTGAT CCGGTGCACT 53
ATGGTCATCT AAAACCCGTT GGAAGCGTGG CCGAAGTTTT GATTGGTCTG AC 105
[0073] The holA gene translates into the amino acid sequence:
7 Met Ile Arg Leu Tyr Pro Glu Gln Leu Arg Ala Gln Leu Asn Glu 5 10
15 Gly Leu Arg Ala Ala Tyr Leu Leu Leu Gly Asn Asp Pro Leu Leu 20
25 30 Leu Gln Glu Ser Gln Asp Ala Val Arg Gln Val Ala Ala Ala Gln
35 40 45 Gly Phe Glu Glu His His Thr Phe Ser Ile Asp Pro Asn Thr
Asp 50 55 60 Trp Asn Ala Ile Phe Ser Leu Cys Gln Ala Met Ser Leu
Phe Ala 65 70 75 Ser Arg Aln Thr Leu Leu Leu Leu Leu Pro Glu Asn
Gly Pro Asn 80 85 90 Ala Ala Ile Asn Glu Gln Leu Leu Thr Leu Thr
Gly Leu Leu His 95 100 105 Asp Asp Leu Leu Leu Ile Val Arg Gly Asn
Lys Leu Ser Lys Ala 110 115 120 Gln Glu Asn Ala Ala Trp Phe Thr Ala
Leu Ala Asn Arg Ser Val 125 130 135 Gln Val Thr Cys Gln Thr Pro Glu
Gln Ala Gln Leu Pro Arg Trp 140 145 150 Val Ala Ala Arg Ala Lys Gln
Leu Asn Leu Glu Leu Asp Asp Ala 155 160 165 Ala Asn Gln Val Leu Cys
Tyr Cys Tyr Glu Gly Asn Leu Leu Asn 170 175 180 Leu Ala Gln Ala Leu
Glu Arg Leu Ser Leu Leu Trp Pro Asp Gly 185 190 195 Lys Leu Thr Leu
Pro Arg Val Glu Gln Ala Val Asn Asp Ala Ala 200 205 210 His Phe Thr
Pro Phe His Trp Val Asp Ala Leu Leu Met Gly Lys 215 220 225 Ser Lys
Arg Ala Leu His Ile Leu Gln Gln Leu Arg Leu Gly Gly 230 235 240 Ser
Glu Pro Val Ile Leu Leu Arg Thr Leu Gln Arg Glu Leu Leu 245 250 255
Leu Leu Val Asn Leu Lys Arg Aln Ser Ala His Thr Pro Leu Arg 260 265
270 Ala Leu Phe Asp Lys His Arg Val Trp Gln Asn Arg Arg Gly Met 275
280 285 Met Gly Glu Ala Leu Asn Arg Leu Ser Gln Thr Gln Leu Arg Gln
290 295 300 Ala Val Gln Leu Leu Thr Arg Thr Glu Leu Thr Leu Lys Gln
Asp 305 310 315 Tyr Gly Gln Ser Val Trp Ala Glu Leu Glu Gly Leu Ser
Leu Leu 320 325 330 Leu Cys His Lys Pro Leu Ala Asp Val Phe Ile Asp
Gly 335 340 343
[0074] The holA gene is located in an area of the chromosome
containing several membrane protein genes. They are all transcribed
in the same direction. The mrdA and mrdB genes encode proteins
responsible for the rod shape of E. coli and the r1pA and r1pB
genes encode rare lipoproteins which are speculated to be important
to cell duplication. The position of the .delta. gene within a
cluster of membrane proteins may be coincidental or may be related
to the putative attachment of the replisome to the membrane.
[0075] As noted, the termination codon of the r1pB protein overlaps
one nucleotide with the initiating ATG of holA leaving a gap of
only 2 nucleotides between these genes. holA may be an operon with
r1pB or there may be a promoter within r1pB. The nearest possible
initiation signals for transcription (the putative RNA polymerase
signals) and translation (Shine-Dalgarno) are underlined in the
sequence given above; the match to their respective consensus
sequences is not strong suggesting a low utilization efficiency.
Inefficient transcription and/or translation may be expected for a
gene encoding a subunit of a holoenzyme present at only 10-20
copies/cell. The .delta. gene uses several rare codons [CCC(Pro),
ACA(Thr), GGA(Gly), AGT(Ser), AAT(Asn), TTA, TTG, CTC(all Leu)] 2-5
times more frequently than average which may decrease translation
efficiency. ATP binding to .delta. within the holenzyme has been
detected previously by UV crosslinking. The DNA sequence of the
.delta. shows a near match to the ATP binding site consensus motif
(i.e. AX.sub.3GKS for .delta. at residues 219-225 compared to the
published consensus G/AX.sub.4GKS/T, G/AXGKS/T or G/AX.sub.2GXGKS/T
[see Nuc. Acids Res 17:8413 (1989)]. Whether .delta. binds with ATP
specifically at this site remains to be determined. Of the 33
arginine and lysine residues in .delta., 16 of them (50%) are
within amino acids 225-307. This same region contains only 5 (14%)
of the 35 glutamic and aspartic acid residues. Whether this
concentration of basic residues is significant to function is
unknown. There are no strong matches to consensus sequences to
motifs encoding: zinc fingers or helix-loop-helix DNA binding
domains.
[0076] The holA gene was cloned into M13mp18 and an NdeI site was
created at the initiating methionine by the site directed
mutagenesis technique in order to study the overproduction of this
gene. The .delta. gene was then excised from M13delta and inserted
into the NdeI site of the pET-3c expression vector [see Methods
Enzymol 185:60 (1990)] which places .delta. under control of a
strong T7 RNA polymerase promotor ,see FIG. 3-1. Upon
transformation into BL21(DE3) cells and induction of T7 RNA
polymerase with IPTG, the .delta. protein was expressed to 27%
total cell protein. For reasons unknown, .delta. was not produced
in BL21(DE3) cells containing the pLysS plasmid. Induction at
25.degree. C. yielded approximately 2-fold more .delta. and
increased the solubility of the overproduced .delta. relative to
induction at 37.degree. C. Twelve liters of induced cells were
lysed using lysozyme and 141 mg of pure .delta. was obtained in 28%
overall yield upon column fractionation using Hexylamine Sepharose,
Heparin Agarose, and Q Sepharose. Delta protein tended to
precipitate upon standing in low salt (<70 mM), especially
during dialysis. Therefore, low salt was avoided except for short
periods of time and column fractions containing .delta. were
sometimes diluted in preparation for the next column rather than
dialyzed overnight. The .delta. subunit was assayed by its ability
to reconstitute efficient replication of a singly primed M13mp18
ssDNA "coated" with SSB in the presence of .alpha., .epsilon.,
.beta., and .gamma. subunits. Cell lysate prepared from induced
cells containing pETdelta were more active in the replication assay
than cell lysate prepared from induced cells containing the pET-3c
vector.
[0077] The expressed .delta. protein comigrated with the authentic
.delta. subunit contained within the .gamma. complex of the
holoenzyme. The N-terminal sequence analysis of the pure cloned
.delta. was identical to that predicted from the holA sequence
according to the present invention provided that the protein
encoded by the gene had been purified. Furthermore, the
overproduced .delta. subunit was active with only the .alpha.,
.epsilon., .gamma. and .beta. subunits of the holenzyme (FIG. 5-1).
in the presence of a sixth subunit, .delta.', activity was
enhanced. The amount of the cloned .delta. required to reconstitute
the efficient DNA synthesis characteristic of the holoenzyme Using
the 5 or 6 subunit combination according to the present invention
is in the range shown previously for the naturally purified .delta.
resolved from the .gamma. complex. As shown below, addition of more
.gamma. to the replication assay brings the amount of .delta. down
even further to about 1 ng for a stoichiometry of about 1-2 .delta.
monomers per DNA circle replicated.
[0078] Electrospray mass spectrometry of the cloned .delta. protein
yielded a molecular mass of 38,704 da. This mass is within 0.0015
of the mass predicted from the gene; well within the 0.01% error of
the mass spectrometry technique. This is evidence that the DNA
sequence above according to the present invention contains no
errors and indicates the overproduced .delta. is not modified
during or after translation. The .epsilon..sub.280 calculated from
the amino acid composition of .delta. is 46,230 M.sup.-1cm.sup.-1.
The measured absorbance of .delta. in 6M guanidine hydrochloride is
only 0.2% higher than in buffer A. Hence, the .epsilon..sub.280 of
native .delta. is 46,137 M.sup.-1 cm.sup.-1.
[0079] Further understanding of the individual subunits the present
invention also determines whether .delta. and .delta.' are
monomeric, dimeric or higher order structures. The .delta. and
.delta.' subunits were also each analyzed in a gel filtration
column, and they migrated in essentially the same position as one
another (fractions 30-32). As discussed below the .delta.' appears
as two proteins, .delta.'.sub.L and .delta.'.sub.S, which differ by
approximately 0.5 kda. Comparison with protein standards of known
Stokes radius yielded a Stokes radius of 26.5 .ANG. for .delta. and
25.8 .ANG. for .delta.', slightly smaller than the 27.5 .ANG.
radius of the 43.5 kDa ovalbumin standard indicating both .delta.
and .delta.' are both monomeric (their gene sequences predict:
.delta., 38.7 kDa .delta.', 36.9 kDa). in a glycerol gradient
sedimentation analysis both .delta. and .delta.' migrated in the
same position as one another with an S value of 3.0 relative to
protein standards, a slightly lower sedimentation value than the
43.5 kda ovalbumin standard, again indicating a monomeric state for
the .delta. and .delta.'. Besides protein mass, the protein shape
is also a determinant of both the Stokes radius and the S value
obtained by these techniques. The shape however, causes opposite
behavior in these two techniques, a protein with an asymmetric
shape behaves in gel filtration as a larger protein than if it were
spherical (elutes early) and behaves in sedimentation like a
smaller protein than if it were spherical (sediments slower). The
Stokes radius and S value can be combined in the equation of Siegel
and Monty whereupon the protein shape factor cancels. Therefore,
the native mass of the protein obtained from such treatment is more
accurate than calculating the mass from only the S value or the
Stokes radius and assuming a spherical shape. This calculation
yielded a native mass of 34.7 kDa for .delta. and 33.8 kDa for
.delta.'; values similar to the monomer molecular mass predicted
from the gene sequences of .delta. and .delta.', further evidence
they are monomers. Their frictional coefficients are each
significantly greater than 1.0 indicating they are not spherical
but have some asymmetry to their shape. One can also conclude from
this work that the two .delta.' subunits are a mixture of
.delta.'.sub.L and .delta.'.sub.S rather than a complex of
.delta.'.sub.L and .delta.'.sub.S.
[0080] In initial studies using the cloned .delta., .delta. forms
only a weak complex with .gamma. but, together with .delta.' a
stable .gamma..delta..delta.' complex can be reconstituted which
remains intact in gel filtration and ion exchange chromatography.
Likewise, .delta.' forms only a weak complex with .gamma., and
requires the .delta. subunit to bind .gamma. tightly. Both .delta.
and .delta.' appear monomeric and bind to each other to form a
.delta..delta.' heterodimer.
[0081] Availability of the .delta. subunit in large quantity will
allow detailed studies of the mechanism of the .gamma. complex in
.beta. clamp formation. Further, identification of the .delta. gene
will provide for genetic analysis (essentiality) of .delta. in E.
coli replication and possibly other roles of .delta. in DNA
metabolism.
[0082] The second subunit according to the present invention, that
of .delta.', was also identified from the .delta..delta.' fraction
in like manner. The N-terminal sequence, comprising the first 18
amino acids in the peptide, and the tryptic peptide sequence were
obtained. The amino acid sequence determined from the initial
sequence studies for the .delta.' peptide is:
8 Met Arg Trp Tyr Pro Trp Leu Arg Pro Asp Phe Glu Lys Leu Val 5 10
15 Ala Ser Tyr Gln Ala Gly Arg Gly His His Ala Leu Leu Ile Gln 20
25 30 Ala Leu Pro Gly Met Gly Asp Asp Ala Leu Ile Tyr Ala Leu Ser
35 40 45 Arg Tyr Leu Leu Cys Gln Gln Pro Gln Gly His Lys Ser Cys
Gly 50 55 60 His Cys Arg Gly Cys Gln Leu Met Gln Ala Gly Thr His
Pro Asp 65 70 75 Tyr Tyr Thr Leu Ala Pro Glu Lys Gly Lys Asn Thr
Leu Gly Val 80 85 90 Asp Ala Val Arg Glu Val Thr Glu Lys Leu Asn
Glu His Ala Arg 95 100 105 Leu Gly Gly Ala Lys Val Val Trp Val Thr
Asp Ala Ala Leu Leu 110 115 120 Thr Asp Ala Ala Ala Asn Ala Leu Leu
Lys Thr Leu Glu Glu Pro 125 130 135 Pro Ala Glu Thr Trp Phe Phe Leu
Ala Thr Arg Glu Pro Glu Arg 140 145 150 Leu Leu Ala Thr Leu Arg Ser
ARg Cys Arg Leu His Tyr Leu Ala 155 160 165 Pro Pro Pro Glu Gln Tyr
Ala Val Thr Trp Leu Ser Arg Glu Val 170 175 180 Thr Met Ser Gln Asp
Ala Leu Leu Ala Ala Leu Arg Leu Ser Ala 185 190 195 Gly Ser Pro Gly
Ala Ala Leu Ala Leu Phe Gln Gly Asp Asn Trp 200 205 210 Gln Ala Arg
Glu Thr Leu Cys Gln Ala Leu Ala Tyr Ser Val Pro 215 220 225 Ser Gly
Asp Trp Tyr Ser Leu Leu Ala Ala Leu Asn His Glu Gln 230 235 240 Ala
Pro Ala Arg Leu His Trp Leu Ala Thr Leu Leu Met Asp Ala 245 250 255
Leu Lys Arg His His Gly Ala Ala Gln Val Thr Asn Val Asp Val 260 265
270 Pro Gly Leu Val Ala Glu Leu Ala Asn His Leu Ser Pro Ser Arg 275
280 285 Leu Gln Ala Ile Leu Gly Asp Val Cys His Ile Arg Glu Gln Leu
290 295 300 Met Ser Val Thr Gly Ile Asn Arg Glu Leu Leu Ile Thr Asp
Leu 305 310 315 Leu Leu Arg Ile Glu His Tyr Leu Gln Pro Gly Val Val
Leu Pro 320 325 330 Val Pro His Leu 334
[0083] From these sequences, two DNA oligonucleotide probes were
made and used (after end-labelling with .sup.32P for use in
Southern blot analysis) to probe a Southern blot of E. coli DNA
which was grown, isolated and restricted as above. The sequences of
the two probes were:
9 probe 1: ACT CTG GAA GAA CCG CCG GCT GAA ACT TGG TTT TTT CTG GCT
42 ACT CGT GAA CCG GAA 57; and
[0084]
10 probe 2: GCT GGT TCT CCG GGT GCT GCT CTG GCT CTG TTT CAG GGT GAT
42 GAC TGG CAG GCT 54.
[0085] Of the two Southern blots analyzed (one with the 57-mer
probe and the other with the 54-mer probe), the patterns from the
blots had one set of bands in common, and these were sized by
comparison with size standards in the same gel following recognized
techniques. The size of these 8 common "bands" or DNA fragments
produced by digestion with 8 restriction enzymes were used to scan,
by eye, the restriction map of the E. coli genome [see Cell 50:495
(1987)]. One unique location on the genome was located which was
compatable with all 8 restriction fragment sizes.
[0086] Phage .lambda..sub.236 was selected as a phage containing
the "unique location" in the E. coli genome. The .delta.' gene was
excised from the .lambda..sub.236 phage using restriction enzymes
EcoRV and KpnI to yield a 2.3 kb fragment of DNA. This fragment was
then ligated into pUC18 and sequenced using a sequenase kit (US
Biochemicals) in accordance with the manufacturer's instructions.
The fragment was also ligated into a M13mp18 vector for making a
site specific mutation, as described above, at the ATG start codon
(i.e., changing the CGCATG to CATATG; thereby allowing NdeI to
cleave the nucleotide at CATATG, whereas it could not cleave the
nucleotide using the normal CGCATG sequence).
[0087] The nucleic acid sequence obtained from these studies
predicted the amino acid sequence determined for .delta.' peptide
in frame, and thus the selected sequence was that for the .delta.'
gene. The nucleic acid sequence, according to the present
invention, for this second subunit, .delta.', is:
11 ATG AGA TGG TAT CCA TGG TTA CGA CCT GAT TTC GAA AAA 39 CTG GTA
GCC AGC TAT CAG GCC GGA AGA GGT CAC CAT GCG 78 CTA CTC ATT CAG GCG
TTA CCG GGC ATG GGC GAT GAT GCT 117 TTA ATC TAC GCC CTG AGC CGT TAT
TTA CTC TGC CAA CAA 156 CCG CAG GGC CAC AAA AGT TGC GGT CAC TGT CGT
GGA TGT 195 CAG TTG ATG CAG GCT GGC ACG CAT CCC GAT TAC TAC ACC 234
CTG GCT CCC GAA AAA GGA AAA AAT ACG CTG GGC GTT GAT 273 GCG GTA CGT
GAG GTC ACC GAA AAG CTG AAT GAG CAC GCA 312 CGC TTA GGT GGT GCG AAA
GTC GTT TGG GTA ACC GAT GCT 351 GCC TTA CTA ACC GAC GCC GCG GCT AAC
GCA TTG CTG AAA 390 ACG CTT GAA GAG CCA CCA GCA GAA ACT TGG TTT TTC
CTG 429 GCT ACC CGC GAG CCT GAA CGT TTA CTG GCA ACA TTA CGT 468 AGT
CGT TGT CGG TTA CAT TAC CTT GCG CCG CCG CCG GAA 507 CAG TAC GCC GTG
ACC TGG CTT TCA CGC GAA GTG ACA ATG 546 TCA CAG GAT GCA TTA CTT GCC
GCA TTG CGC TTA AGC GCC 585 GGT TCG CCT GGC GCG GCA CTG GCG TTG TTT
CAG GGA GAT 624 AAC TGG CAG GCT CGT GAA ACA TTG TGT CAG GCG TTG GCA
663 TAT AGC GTG CCA TCG GGC GAT TGG TAT TCG CTG CTA GCG 702 GCC CTT
AAT CAT GAA CAA GTC CCG GCG CGT TTA CAC TGG 741 CTG GCA ACG TTG CTG
ATG GAT GCG CTA AAA CGC CAT CAT 780 GGT GCT GCG CAG GTG ACC AAT GTT
GAT GTG CCG GGC CTG 819 GTC GCC GAA CTG GCA AAC CAT CTT TCT CCC TCG
CGC CTG 858 CAG GCT ATA CTG GGG GAT GTT TGC CAC ATT CGT GAA CAG 897
TTA ATG TCT GTT ACA GGC ATC AAC CGC GAG CTT CTC ATC 936 ACC GAT CTT
TTA CTG CGT ATT GAG CAT TAC CTG CAA CCG 975 GGC GTT GTG CTA CCG GTT
CCT CAT CTT 1002
[0088] The underlined portions of this sequence refer to subunits
which are .delta.'-1 (283-315), .delta.'-2 (316-327), .delta.'-3
(328-390), .delta.'-4 (391-462), .delta.'-5 (481-534), and
.delta.'-6 (577-639). In addition, the upstream sequence:
12 AAGAATCTTT CGATTTCTTT AATCGCACCC GCGCCCGCTA TCTGGAACTG 50
GCAGCACAAG ATAAAAGCAT TCATACCATT GATGCCACCC AGCCGCTGGA 100
GGCCGTGATG GATGCAATCC GCACTACCGT GACCCACTGG GTGAAGGAGT 150 TGGACGC
157
[0089] contains an underlined putative translational signal:
Shine-Dalgarno.
[0090] In addition, the downstream nucleic acid sequence for
.delta.' begins with a stop codon:
13 TTA GAGAGACATC ATGTTTTTAG TGGACTCACA CTGCCATCTC 43 GATGGTCTGG
ATTATGAATC TTTGCATAAG GACGTGGATG ACGTTCTGGC 93 GAAAGCCGCC
GCACGCGATG TGAAATTTTG TCTGGCAGTC GCCACAACAT 143
[0091] The .delta.' gene (holB) was then subcloned into M13mp18,
and a NdeI site was created at the initiating codon as described
above. The .delta.' gene was then excised from M13 using NdeI
restriction enzyme and a second enzyme which cut downstream of
.delta.', and the excised gene was subcloned into the pET-3c
overexpression plasmid using the same techniques described above.
Following overexpression of the .delta.' protein, the protein was
purified using a Fast flow Q--Heparin--Hexylamine techique as
described herein. Ninety mg of .delta.' protein was obtained from 4
liters of cells.
[0092] Further studies on the .delta.' gene were conducted to make
certain that the gene sequence obtained from these research was
actually the .delta. genL and not some artifact. These studies
showed that the gene sequence according to the present invention
predicted all the peptide sequence information, that the cloned
.delta.' gene comigrates with the naturally occuring gene on a 13%
SDS-PAG gel, that the cloned .delta.' gene stimulates the 5 protein
system as does the naturally occuring .delta.', and that .delta.'
forms a .delta.'.delta. complex with .delta. in a similar manner to
that which occurs with the naturally occurring .delta.' and
.delta..
[0093] With specific regard to the isolation and characterization
of .delta.' and holB according to the present invention, the amino
acid sequencing was conducted using .delta. and .delta.' subunits
purified to apparent homogenicity through the ATP-agarose column
step [see J. Biol. Chem. 265:1179 (1990)] from 1.3 kg of the
.gamma./.tau. overproducing strain of E. coli: HB101(pNT203,
pSK100), [see J. Biol. Chem. 263:6555 (i988)]. The .delta. and
.delta.' subunits were separated on a 13% SDS polyacrylamide gel
whereupon the .delta.' resolved into two bands. The slower and
faster migrating .delta.' bands are referred to as .delta.'.sub.L
(large) and .delta.'.sub.S (small), respectively; .delta.'.sub.S
was approximately 2 times the abundance of .delta.'.sub.L. Both
.delta.'.sub.L and .delta.'.sub.S were electroblotted onto PVDF
membrane (Whatman) [see J. Biol. Chem. 262:10035 (1987)] for
N-terminal sequencing (50 pmol each of .delta.'.sub.L and
.delta.'.sub.S), and onto nitrocellulose membrane (Schleicher and
Schuell) [see Proc. Natl. Acad. Sci. USA 84:6970 (1987)] for
tryptic analysis (90 pmol of .delta.'.sub.L and 180 pmol of
.delta.'.sub.S). Proteins were visualized by Ponceau S stain
(Sigma).
[0094] Analysis of the more abundant .delta.'.sub.S was as follows:
the N-terminal sequence was: 1
[0095] and the tryptic peptides were:
14 .delta.'-1 NH.sub.2-Glu Val Thr Glu Lys Leu Asn Glu His Ala Arg;
5 10 .delta.'-3: NH.sub.2-Val Val Trp Val Thr Asp Ala Ala Leu Leu
Thr Asp 5 10 Ala Ala Ala Asn Ala Leu Leu Lys 15 20; .delta.'-4:
NH.sub.2-Thr Leu Glu Glu Pro Pro Ala Glu Thr Trp Phe Phe Leu Ala 5
10 Thr Arg Glu Pro (Glu) (Arg) Leu Leu Ala Thr (Leu); 15 20
.delta.'-5: NH.sub.2-Leu His Tyr Leu Ala Pro Pro (Pro) Glu Gln Tyr
Ala Val 5 10 Thr (Thr) Leu Ser Arg; and 15 .delta.'-6: NH.sub.2-Leu
Ser Ala Gly Ser Pro Gly Ala Ala Leu Ala Leu Phe Gln 5 10 Gly Asp
Asn Trp Gln Ala Arg. 15 20 Sequence analysis of tryptic peptides of
the less abundant .delta.'.sub.L were: .delta.'-2: NH.sub.2-Leu Gly
Gly Ala Lys; and 5 .delta.'-7 (same as .delta.'-3): NH.sub.2-Val
Val Trp Val Thr Asp Ala Ala Leu Leu Thr Asp 5 10 Ala Ala Ala Asn
Ala Leu Leu Lys 15 20;
[0096] Parenthesis in the above sequences indicate uncertain
assignments.
[0097] Two synthetic oligonucleotide probes (DNA oligonucleotides,
Oligos etc. Inc.) were designed from the sequence of two of the
tryptic peptides and the codon usage of E. coli with allowance for
a T-G mispair at the wobble position. A synthetic DNA 57-mer probe
was based on the sequence of .delta.'-4 (amino acids 131-149):
15 Ala Cys Thr Cys Thr Gly Gly Ala Ala Gly Ala Ala Cys Cys Gly 5 10
15 Cys Cys Gly Gly Cys Thr Thr Gly Ala Ala Ala Cys Thr Thr Gly 10
25 30 Gly Thr Thr Thr Thr Thr Thr Cys Thr Gly Gly Cys Thr Ala Cys
35 40 45 Thr Cys Gly Thr Gly Ala Ala Cys Cys Gly Gly Ala Ala 50
55
[0098] (after identification and sequencing of holB this probe was
incorrect at 11 positions). A DNA 54-mer probe was based on the
sequence of .delta.'-6 (amino acids 195-212):
16 Gly Cys Thr Gly Gly Thr Thr Cys Thr Cys Cys Gly Gly Gly Thr 5 10
15 Gly Cys Thr Gly Cys Thr Cys Thr Gly Gly Cys Thr Cys Thr Gly 20
25 30 Thr Thr Thr Cys Ala Gly Gly Gly Thr Gly Ala Thr Ala Ala Cys
35 40 45 Thr Gly Gly Cys Ala Gly Gly Cys Thr 50
[0099] (after identification and sequencing of holB the probe was
incorrect at 9 positions. These probes (100 pmol each) were 5'
end-labelled with 1 .mu.M [.gamma.-.sup.32P]ATP (radionucleotides,
Dupont-New England Nuclear) and polynucleotide kinase. E. coli
genomic DNA (strain C600) was extracted [see J. Mol. Bio. 3:208
(1961)] and restricted with either BamHI, HindIII, EcoRI, EcoRV,
BglI, KpnI, PstI or PvuII (DNA modification enzymes, New England
Biolabs) and then each digest was electrophoresed in a 0.8% native
agarose gel followed by depurination (0.25 M HCl), denaturation
(0.5 M NaCl) and then neutralized (1 M Tris, 2 M NaCl, pH %0.0)
prior to transfer to Gene Screen Plus (DuPont-New England Nuclear)
for Southern analysis using a Vacugene appartus (Pharmacia) in the
presence of 2.times.SSC (0.3 M NaCl. 0.3M sodium acetate, pH 7.0).
Conditions for hybridization and washing using these
oligonucleotide probes were determined empirically and the desired
results were obtained using a hybridization temperature of
42.degree. C. then washing with 2.times.SSC and 0.2% SDS at
successively higher temperature until evaluation by autoradiography
showed a single band in each lane for the 57-mer, and two bands in
each lane for the 54-mer (this occurred at 53.degree. C. for both
probes). Although the 54-mer showed two bands in each lane, one
band always matched the position of the band probed with the
57-mer.
[0100] The 2.1 kb KpnI/EcoRV fragment containing holB was excised
from .lambda. E9G1(236) [see Cell 50:495(1987)] and directionally
ligated into PUC18 (KpnI/HincII) to yield pUC-.delta.'. Both
strands of DNA were sequenced by the chain termination method of
Sanger using the United States Biochemicals sequenase kit,
[.alpha.-.sup.35S]dATP, and synthetic DNA 18-mers.
[0101] A 2.1 kb KpnI/HindIII fragment containing the holB gene was
excised from pUC-.delta.' and directionally ligated into M13mp18 to
yield M13-.delta.'. An NdeI site was generated at the start codon
of holB by oligonucleotide site directed mutagenesis [see Methods
Enzymol 154:367 (1987)] using a DNA 33-mer:
17 Gly Gly Thr Gly Ala Ala Gly Gly Ala Gly Thr Thr Gly Gly Ala 5 10
15 Cys Ala Thr Ala Thr Gly Ala Gly Ala Thr Gly Gly Thr Ala Thr 20
25 30 Cys Cys Ala
[0102] containing the NdeI site (underlined) at the start codon of
holB to prime replication of M13-.delta.' viral ssDNA and using SSB
and DNA polymerase III holoenzyme (in place of DNA polymerase I) to
replicate the circular template without strand displacement. The
M13 chimera is called M13-.delta.'-NdeI. And NdeI fragment (1160
bp) containing the holB gene was excised from M13-.delta.'-NdeI and
ligated into pET3c, linearized using NdeI, to yield pET-.delta.'.
The orientation of the holB gene in pET-.delta.' was determined by
sequencing.
[0103] Reconstitution assays contained 108 ng M13mp18 ssDNA (0.05
pmol as circles) uniquely primed with a DNA 30-mer [see J. Biol.
Chem 266:11328 (1991)], 1.5 .mu.g SSB (21 pmol as tetramer), 30ng
.beta. (0.39 pmol as dimer), 22.5 ng .alpha..epsilon. complex (0.14
pmol), 20 ng .gamma. (0.12 pmol as dimer), 2 ng .delta. (0.5 pmol
as monomer) and the indicated amount of .delta.' (or 1-5 ng of
column fraction during purification) in 20 mM Tris-HCl (pH 7.5), 8
mM MgCl.sub.2, 5 mM DTT, 4% glycerol, 40 .mu.g/ml BSA, 0.5 mM ATP,
60 .mu.M dGTP, and 0.1 mM EDTA in a final volume of 25 .mu.l (after
the addition of the remaining proteins). Assays of .gamma. or .tau.
activity with either .delta., .delta.' or .delta..delta.',
contained either 2 ng .delta. (0.05 pmol as monomer), 2 ng .delta.'
(0.05 pmol as monomer), or 1 ng (0.025 pmol) each of .delta. and
.delta.', and the indicated amount of .gamma. or .tau.. All
proteins were added to the assay on ice and then shifted to
37.degree. C. for 8 minutes to allow reconstitution of the
pocessive polymerase on the primed ssDNA. DNA synthesis was
initiated upon rapid addition of 60 .mu.M dATP and 20 .mu.M
[.alpha..sup.32P]TTP, then quenced after 20 seconds and quantitated
using DE81 paper. When needed, proteins were diluted in 20 mM
Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM EDTA, 20% glycerol, and 50
.mu.g/ml BSA. Proteins used in the reconstitution assays were
purified [see J. Biol. Chem 266:9833 (1991). The concentration of
.beta. and .delta. were determined by absorbance using an
.epsilon..sub.280 value if 17,900M.sup.-1cm.sup.-1, and
46,137M.sup.-1cm.sup.-1, respectively. Concentrations of .alpha.,
.epsilon., .gamma., .tau. and SSB were then determined [see Anal.
Biochem 72:248 (1976)] using BSA as a standard. The concentration
of .delta.' was determined by absorbance using an .epsilon..sub.280
value of 60,136 M.sup.-1cm.sup.-1.
[0104] ATPase assays were performed in a final volume of 20 .mu.l
containing 20 mM Tris-HCl (pH 7.5), 8 mM MgCl.sub.2 and contained
285 ng M13mp18 ssDNA. ATPase assays of .gamma., .delta.,
.delta.'.delta..delta.'- , .gamma..delta. and .gamma..delta.' with
and without .beta. contained 100 .mu.M [.gamma.-.sup.32P] ATP and
when present 376 ng .gamma. (4 pmol as dimer), 304 ng .delta. (7
pmol as monomer). 296 ng .delta.' (8.0 pmol as monomer), and 320 ng
.beta. (4.2 pmol as dimer). Proteins were added on ice, shifted to
37.degree. C. for 30 minutes, then 0.5 ml was spotted on a plastic
backed thin layer of chromatography (TLC) sheet coated with Cel-300
polyethyleneimine (Brinkman instruments Co.). To assay the more
active ATPase activity of .gamma..delta..delta.' and .tau., 300
.mu.M ATP was used, less total protein and less time at 37.degree.
C. inorder to assess the initial rate of reaction. Therefore,
ATPase assays of .gamma..delta..delta., .tau., .tau..delta.,
.tau..delta.' and .tau..delta..delta.' with and without .beta.
contained 300 mM [.gamma.-.sup.32P] ATP and when present, 47 ng
.gamma. (0.5 pmol as dimer), 71 ng .tau. (0.5pmol as dimer), 38 ng
.delta. (1 pmol as monomer), 37 ng .delta.' (1 pmol as monomer) and
40 ng .beta. (0.5 pmol as dimer. Proteins were added on ice,
shifted to 37.degree. C. for 10 minutes, then analyzed by TLC as
described above.
[0105] TLC sheets were developed in 0.5 M lithium chloride, 1 M
formic acid. An autoradiogram of the TLC chromatogram was used to
visualize the free phosphate at the solvent front and ATP at the
origin which were then cut from the TLC sheet and quantitated by
liquid scintillation. The amount of ATP hydrolyzed was calculated
as the percent of total radioactivity located at the solvent front
(P.sub.i) times the total moles of ATP added to the reaction.
[0106] The results of the .delta.' studies appear below:
[0107] The naturally purified .delta.' (resolved from the .gamma.
complex) appears in a 13% SDS polyacrylamide gel as two bands of
approximately 37 kDa that differ in size by about1 kDa. The larger
protein (.delta.'.sub.L) is approximately one half the abundance of
the smaller one (.delta.'.sub.S). Both .delta.'.sub.L and
.delta.'.sub.S are believed encoded by the same gene as there was
no noticeable difference in their HPLC profiles upon digestion with
trypsin. In support of this, peptides from .delta.'.sub.S and
.delta.'.sub.L that had the same retention time on HLPC analysis
also had identical amino acid sequences (peptide .delta.'-7 from
.delta.'.sub.S and .delta.'-3 from .delta.'.sub.L were identical).
The N-terminus of .delta.'.sub.S and five tryptic peptides of
.delta.'.sub.S and two tryptic peptides of .delta.'.sub.L were
sequenced.
[0108] A search of the GenBank revealed no match to the N-terminal
sequence or to any of the tryptic peptides from either
.delta.'.sub.L or .delta.'.sub.S. Two best-guess oligonucleotide
probes (a 57-mer and a 54-mer) were designed from tryptic peptides
.delta.'-4 and .delta.'-6 based on the codon usage frequency in E.
coli [see PNASUSA 80:687 (1983)]. The oligonucleotide probes were
used in a Southern analysis of E. coli genomic DNA digested with
each of the eight Kohara restriction map enzymes. Imposing the
restraint that the eight restriction fragments from the Southern
analysis must overlap the holB gene, the Kohara map of the E. coli
chromosome was searched and only one position of overlap at 24.3
minutes (1,174 kb on the E. coli chromosome starting from thrA) was
found which satisfied the fragment sizes. The fragment sizes in the
Kohara map and from the Southern analysis are given in the
following table which depicts the correspondence of the observed
size of genomic DNA restriction fragments with the Kohara
restriction map of the E. coli chromosome in the region of 24
minutes. E. coli genomic DNA was digested with the restriction
enzymes indicated. The size of the restriction fragments that were
in common for both the 57-mer and 54-mer probes in the, Southern
analysis and also the corresponding sizes of the restriction
fragments on the Kohara restriction map of the E. coli chromosome
at 24.5 minutes are listed below.
18 Restriction Size of restriction fragment (kb) enzyme Southern
Kohara map PstI 1.7 1.9 BglI 4.25 4.2 KpnI 6.6 6.4 EcoRV 7.0 6.8
PvulI 6.2 6.2 EcoRI >15 16.2 HindIII >20 30 BamHI >25
38
[0109] The Kohara .lambda. phage E9G1(236) contains 16.2 kb of DNA
surrounding the putative holB gene. A 2.1 KpnI/EcoRV fragment
containing holB was excised from .lambda. E9G1(236), cloned into
pUC18 and sequenced. The sequence of the KpnI/EcoRV fragment
revealed an open reading frame of 1002 nucleotides which predicts a
334 amino acid protein of 36.9 kDa (predicted pl of 7.04),
consistent with the mobility of .delta.' in a SDS polyacrylamide
gel. The open reading frame encodes the N-terminal sequence and all
six tryptic peptide sequences obtained from .delta.'.sub.L and
.delta.'.sub.S.
[0110] Analysis of the DNA sequence upstream of the open reading
frame revealed a putative translation initiation signal
(Shine-Dalgarno sequence) 8 nucleotides upstream of the ATG
initiating codon. No obvious transcription initiation signals were
detected upstream of the initiation codon leaving open the
possibility that holB is in an operon with an upstream gene(s).
Alternatively, the transcription initiation signals may poorly
match the consensus signals and thereby be unrecognizable, as a low
level of transcription would not be unexpected for a gene encoding
a subunit of the holoenzyme present at only 10-20 copies/cell. The
holB gene uses several rare codons [TTA (Leu), ACA (Thr), GGA
(Gly), AGC, TCG (Ser)] 2-4 times more frequently than average which
may decrease translation efficiency.
[0111] The holB sequence contains a helix-turn-helix consensus
motif (Ala/GlyX.sub.3GlyX.sub.5Ile/Val) at
Ala.sub.80Gly84Val.sub.90 although ability of .delta.' to bind DNA
has yet to, be examined. There is also a possible leucine zipper
(Leu.sub.7X.sub.6Leu.sub.14X.sub.6Gly.sub.21X.sub- .6Leu.sub.28) in
the N-terminus although Gly interrupts the Leu pattern. The holB
sequence does not contain consensus sequences for motifs encoding
an ATP-binding site or a zinc finger. The molar extinction
coefficient of .delta.' calculated from its 8 Trp and 11 Tyr
residues is 59,600M.sup.-1cm.sup.-1 which is only 0.9% lower than
that observed in the presence of 6M guanidine hydrochloride for a
native extinction coefficient of 60,136M.sup.-1cm.sup.-1.
[0112] To obtain the .delta.' subunit in large quantity, an
expression plasmid was constructed. The holB gene was first cloned
into M13mp18 followed by site directed mutagenesis to create an
NdeI site at the initiating methionine to allow precise subcloning
of holB into the pET3c expression vector. The holB gene was excised
from the M13-.delta.'-NdeI mutant using NdeI followed by insertion
into the NdeI site of the pET3c expression vector [see Methods
Enzymol 185:60 (1990)] which places holB under the control of the
T7 RNA polymerase promotor of T7 gene 10 and the efficient
Shine-Dalgarno sequence of gene 10. The pET-.delta.' construct was
transformed into BL21(DE3)plysS cells which harbor a .lambda.
lysogen containing the T7 RNA polymerase gene controlled by the lac
UV5 promoter. Upon induction of T7 RNA polymerase with IPTG, the
.delta.' protein was expressed to 50% of total cell protein. Cell
lysate prepared from the induced cells containing pET-.delta.' was
5600-fold more active in the replication assays than cell lysate
prepared from induced cells containing the pET3c vector as
described below.
[0113] Three hundred liters of BL21(DE3)plysS cells harboring
pET-.delta.' were grown at 37.degree. C. in LB media supplemented
with 5 mg/ml glucose, 10 .mu.g/ml thiamine, 50 .mu.g/ml thymine
containing 100 .mu.g/ml ampicillin and 25 .mu.g/ml chloramphenicol.
Upon growth to an OD.sub.600 of 0.6, IPTG was added to 0.2 mM.
After further growth for two hours the cells (940 g) were collected
by centrifugation, resuspended in an equal weight of 50 mM Tris-HCl
(pH 7.5), 10% sucrose (Tris-Sucrose) and stored at -70.degree. C.
100 g of cells (30 liters of cell culture) were thawed whereupon
they lysed (due to lysozyme produced by plysS) and to this was
added 250 ml Tris-Sucrose, DTT to 2 mM and 40 ml of 10.times.heat
lysis buffer (50 mM Tris-HCl (pH 7.5), 10% sucrose, 0.3M
spermidine, 1M NaCl). The cell debris was removed by centrifugation
to yield the cell lysate (Fraction I, 4.41 g in 325 ml). The
purification steps that followed were performed at 4.degree. C. The
reconstitution activity assay for .delta.' is as described
previously. Ammonium sulphate (0.21 g/ml) was dissolved in the
clarified cell lysate and stirred for 90 minutes. The precipitated
protein containing .delta.' was pelleted (Fraction II, 1.58 g) and
redissolved in 660 ml of 30 mM Hepes-NaOH (pH 7.2), 10% glycerol,
0.5 mM EDTA, 2 mM DTT (buffer A) and dialyzed against two
successive changes of 2 liters each of buffer A to a conductivity
equal to 40 mM NaCl. The Fraction II was loaded onto a 300 ml
heparin agarose column (BioRad) equilibrated with buffer A. The
heparin column was washed with 450 ml buffer A plus 20 mM NaCl,
then eluted over a period of 14 hours using a 2.5 liter linear
gradient of 20 mM NaCl to 300 mM NaCl in buffer A. One hundred
fractions were collected. Fractions 36-53 were pooled (Fraction
111, 550 ml, 990 mg) and dialyzed twice against 2 liters of 20 mM
Tris-HCl (pH 7.5), 10% glycerol, 0.5 mM EDTA, 2 mM DTT (buffer B)
to a conductivity equal to 60 mM NaCl. The Fraction III was loaded
onto a 100 ml Q sepharose column (Pharmacia) equilabrated with
buffer B. The loaded Q sepharose column was washed with 150 ml of
buffer B plus 20 mM NaCl then eluted over a period of 12 hours
using a 1.2 liter linear gradient of 20 mM NaCl to 300 mM NaCl in
buffer B. Eighty fractions were collected. Fractions 34-56 were
pooled (Fraction IV, 781 mg in 370 ml) and dialyzed twice against 2
liters each of buffer B to a conductivity equal to 60 mM NaCl just
prior to loading onto a 60 ml EAH sepharose column (Pharmacia) that
was equilibrated with buffer B. The loaded EAH sepharose column was
washed with 60 ml of buffer B plus 40 mM NaCl then eluted over a
period of 10 hours using a 720 ml linear gradient of 40 mM NaCl to
500 mM NaCl in buffer B. Eighty fractions were collected. Fractions
18-30 (Fraction V, 732 mg in 130 ml), which contained homogeneous
.delta.' were pooled and dialyzed against 2 L buffer B (lacking DTT
to allow an absorbance measurement, see below) to conductivity of
40 mM NaCl. Fraction V was passed over a 5 ml ATP-agarose column
(Pharmacia, Type II, N-6 linked) to remove any .gamma. complex
contaminant followed by addition of DTT to 2 mM and then was
aliquoted and stored at -70.degree. C. Protein concentration was
determined rising BSA as a standard except at the last step in
which concentration was determined by absorbance using
.epsilon..sub.280=60,136M.sup.-1cm.sup.-1.
19 total fold total units.sup.1 specific purification % Step
protein (mg) activity (units/mg) yield I Lysate.sup.2,3 4414 3.0
.times. 10.sup.1 7 .times. 10.sup.6 1.0 100 II Ammonium 1584 2.5
.times. 10.sup.10 16 .times. 10.sup.6 2.3 83 Sulfate III Heparin
990 2.6 .times. 10.sup.10 26 .times. 10.sup.6 3.7 87 IV Q Sepharose
781 2.6 .times. 10.sup.10 33 .times. 10.sup.6 4.7 87 V EAH- 732 2.5
.times. 10.sup.10 34 .times. 10.sup.6 4.9 83 Sepharose.sup.4
.sup.1One unit is defined as pmol nucleotide incorporated in 20
seconds .sup.2Lysate of BL21(DE3)plysS cells harboring the pET3c
vector yielded a specific activity of 1252 units/mg. .sup.3Omission
of .gamma. and .delta. from the assay of the lysate resulted in a
7650-fold reduction of specific activity (915 units/mg).
.sup.4Using pure .delta.', omission of .gamma. from the assay gave
no detectable synthesis under the conditions of the assay.
[0114] The purified overproduced .delta.' stimulated .gamma..delta.
30-fold in its action in reconstituting the processive holoenzyme
from the ae polymerase and the .beta. clamp accessory protein. In
this assay the .delta.' is titrated into a reaction containing a
low concentration of .gamma. and .delta. and also contains the
.beta. subunit, .alpha..epsilon. polymerase and M13mp18 ssDNA
primed with a synthetic olignucleotide and coated with SSB. The
proteins were preincubated with the DNA for 8 minutes to allow time
for the accessory proteins to form the preinitiation complex which
contains the .beta. clamp and for .alpha..epsilon. to bind the
preinitiation complex. DNA synthesis is initiated upon addition of
deoxyribonucleoside triphosphates and the reaction is stopped after
20 seconds which is sufficient time for the processive
reconstituted polymerase to complete the circular DNA. Although a
processive polymerase can be reconstituted without the
.delta.']subunit, under the conditions used in the present
invention in which .gamma. and .delta. are at low concentration,
the .delta.' subunit stimulates the reaction greatly (30-fold). The
.delta.' subunit saturated this assay at a level of approximately
one molecule of .delta.' to one molecule of .delta..
[0115] Both the .tau. and .gamma. subunits of the holoenzyme are
encoded by the same gene (dnaX). The .gamma. subunit is formed as a
result of a -1 frameshift during translation with, the result that
.gamma. is only 2/3 the length of .tau. due to an earlier
translational stop codon (within 2 codons) in the -1 reading frame.
The activity of the .gamma. and .tau. proteins in reconstituting
the processive polymerase was compared using either the .delta.,
.delta.' or both .delta..delta.' subunits in the presence of
.alpha..epsilon. complex and .beta. subunit (FIGS. 6A and 6B). In
the absence of .delta. and .delta.', the .gamma. subunit alone
displays insignificant activity in the reconstitution assay
although when a large amount of .gamma. was present it had very
little, but detectable, activity (FIG. 6A). The .delta. subunit
provides .gamma. with activity in the reconstitution assay, but
.delta.' does not provide .gamma. with activity. However, the
cloned .delta.' subunit, when present with .delta., markedly
stimulated the activity of the .gamma. and .delta. mixture such
that maximal activity was achieved at much lower concentrations of
added .gamma..
[0116] The .tau. subunit alone, like .gamma., was also essentially
inactive in the reconstitution assay, although at very high amounts
of .tau. a slight, but reproducible amount of activity was
observed. .tau. is active with .delta. in this assay although more
.tau. (50-fold) than .gamma. is needed for comparable activity.
Previously it was observed that .tau. was unlike .gamma. in that
.tau. was active with .delta.' in the reconstitution assay in the
absence of any .delta. subunit (only .tau., .delta.' and .alpha.,
.epsilon., .beta. were needed). Consistent with these previous
results, the .delta.' subunit is active with .tau. in the absence
of .delta. (similar to the activity of .tau. and .delta. in the
absence of .delta.'). With both .delta. and .delta.' present, only
a small amount of .tau. subunit is required for maximal activity in
the reconstitution assay. The activity of .tau..delta..delta.'
parallels that of .gamma..delta..delta.' and requires 500-fold less
.tau. for maximal activity than either .tau..delta. or
.tau..delta.'. Hence, both the .gamma. subunit and the .tau.
subunit are highly active in this reconstitution assay when both
.delta. and .delta.' are present.
[0117] The effect of the .delta., .delta.' and .beta. subunits on
the DNA dependent ATPase activity of .tau. was quite different from
their effect on .gamma., the close relative of the .tau. subunit.
The .tau. subunit, by itself, is a much more active DNA dependent
ATPase than .gamma. and, in fact turns over two times more ATP than
the .gamma..delta..delta.' complex. Unlike the .gamma. ATPase, the
.tau. ATPase was essentially unaffected by .beta. or by .delta.
with or without .beta. or by .delta.' with or without .beta..
However, like the .gamma. ATPase, the presence of both .delta. and
.delta.' stimulated the .tau. ATPase, although the effect was only
4-fold compared to the 30-fold stimulation of .gamma. by
.delta..delta.'. Whereas .beta. stimulated the
.gamma..delta..delta.' ATPase 3-fold, the .beta. subunit did not
stimulate the .gamma..delta..delta.' ATPase at all, in fact .beta.
slightly inhibited it, yet the .tau..delta..delta.' complex is as
active as .gamma..delta..delta.' in reconstituting a processive
polymerase with .beta. and .alpha..epsilon..
[0118] The cloned .delta.' preparation appears as a doublet in a
13% SDS polyacrylamide gel and the two polypeptides are of the same
size and molar ratio (2:1, lower band-to-upper band) as the
.delta.' doublet purified from the .gamma. complex. Electrospray
mass spectometry revealed that the smaller polypeptide
(.delta.'.sub.S) was the size predicted from the gene sequence and
the larger polypeptide (.delta.'.sub.L) was increased in size by
521 Da. The nature of the larger polypeptide is presently under
investigation. Possibilities include mRNA splicing, use of an
upstream translational tart signal, read through of the stop codon,
translational frameshifting, and posttranslational modification.
Whatever the mechanism which produces .delta.'.sub.L it must be
efficient since the highly overproduced .delta.' still produces the
same level of .delta.'.sub.L relative to the .delta.'.sub.S and
.delta.'.sub.L within the holoenzyme. Irrespective of how
.delta.'.sub.L is synthesized, the fact remains that .delta.'.sub.L
and .delta.'.sub.S are different. Presumably they also have
functional differences as in the case of the related .gamma. and
.tau. subunits. Whereas .tau. and .gamma. both appear to be within
each holoenzyme molecule, it reamins to be shown whether the
.delta.'.sub.L and .delta.'.sub.S subunits are on one or on
different holoenzyme molecules.
[0119] Sequence analysis of .delta.'.sub.L and .delta.'.sub.S show
they have identical N-termini proving .delta.'.sub.L is not derived
from an alternate upstream ATG start site. Translational read
through of the stop codon was considered as an explanation which
would produce a protein containing 19 additional amino acids before
the next stop codon in the open reading frame, but this would
increase the size of .delta.' by 2130 Da, much larger than the
observed mass of .delta.'.sub.L. Treatment of .delta.' with calf
intestinal and bacterial alkaline phosphatases did not effect the
mobility of either .delta.'.sub.S or .delta.'.sub.L suggesting that
serine and threonine phosphorylation is not involved in, the
formation of .delta.'.sub.L; attachment of other groups remains a
possibility. Hence, translational frameshifting (or lumping),
covalent modification (other than phosphate on Ser or Thr) and mRNA
splicing remain possible.
[0120] It seems most pertinent to consider translational
frameshifting as a source of .delta.'.sub.L since such a mechanism
has precedent in holoenzyme structure. The dnaX gene encoding the
.tau. subunit of the holoenzyme generates the .gamma. subunit by a
translational frameshift into the -1 reading frame. If
.delta.'.sub.L is produced by a -1 frameshift, the frameshift would
have to occur upstream of the holB stop codon but not so far
upstream that a -1 frameshift would produce a truncated protein due
to running into an early -1 frame stop codon. Thus the -1
frameshift would have to occur at or after the last -1 frame stop
codon near Glu.sub.320 after which translation would proceed past
the normal stop codon in the open reading frame to produce a
protein which is 7 amino acids larger than that predicted by the
open reading frame of holB.
[0121] The .gamma. complex expends ATP energy to clamp the .beta.
subunit onto a primer and it is this .beta. dimer clamp that
tethers the .alpha..epsilon. polymerase to the template for rapid
and highly processive DNA synthesis by the .alpha..epsilon.
polymerase which is only efficient after the .beta. subunit has
been clamped onto the DNA by .gamma. complex action. A mixture of
the .gamma. and .delta. subunits is sufficient in this assay to
clamp .beta. onto DNA, however much more .gamma. and .delta. is
heeded relative to the amount of .gamma. complex. The .delta.'
subunit stimulates .gamma. and .delta. in this assay such that the
amounts of .gamma., .delta. and .delta.' are nearly comparable with
the amount of .gamma. complex that is required (the .lambda. and
.sub..psi. subunits give another 3-8 fold stimulation of activity
at low concentrations of .gamma..delta..delta.', as described in
the accompanying report. Likewise, neither .delta. or .delta.' have
a large effect on the ATPase activity of .gamma. but addition of
both .delta. and .delta.' to .gamma. gives a 30-fold stimulation of
the .gamma.ATPase activity. The requirement of both .delta. and
.delta.' for efficient replication activity and for maximal ATPase
activity of .gamma. correlates with the physical studies in the
accompanying report which show that .delta. and .delta.' form a
complex and the .delta..delta.' complex binds tightly to .gamma.,
whereas when .delta. and .delta.' are added separately with .gamma.
they do not form a strong .gamma..delta. or .gamma..delta.'
complex.
[0122] The .tau. subunit contains the sequence of the
.gamma.subunit (.gamma. is produced from .tau.) plus, an extra
domain of 212 amino acids which binds to .alpha. and to DNA.
[0123] A homology search of the translated GenBank indicated that
the most homologous protein to .delta.' of the present invention
was another E. coil protein, the .gamma./.tau. subunit(s) of DNA
polymerase III holoenzyme. There is 27% identity and 44% similarity
including conservative substitutions over the entire length of
.delta.' and .gamma./.tau.. One particular region in .delta.' of 50
amino acids (amino acids 110-159) is strikingly similar to
.gamma./.tau. (amino acids 121-170) having 49% identity. A putative
helix-turn-helix motif in .gamma./.tau.
(Ala.sub.114X.sub.3Gly.sub.118X.sub.5Leu.sub.124) is positioned
just 19 residues downstream of the helix-turn-helix motif in
.delta.'.
[0124] The extent of sequence homology between .delta.' and the
.gamma./.tau. subunit is above the level required to speculate that
they have similar three dimensional structures; When both
.delta.'.sub.S and .delta.'.sub.L are taken into account, four of
the eleven subunits within the holoenzyme, according to the present
invention, may have similar structures.
[0125] The interactions between .delta. and .delta.' were also
studied as part of the present invention.
[0126] Equal amounts of .delta. and .delta.' were incubated
together for 30 minutes at 15.degree. C. and then analyzed by gel
filtration and glycerol gradient sedimentation. Gel filtration
analysis showed .delta. and .delta.' subunits comigrate and elute
approximately six-to-eight fractions earlier than either .delta.
or.delta.' alone indicating that they form a .delta..delta.'
complex. Comparison with protein standards yields a Stokes radius
of 31.1 .ANG.. The .delta. and .delta.' also comigrated during
glycerol gradient analysis and sedimented faster than either
.delta. or .delta.' alone, again consistent with formation of a
.delta..delta.' complex with an S value of 3.9S. Combining the S
value and Stokes radius yields a native mass of 53 kDa for the
.delta..delta.' complex, most consistent with the mass of a 1:1
complex of .delta..sub.1.delta.'.sub.1 (75.6 kDa) then of a higher
order aggregate of .delta..delta.'. Both .delta.'.sub.L and
.delta.'.sub.S are visible in the .delta..delta.' conplex
indicating they are present as a mixture of .delta..delta.'.sub.L
and .delta..delta.'.sub.S. Formation of a trimeric
.delta..delta.'.sub.L.delta.'.sub.S complex Is unlikely as the
combined mass would be 113 kDa, twice the observed mass. However,
if free .delta. and .delta.' were in a rapid equilibrium with the
.delta..delta.' complex then the observed mass of the
.delta..delta.' complex would be a weighted average of the amount
of complex and amount of free subunits and therefore the
possibility of a higher order aggregate such as a
.delta..delta.'.sub.L.delta.'.sub.S complex can not be rigorously
excluded.
[0127] Densitometry analysis of the Coomassie Blue stained gel
yielded a molar ratio of .delta.:.delta.' of 1.1:1.0, respectively
( the two .delta.' bands were considered together as one .delta.')
further supporting the .delta..sub.1.delta.'.sub.1 composition.
Different proteins may take up different amounts of Coomassie Blue
stain and therefore molar rates determined by densitometry must be
regarded as tentative. A dynamic light scattering analysis of
.delta., .delta.' and .delta..delta.' complex is also presented in
the table below.
[0128] The Stokes radius and sedimentation coefficient of .delta.,
.delta.' and .delta..delta.' complex were determined from the gel
filtration and glycerol gradient sedimentation analyses; and the
native molecular mass and the frictional coefficient were
calculated from the Stokes radius and S value. These calculations
require the partial specific volume of .delta. and .delta.'; these
volumes were calculated by summation of the partial specific
volumes of the individual amino acids for each .delta. and
.delta.'. Molecular weights of .delta., .delta.' and the
.delta..delta.' complex (assuming a composition of
.delta..sub.1.delta.'.sub.1) were calculated from the gene
sequences of .delta. and .delta.'.
20 .delta. .delta.' .delta..delta.' Stokes radius 26.5 25.8 31.1
Sedimentation coefficient 3.0 3.0 3.9 Partial specific volume 0.74
0.74 0.74 Native mass (radius and S value) 34,708 33,791 52,952
Native mass (gene sequence) 38,704 36,934 75,630 Frictional
coefficient 1.22 1.20 1.25 Diffusion coefficient (light scattering)
7.60 8.16 6.61 Radius calculated (D) 28.2 26.3 32.5
[0129] The diffusion coefficient obtained from the light scattering
analysis can be used to calculate the Stokes radius and these
values were within 6% of the Stokes radius of .delta., .delta.' and
.delta..delta.' complex determined in gel filtration.
[0130] In the .gamma. complex, the .gamma., .delta. and .delta.'
subunits are bound together along with the .sub..chi. and
.sub..psi. subunits. The activity analysis described herein
indicates that .gamma. and .delta. interact since both are
necessary and sufficient to assemble the .beta. clamp onto DNA.
Further, the .delta.' subunit stimulates the DNA dependent ATPase
activity of .gamma. indicating that .gamma. and .delta.'
interact.
[0131] The physical interaction between .delta., .delta.' and
.gamma. were examined using the gel filtration technique which
detects tightly bound protein-protein complexex, but since
components are not at equilibrium during gel filtration, weak
protein complexes will dissociate. The .gamma. subunit (47 kda) is
larger than .delta. and .delta.', and is at least a dimer in its
native state with a large Stokes radius and quite an asymmetric
shape (.gamma. runs as a trimer or tetramer in gel filtration and
as a dimer in a glycerol gradient. The .gamma. was mixed with a
4-fold molar excess of .delta. and .delta.' then gel filtered. A
complex of .gamma..delta..delta.' was formed as indicated by the
comigration of both the .delta. and .delta.' subunits with .gamma..
The excess .delta..delta.' complex eluted much later (fraction
40-46). Since .delta. binds .delta.', it is possible that only one,
for example .delta., binds .gamma. and the other (eg. .delta.') is
part of the complex by virtue of binding .delta. instead of
directly interacting with .gamma.. To determine which subunit,
.delta. or .delta.', binds directly to .gamma., the .gamma. subunit
was mixed with either .delta. or .delta.' then gel filtered. The
mixture of .gamma. and .delta. showed that .gamma. and .delta. did
not form a gel filterable .gamma..delta. complex as indicated by
the absence of .delta. in fractions 24-32 containing .gamma.. The
mixture of .gamma. and .delta.' showed that .delta.' did not form a
complex with .gamma. either as indicated by the absence of .delta.'
in fractions containing .gamma.. Therefore both .delta. and
.delta.' must be present to form a gel filterable complex with
.gamma.. Using pure cloned .delta. no .gamma..delta. complex in gel
filtration (or in glycerol gradient analysis) was seen.
[0132] The gel filtration column fractions of the
.gamma..delta..delta.' complex were analyzed for their activity in
assembly of the .beta. clamp on primed DNA. Fractions containing
the .gamma..delta..delta.' complex were quite active. The
.delta..delta.' complex, even at high concentration, is not active
in assembly of the .beta. clamp and therefore the slight amount of
activity in following fractions wag probably due to a slight amount
of .gamma. which trailed into the peak of the .delta..delta.'
complex thus giving activity in the assay. The column fractions of
the .gamma..delta. and .gamma..delta.' mixtures were inactive
except for the peak fraction of .gamma. in the .gamma..delta.'
analysis which supported weak activity. There was a slight, barely
detectable amount of .delta.' (but not .delta.), in the fractions
containing .gamma. as though a slight amount of .gamma..delta.'
complex was formed and survived the column.
[0133] Following these studies with .delta. and .delta.', the
present invention has found that .delta. behaved as a monomer in
gel filtration and glycerol gradient sedimentation. The .delta.'
subunit also appeared monomeric. Neither .delta. or .delta.', when
separate, formed a gel filterable complex with the .gamma. subunit.
Yet they most likely bind to .gamma. (at least weakly) as indicated
by activity assays in which .gamma..delta. is active (without
.delta.') in assembly of the .beta. clamp, and .delta.' (without
.delta.) stimulates the DNA dependent ATPase activity of .gamma..
The .delta. and .delta.' subunits bound each other to form a gel
filterable 1:1 .delta..sub.1.delta.'.sub.1 complex and when mixed
with .gamma. they efficiently formed a tight gel filterable
.gamma..delta..delta.' complex. Hence, the binding of .delta. and
.delta.' to .gamma. is cooperative.
[0134] The .delta.' subunit is a mixture of two related proteins,
.delta.'.sub.L and .delta.'.sub.S which are encoded by the same
gene; .delta.'.sub.L is 521 da larger than the gene sequence
predicts. The functional and structural difference between them is
presently unknown. In these binding studies, both .delta.'.sub.S
and .delta.'.sub.L bound to .delta. and they both assembled into
the .gamma..delta..delta.' and .tau..delta..delta.' complexes,
consistent with the fact that both .delta.'.sub.L and
.delta.'.sub.S are observed within polIII and the .gamma.
complex.
[0135] No single subunit of the .gamma. complex is active in
assembling the .beta. clamp on DNA. Presumably this reacton is to
complicated for just one protein. A mixture of .gamma. and .delta.
is capable of assembling .beta. onto DNA although they are
inefficient and require .delta.' for efficient activity. Perhaps
.delta.' increases the efficiency of .gamma..delta. by physically
bringing .gamma. and .delta. together in the .gamma..delta..delta.'
complex, although it is also possible that .delta.' participates
directly in the chemistry of the reaction. The .gamma. subunit has
a low level of DNA dependent ATPase activity, and described above,
.delta. binds the .beta. subunit. These two facts allow speculation
that .gamma. binds the primed template, and .delta. brings in the
.beta. subunit, then ATP hydrolysis is coupled to assemble the ring
shaped .beta. dimer around the DNA.
[0136] Since .gamma. is known to bind ATP and has a low level of
DNA dependent ATPase activity, it is an obvious candidate as the
subunit which interacts with the ATP in the .beta. clamp assembly
reaction. Two molecules of ATP are hydrolyzed in the initiation
reaction in which the holoenzyme becomes clamped onto a primed
template to form the initiation complex. This initiation reaction
has its basis in the assembly of the .beta. clamp on DNA. The
stoichiometry of two ATP hydrolyzed in formation of one initiation
complex suggests two proteins hydrolyze ATP. These two proteins may
be the two halves of a .gamma. dimer. However it is also possible
that .delta. interacts with ATP. The sequence of .delta. shows a
very close match to the consensus for an ATP binding site and UV
induced cross-linking studies suggest that .delta. binds ATP. The
availability of .delta. in quantity should now make possible a full
description of the mechanism by which ATP is coupled to assemble
the ring shaped .beta. dimer around DNA.
[0137] The third subunit according to the present invention, that
of .theta., was also identified, purified, cloned and sequenced.
N-terminal analysis of the .theta. peptide yielded the following
sequence of 40 amino acids:
21 Met Leu Lys Asn Leu Ala Lys Leu Asp Gln Thr Glu Met Asp Lys 5 10
15 Val Asn Val Asp Leu Ala Ala Ala Gly Val Ala Phe Lys Glu Arg 20
25 30 Tyr Asn Met Pro Val Ile Ala Glu Ala Val 35 40
[0138] Based upon this sequence, two DNA probes were fashioned.
These probes had the sequences of:
22 ATG CTG AAA AAC CTG GCT AAA CTG GAT CAG ACT GAA ATG GAT AAA 45
GTT AAC GTT GAT; and 57 CTG GCT GCT GCT GGT GTT GCT TTT AAG GAA CGT
TAT AAC ATG CCG 45 GTT ATT GCT GAA. 57
[0139] These two probes were also end-labelled with .sup.32P for
use with Southern blot procedures.
[0140] For Southern blot analysis, E. coli DNA was cut with the 8
Kohara map enzymes [see Cell 50:495 (1987)]. The two probes
described above were used to probe two Southern blots of E. coli
DNA. The bands (DNA fragments) in common with the two blots were
noted, as was their size. At least 3 positions on the Kohara map of
the E. coli chromosome were consistent with the Southern blot
fragmentation pattern.
[0141] Thus, based upon these findings, E. coli Dna digested with
either EcoRV or PvuII following DNA extraction [see J.M.B. 3:208
(1961)] was run out in an agarose gel, and all the DNA in the size
region of the gel corresoponding to the fragment size containing
.theta. for that enzyme (PvuII or EcoRV) from the Southern blot
analysis, was extracted from the gel and cloned into M13mp18 and
M3mp19 using conventional techniques. The M13 transformant DNAs
were analyzed by Southern blot and probed using the two probes
described above. One M13 DNA was obtained with the .theta.
sequence. When this M13 .theta. was sequenced, however, not all the
theta gene was present; the gene extended beyond the PvuII
restriction site. The M13 .theta. was then used as a reagent to
obtain the complete .theta. gene.
[0142] A Kohara .lambda. phage (.lambda..sub.336) was grown and the
.theta. gene in E. coli was excised using an EcoRV cut 2.7 kb
fragment. Next, a filter containing all the Kohara .lambda. phage
was probed using the partial .theta. gene as the probe. Thus, it
was possible to identify the .lambda. phage containing the full
.theta. gene.
[0143] The holE gene was then cloned from the .lambda. phage into
pUC18 and subsequently sequenced. The full genetic sequence for the
.theta. gene was thus determined to be:
23 ATG CTG AAG AAT CTG GCT AAA CTG GAT CAA ACA GAA ATG 39 GAT AAA
GTG AAT GTC GAT TTG GCG GCG GCC GGG GTG GCA 78 TTT AAA GAA CGC TAC
AAT ATG CCG GTG ATC GCT GAA GCG 117 GTT GAA CGT GAA CAG CCT GAA CAT
TTG CGC AGC TGG TTT 156 CGC GAG CGG CTT ATT GCC CAC CGT TTG GCT TCG
GTC AAT 195 CTG TCA CGT TTA CCT TAC GAG CCC AAA CTT AAA 228
[0144] The open reading frame above predicts that .theta. is a 76
amino acid protein of 8,629 Da. The underlined nucleotide sequence
exactly matches the corresponding N-terminal sequence of .theta..
In addition, the upstream sequence contains two putative RNA
polymerase promoter signals and a Shine-Dalgarno sequence. This
upstream sequence is:
24 AG GCGTAGCGAA GGGAGCGTGC AGTTGAAGCC ATATTATCTA TTCCTTTTTG 52
TAATAACTT TTTACAGACG ATAACCTTGT CTAATGTCTG AGTCGAGGAT 102
CATCAATTCC GGCTTGCCAT CCTGGCTCAC TCTTAGTAAC TTTTGCCCGC 152
GAATGATGAG GAGATTAAGA 172
[0145] The downstream sequence begin with a stop codon:
25 TAA AACTTATAC AGAGTTACAC TTTCTTACAT AACCCCTGCT AAATTATGAG 52
TATTTTCTAA ACCGCACTCA TAATTTGCAG TCATTTTGAA AAGGAAGTCA 102 TTATG
107
[0146] This translated into the peptide sequence:
26 Met Leu Lys Asn Leu Ala Lys Leu Asp Gln Thr Glu 5 10 Met Asp Lys
Val Asn Val Asp Leu Ala Ala Ala Gly 15 20 Val Ala Phe Lys Glu Arg
Tyr Asn Met Pro Val Ile 25 30 35 Ala Glu Ala Val Glu Arg Glu Gln
Pro Glu His Leu 40 45 Arg Ser Trp Phe Arg Glu Arg Leu Ile Ala His
Arg 50 55 60 Leu Ala Ser Val Asn Leu Ser Arg Leu Pro Tyr Glu 65 70
Pro Lys Leu Lys 75 76
[0147] Using site-directed mutagenesis, the initial Met codon (AGA
ATG) was mutated to CAT AtG (NdeI site) using an oligonucleotide
with 15 bases on either side of the mutation. This was then used to
obtain the overproduction of the .theta. gene in which the
mp19.theta. (a 2700 bp insert) was grown in strain CJ236 cells in
the presence of uridine. The purified single stranded DNA from
these cells was purified and hybridized with the NdeI mutation and
replicated with the holoenzyme in vitro. XLI-Blue cells were
transformed with the double stranded DNA product and ten plaques
were selected for miniprep sequencing; all 10 plaques contained the
mutation. The .theta. sequence was excised from the DNA with
HindIII, NdeI, and the resulting 1 kbp fragment was inserted into
pET-3C [see Methods in Enzymology 185:60 (1990)]. The resulting
pET-3C.theta. was used to transform competent cells [BL21(DE3)).
Single colonies of the transformed cells were grown in liquid media
at 37.degree. C. to an OD of about 0.6, induced with IPTG generally
as described previously, and harvested post induction. Successful
overexpression of the .theta. peptide was obtained using this
system.
[0148] The N-terminal sequence analysis of .theta. was examined as
follows: PolIII was purified [see J. Biol. Chem. 263:6570, (1988)]
except that the last step using Seperose 6 was replaced with an
ATP-agarose column (Pharmacia, type II) which was eluted with a
linear salt gradient. After the .delta..delta.' eluted from the ATP
agarose column, a mixture of pure polIII' and
.gamma..sub..chi..psi. complex eluted together. This mixture was
separated by column chromatography on MonoQ using a linear gradient
of 0-0.4 M NaCl in buffer A. The polIII' which was eluted after the
.gamma..sub..chi..psi. complex was used as the source of .theta.
subunit. The subunit was separated from .alpha., .tau. and
.epsilon. subunits of polIII' by electrophoresis in a 15% SDS
polyacrylamide gel, and was electroblotted (110 pmol) onto PVDF
membrane. The .theta. subunit was visualized by Ponceau S stain,
and the N-terminal sequence was determined to be:
27 NH.sub.2-Met Leu Lys Asn Leu Ala Lys Leu Asp Gln Thr 5 10 Glu
Met Asp Lys Val Asn Val Asp Leu Ala Ala Ala 15 20 Gly Val Ala Phe
Lys Glu Ala Tyr Asn Met Pro Val 25 30 35 Ile Ala Glu (Ala)
(Val)
[0149] in which the parenthesis indicate uncertain amino acid
assignments.
[0150] The .theta. was isolated using E. coli genomic DNA isolated
from strain C600 [see J. Mol. Biol. 3:208 (1961)], cut with the
Kohara panel of restriction enzymes (BamHI, HindIII, EcoRI, EcoRV,
BglI, KpnI, PstI and PvuII), and separated in a 0.8% native agarose
gel. The gel was depurinated (0.25 M HCl), denatured (0.5 M NaOH,
1.5 M NaCl) and neutralized (1 M Tris, 2 M NaCl, pH 5.0) prior to
transfer of the DNA to Genescreen plus (DuPont New England Nuclear)
using a Vacugene (Pharmacia) apparatus in the presence of
2.times.SSC (0.3 M NaCl, 0.3 M sodium citrate, pH 7.0). The
membrane was air dried prior to hybridization. Two synthetic
oligonucleotide DNA 57-mer probes were designed based on the
N-terminal sequence of .theta. assuming the highest frequency of
codon usage and favoring T over C in the wobble position. The two
probes (5'->3') were:
28 Theta 1 (codons 1-19): ATG CTG AAA AAC CTG GCT AAA CTG GAT CAG
ACT GAA ATG GAT 42 AAA GTT AAC GTT GAT 57; and Theta 2 (codons
20-38): CTG GCT GCT GCT GGT GTT GCT TTT AAA GAA CGT TAT AAC ATG 42
CCG GTT ATT GCT GAA 57.
[0151] The DNA 57-mers (100 pmol each) were 5' end-labelled using 1
.mu.M [.gamma.-.sup.32P] ATP and T4 polynucleotide kinase, and then
used to probe Southern blot of the restricted E. coli genomic DNA.
Two Southern blots were hybridized individually using one or the
other of the 57-mer probes overnight in the same buffer as above
except with an additional 200 .mu.g/ml of denatured salmon sperm
DNA. The Southerns were washed in 2.times.SDS at room temperature
for 30 minutes, then 3 hours at 42.degree. C. (changing the buffer
each hour), then exposed to X-ray film. The Theta 1 probe showed a
single bnd in 7 of the 8 restriction digests; the Theta 2 probe
consistently showed many bands in each lane which were eliminated
equally as the hybridization and washing conditions were gradually
increased in stringency, suggesting that Theta 2 did not match the
true sequence of the holE gene. After holE was cloned and
seqeunced, it was found that 7 nucleotides of Theta 1 and 12
nucleotides of Theta 2 did match the holE sequence.
[0152] To clone the holE gene, 100 .mu.g of E. coli DNA was
digested with PvuII, and the small population of DNA fragments
migrating in the 400 to 600 bp range (the Southern blot using Theta
1 probe indicated holE was on a 500 bp PvuII fragment) was
extracted from the agarose gel, blunt-end ligated into M13mp18
digested with HincII, and transformed into competent XL1-Blue
cells. Presence of the holE gene was determined by Southern blot
analysis of minilysate DNA prepared from recombinant colonies using
the 5' end-labelled Theta 1 as a probe. One positive clone was
obtained and sequenced; it contained approximately one-half of the
holE gene (a PvuII site lies in the middle of holE). This fragment
of holE was uniformly labelled using the random primer labelling
method, and used to screen the complete Kohara ordered lambda phage
library of E. coli chromosomal DNA transferred onto a nylon
membrane. Prehybridization and hybridization were conducted as
described above except that the temperature was increased to
65.degree. C. and the wash steps were more stringent (2.times.SSC,
0.2% SDS ,next 1.times.SSC, and then 0.5.times.SSC at 65.degree.
C.). A single phage clone .lambda. 19H3 (336 of the miniset) [see
Cekk 50:495 (1987)] hybridized with both the genomic fragment and
the Theta 1 probe.
[0153] The phage and a 2.7 kb EcoRV fragment containing the .theta.
gene was excised, purified from a native agarose gel, and blunt-end
ligated into the HincIII site of M13mp19 to yield M13mp19-.theta..
The 2.7 kb EcoRI-HindIII fragment from M13mp19-.theta. was excised,
gel purifeid, and directionally ligated into the corresponding
sites of pUC18 to generate pUC-.theta.. Both strands of the holE
gene in pUC-.theta. were sequenced using the sequenase kit
[.alpha.-.sup.35S]dATP, and synthetic DNA 20-mers. This time the
entire holE gene was present.
[0154] An NdeI site was, generated at the start codon of the holE
gene by the oligonucleotide site directed mutagenesis using a DNA
33-mer
[0155] AGATGAGGA GATTACATAT GCTGAAGAAT CTG 33
[0156] containing an NdeI site (underlined) at the start codon of
holE to prime replication of M13mp19-.theta. viral ssDNA and using
SSB and DNA polymerase III holoenzyme in place of DNA polymerase I.
The NdeI site in the resultant phage (M13mp19-.theta.-NdeI) was
verified by DNA sequencing. An approximately 1 kb NdeI-HindIII
fragment was excised from M13mp19-.theta.-NdeI and directionally
ligated into the corresponding sites of pUC18 to yield
pUC-.theta.-NdeI. A 1 kb NdeI/BamHI fragment from pUC-.theta.-NdeI
was then subcloned directionally into pET3c digested with both NdeI
and BamHI to generate the overproducing plasmid, pET-.theta..
[0157] Reconstitution assays contained 72 ng phage.times.174 ssDNA
(0.04 pmol as circles) uniquely primed with a DNA 30-mer, 0.98 SSB
(13.6 pmol as tetramer) 10 ng .beta. (0.13 pmol as dimer), and 4 ng
.gamma. complex (0.02 pmol) in 20 mM Tris-HCl (pH 7.5), 8 mM
MgCl.sub.2, 5 mM DTT, 4% glycerol, 40 .mu.g/ml BSA, 0.5 mM ATP, 60
.mu.M dGTP, and 0.1 mM EDTA in a final volume of 25 .mu.l (after
addition of .alpha..epsilon. or .alpha..epsilon..theta.). The
.alpha..epsilon. and .alpha..epsilon..theta. complexes were each
preformed upon mixing 38 pmol each of .alpha. and .epsilon., and
when present, 152 pmol of .theta. in 12.5 .mu.l of 25 mM Tris-HCl
(pH 7.5), 2 mM DTT, 1 mM EDTA, 10% glycerol followed by incubation
for 1 hour at 15.degree. C. These protein complexes were diluted
30-fold in the same buffer just prior to addition to the assay on
ice, then the assay tube is shifted to 37.degree. C. for 6 minutes
to allow reconstruction of the processive polymerase or the primed
ssDNA. DNA synthesis was initiated upon rapid addition of 60 .mu.M
dATP and 20 .mu.M [.alpha.-.sup.32P]TTP, then quenched after 15
seconds and quantitated using DE81 paper. Proteins used in the
resconstruction assays were purified, and their concentrations
determined using BSA as a standard.
[0158] The following synthetic DNA 56-mer was designed as a hooked
primer template to assay 3'->5" exonuclease activity:
29 T T TCGGCTTAAGGAG-3' T TGCCGAATTCCTCGGCCCCTAGGAGATCTCAGCT-5'
T
[0159] This DNA 56-mer (75 pmol as 56-mer) was 3' end-labelled with
75 pmol of [.alpha.-.sup.32P] dTTP (3000 Ci/mmol) using 200 units
of terminal transferase under conditions specified by the
manufacturer (Boehrnger) in a total volume of 100 .mu.l followed by
spin dialysis to remove remaining free nucleotide.
[0160] Prior to adding proteins to the assay, .theta. was titrated
into .epsilon. upon incubating 2 .mu.g .epsilon. (70 pmol as
monomer) with .theta. (0-10 .mu.g, 0-1.16 nmol as monomer) in a
total volume of 10 .mu.l buffer A containing 50 .mu.g/ml BSA at
15.degree. C. for 1 hour. The .epsilon..theta. mixture was then
diluted 100-fold using buffer A containing 50 .mu.g/ml BSA. A 2.5
.mu.l sample of diluted complex was added to 200 fmol
3'-.sup.32P-end-labelled mispaired hook DNA in 12.5 .mu.l of 25 mM
Tris-HCl (pH 7.5), 4% sucrose, 5 mM MgCl.sub.2, 8 mM DTT, and 50
.mu.g/ml BSA followed by a 3 minute incubation at 15.degree. C. The
reaction was quenched upon spotting 13 .mu.l of the mixture onto a
DE81 filter. The amount of mispaired nucleotide remaining was
quantitated, and subtracted from the total mispaired template added
to obtain the amount of 3' mispaired nucleotide released.
[0161] Gel filtration was performed using HR 10/30 fast protein
liquid chromatography columns, Superdex 75 and Superose 12, in
buffer C. Samples containing either .theta., .epsilon. or .alpha.
alone, and mixtures of these subunits were incubated at 15.degree.
C. for 1 hour. The entire sample was then injected onto the column
and after collection the first 5.6 ml (Superose 75) or 6.0 ml
(superose 12), fractions of 160 MI were collected and analyzed in
15% SDS,polyacrylamide gels. Protein standards were a mixture of
proteins of known Stokes radius and were also analyzed.
Densitometry of stained gels was performed using a laser
densitometer, Ultrascan XL (Pharmacia-LKB).
[0162] Subunits (.alpha., .theta., .epsilon.) alone and mixtures of
these subunits were incubated 1 hour at 15.degree. C. (with 5%
glycerol), then mixed with protein standards of known S value (50
.mu.g of each protein standard) and immediately layered onto 12.3
ml linear 10%-30% glycerol gradients in 25 mM Tris-HCl (pH 7.5),
0.1 M NaCl, 1 mM EDTA. The gradients were centrifuged at
270,000.times.g for 44 hours (.epsilon., .theta., and
.epsilon..theta. complex) or 26 hours (.alpha..epsilon. and
.alpha..epsilon..theta.) at 4.degree. C. Fractions of 150 .mu.l
were collected from the bottom of the tube and analyzed in a 15%
SDS-polyacrylamide gel stained with Coomassie Blue.
[0163] In summary the sequence of the N-terminal 40 amino acids of
.theta. were obtained from the .theta. subunit within the polIII'
subassembly (.alpha..epsilon..theta..tau.) of holoenzyme. This
sequence did not match any previously identified in GenBank, and
therefore the invention attempted to identify the holE gene using
the Kohara restriction map of the E. coli chromosome. Two 57-mer
DNA probes were made based on the N-terminal amino acid sequence of
.theta. and were used in a Southern analysis of E. coli genomic DNA
digested with the eight Kohara restriction map enzymes. One of the
57-mer probes hybridized to a single band in 7 of 8 bands obtained
upon Southern analysis, indicating that these 7 fragments must
overlap in the holE gene. The Kohara restriction map was searched,
and four near matches were located. Since which of these positions
could not be distinguished in the Kohara map as the true holE gene,
the small 500 bp PvuII fragment from genomic DNA was directly
cloned into M13mp18. The DNA sequence of this PvuII fragment
predicted an amino acid sequence which matched exactly to the 40
residue N-terminal sequence of .theta.. However, this was only a
partial clone of holE due to an internal PvuII site. The PvuII
fragment and one of the synthetic 57-mers were subsequently used to
probe the entire Kohara library of overlapping .lambda. phage on
one membrane which identified the location of holE within .lambda.
19H3 (No. 336 of the miniset).
[0164] The Kohara restriction map of the chromosome in the vicinity
of .lambda. 19H3 shows a close match to the fragment sizes obtained
from the Southern analysis. The overlapping fragments identify the
position of holE at 40.4 minutes on the E. coli chromosome. DNA
analysis showed two BglI sites separated by 122 bp that span the
Theta 1 57-mer probe, thus explaining the absence of a BglI
fragment in the Southern analysis in which a small fragment would
have run off the end of the gel. This small fragment would also
have been missed in the procedure used by Kohara, accounting for
the single BglI site shown on the map.
[0165] A 2.7 kb EcoRV fragment was subcloned from .lambda. 19H3
into M13mp18 and the holE gene was sequenced. The DNA predicts
.theta. is a 76 amino acid protein of 8,647 Da, slightly smaller
than the 10 kDa estimated from the mobility of .theta. in a
SDS-polyacrylamide gel. The pl of .theta. based on the amino acid
composition is 9.79, suggesting it is basic, consistent with its
ability to bind to phosphocellulose, but not to Q Sepharose. The
molar extinction coefficient of .theta. at 280 nm calculated from
its single Trp and the two Tyr residues is 8,250
M.sup.-1cm.sup.-1.
[0166] Site directed mutagenesis was performed ori the holE gene
cloned into M13mp18 to create an NdeI site at the initiator
methionine. The holE gene was excised from the site mutated
M13mp18, inserted into pUC18 (in order to use a convenient BamHI
site), then a 1 kb NdeI-BamHI fragment containing holE was ligated
directionally into the NdeI and BamHI sites of pET3c to yield the
pET-.theta. overproducing plasmid in which holE expression is
driven by T7 RNA polymerase. The pET-.theta. was introduced into
BL21(DE3) cells and upon induction of T7 RNA polymerase by IPTG,
.theta. was expressed to 63% of total cell protein. The induced
subunit was freely soluble upon cell lysis and its purification was
relatively straight-forward. Four liters of cells were lysed and
300 mg of pure .theta. was obtained in 78% overall yield after
column chromatography on Q sepharose, heparin agarose, and
phosphocellulose.
[0167] Specifically, the purification of .theta. was carried out by
utilizing four liters of BL21(DE3) cells harboring the pET-.theta.
expression plasmid were grown in 4 L of LB media containing 50
.mu.l.ml carbenicillin. Upon growth to an OD.sub.600, of 0.6, IPTG
was added to 0.4 mM and the cells were incubated at 37.degree. C.
for 2 hours further before they were harvested by centrifugation
(8.4 g wet weight) at 4.degree. C., resuspended in 15 ml of cold 50
mM Tris-HCl (pH 7.5) and 10% sucrose, and stored at -70.degree. C.
The cells were thawed and lysed by heat lysis. The cell lysate
(Fraction I, 20 ml, 880 mg) was dialyzed (all procedures were
performed at 4.degree. C.) for 2 hours against 2 L of buffer A, and
then diluted 2-fold with buffer A to a conductivity equal to 50 mM
NaCl. The lysate was then applied to a 55 ml Q sepharose fast flow
column equilibrated in buffer A. The .theta. flowed through the
column as analyzed by a Coomassie Blue stained 15% SDS
polyacrylamide gel and confirmed by the stimulation of the
.epsilon. exonuclease activity assay developed for .theta.. The Q
sepharose flow through fraction (Fraction II, 81 ml, 543 mg) was
then applied to a 50 ml column of heparin agarose (BioRad) which
was equilibrated in buffer A containing 50 mM NaCl. The flow
through fraction containing .theta. was approximately 95% pure
.theta.(Fraction III, 110, 464 mg), and was dialyzed overnight
against 2 L buffer B, then applied to a 40 ml phosphocellulose
column (P11, Whatman) equilibrated in buffer B. The column was
washed with buffer B and .theta. was eluted using a 400 ml linear
gradient of 10 mM to 200 mM sodium phosphate (pH 6.5) in buffer B.
Eighty fractions were collected and analyzed for .theta.. Fractions
42-56 were pooled (Fraction IV, 68 ml, 300 mg) and dialyzed against
2 L buffer A prior to aliquoting and storage at -70.degree. C. The
protein concentration was determined using BSA as a standard.
Concentration of pure .theta. determined by absorbance at 280 nm
using .epsilon..sub.280 at 8,250 M.sup.-1cm.sup.-1 was 90% of the
protein concentration.
30 total specific fold protein total activity purifica- % Step (mg)
units.sup.1 (units/mg) tion yield I Cell Lysate 880 2.7 .times.
10.sup.6 3.1 .times. 10.sup.3 1.0 100 II Q Sepharose 543 2.3
.times. 10.sup.6 4.2 .times. 10.sup.3 1.4 85 III Heparin 464 2.6
.times. 10.sup.6 5.6 .times. 10.sup.3 1.8 96 Agarose IV Phospho-
300 2.1 .times. 10.sup.6 7.0 .times. 10.sup.3 2.3 78 Cellulose
.sup.1One unit is defined as the increase in fmol nucleotide
released per minute relative to the same reaction with no .theta.
added (.epsilon. alone).
[0168] Throughout this description of the present invention, buffer
A was 20 mM Tris-HCl (pH 7.5), 10% glycerol, 0.5 mM EDTA, and 2 mM
DTT; Buffer B was 10 mM NaPO.sub.4 (pH 6.5), 10% glycerol, 0.5 mM
EDTA, and 2 mM DTT; and Buffer C was 25 mM Tris-HCl (pH 7.5), 10%
glycerol, 1 mM EDTA and 100 mM NaCl.
[0169] Studies of the purified cloned .theta. showed it had the
same amino terminal sequence as predicted by holE (and .theta.
within polIII' used for electroblotting), proving that the it was
indeed the purified protein encoded by the cloned gene. The
activity of .theta. (stimulation of .epsilon.) co-purified with
.theta. throughout the preparation.
[0170] In searching for activity, the subunit was tested for
polymerase activity and for endonuclease, 3'->5' exonuclease and
5'->3' exonuclease activities on ssDNA and dsDNA. However, no
such activities were observed.
[0171] Since .theta. is one of the subunits of polIII core, it was
examined for any effect it might exert on the DNA polymerase and
3'->5' exonuclease activities of .alpha. and .epsilon.. Previous
work compared the ability of .alpha..epsilon. and polIII core to
form the rapid and processive polymerase with holoenzyme accessory
proteins, but there was no significant difference between
.alpha..epsilon. and the polIII core (.alpha..epsilon..theta.)
suggesting .theta. had no role in the speed and processivity of
synthesis. With pure .theta., assays could be performed by either
adding .theta. to .alpha..epsilon. or omitting .theta.. In a
comparison of the efficiency of .alpha..epsilon. complex and
.alpha..epsilon..theta. complex in their ability to reconstitute
the rapid processive polymerase with accessory proteins, the
.alpha..epsilon. (or .alpha..epsilon..theta.) was mixed with the
.gamma. complex and .beta. subunit in the presence of ATP and phage
X174 ssDNA primed with a synthetic oligonucleotide and "coated"
with SSB. The mixture was preincubated for 6 minutes at 37.degree.
C. to allow the .gamma. complex time to transfer the .beta. ring to
DNA forming the preinitiation complex clamp and time for the
polymerase to associate with the preinitiation complex. The rapid
processive polymerase can fully replicate this template (5.4 kb)
within 12 seconds. Replication was then initiated by the addition
of dATP and [.alpha.-.sup.32P]TTP, which were omitted from the
preincubation, and the reaction was terminated after 15 seconds. In
this assay, the effect of .theta. on the amount of DNA synthesis
will be a reflection of either the speed or processivity of the
polymerase or the binding efficiency of the polymerase to the
preinitiation complex. Based on a previous comparison of
.alpha..epsilon. and core, .theta. was not expected to influence
the speed or processivity of DNA synthesis. However, in the prior
study, the relative affinity of .alpha..epsilon. and polIII core
for the preinitiation complex was not examined.
[0172] The .alpha..epsilon. and .alpha..epsilon..theta. were
titrated into this reconstitution assay and the results indicate
that .theta. had little influence in the assay. Therefore, .theta.
does not significantly increase the affinity of .alpha..epsilon.
for the preinitiation complex. These results are also consistent
with prior conclusions. The accessory protein preinitiation complex
greatly stimulates the activity of the .alpha. subunit (without
.epsilon.) in the reconstitution assay. However, this ".alpha.
holoenzyme" was half as fast as the ".alpha..epsilon. holoenzyme"
and is only processive for 1-3 kb. The ability of .theta. to
stimulate this ".alpha. holoenzyme" was tested in the absence of
.epsilon., but the .theta. subunit had no effect indicating that it
did not increase the speed or processivity of the ".alpha.
holoenzyme" either.
[0173] .theta. was next examined for an effect on the 3'->5'
exonuclease activity of .epsilon. using a synthetic "hooked" primer
template with a 3' terminal G-T mispair. A slight (3-fold), but
reproducible stimulation of .theta. on excision of the 3'
mismatched T residue by .epsilon. was observed. In the absence of
.epsilon., addition of up to 1.0 .mu.g of .theta. released no 3'
terminal nucleotide. These results are compatible with an earlier
study comparing 3' excision rates of polIII core and
.alpha..epsilon. complex in which the polIII core was approximately
3-fold faster than .alpha..epsilon.. Although a 3-fold effect is
not dramatic and may not be the true intracellular role of .theta.,
it is large enough to follow .theta. through the purification
procedure. The stimulation of .epsilon. exonuclease activity
co-purified with .theta. throughout the purification procedure add
the overall activity was recovered in high yield.
[0174] The polIII core subassembly of the holoenzyme consists of
three subunits: .theta., .alpha. (polymerase), and .epsilon.
(3'->5" exonuclease). Gel filtration was used to analyze the
ability of these individual subunits according to the present
invention to assemble into the polIII core assembly. .alpha. and
.theta. were mixed together and gel filtered; however, .theta. did
not comigrate with .alpha.. Upon mixing .epsilon. and .theta., a
stable .epsilon..theta. complex was formed. The results of these
studies are quite consistent with the activity analysis presented
above in which .theta. had no effect on the polymerase but a
noticeable effect on the activity of .epsilon..
[0175] It has been reported that a concentrated preparation of
polIII core (18 .mu.M) was dimeric containing two molecules of
polIII core which were presumed to be dimerized through interaction
between their .theta. subunits since a concentrated solution of ae
complex contained only one .alpha. and one .epsilon.. However, in
the gel filtration experiments of the present invention, the
reconstituted polIII core migrates only slightly larger than the
.alpha. subunit indicating that .theta. did not act as an agent of
polIII core dimerization.
[0176] In gel filtration experiments performed at a concentration
of 73 .mu.M .alpha. and 73 .mu.M .epsilon. in either the absence of
.theta. (.alpha..epsilon. only), the presence of a
substoichiometric amount of .theta. (molar ratio
.alpha.:.epsilon.:.theta. of 1:1:0.5), or with excess .theta.
(molar ratio 1:1:3), showed that the presence of .theta. did not
increase the aggregation state (i.e., monomer to dimer). Thus, it
may be considered that the .alpha..epsilon. complex by itself is a
dimer. However, comparison of .alpha..epsilon. and polIII core with
size standards in the gel filtration analysis show that they elute
near the 158 kDa IgG standard indicating that they are monomeric,
i.e, one of each in the complex. They have a Stokes radius of 49
.ANG. which is substantially the radius determined for the
.alpha..epsilon. complex (50 .ANG.), and similar to the 54 .ANG.
Stokes radius determined in studies of the dilute monomeric polIII
core.
[0177] To increase confidence in the aggregation state of these
reconstituted complexes, the study of the .alpha..epsilon. complex
and reconstituted polIII core was extended to an analysis of their
sedimentation behavior in glycerol gradients using the same
concentration and ratio of subunits as in the gel studies. Again
the .alpha..epsilon. and .alpha..epsilon..theta. essentially
co-sedimented regardless of whether .theta. was present. The
.alpha..epsilon. complex and polIII core each sedimented with an S
value close to that of the 150 kDa IgG size standard further
indicating they are monomeric subassemblies.
[0178] The native molecular weights of .theta., .epsilon. and of
the .epsilon..tau. complex were also determined using gel
filtration and glycerol gradient sedimentation. The .theta. and
.epsilon. subunits were first analyzed separately: .theta., by
itself, elutes after myoglobin which is 17.5 kDa, indicating
.theta. is a monomer (8.6 kDa) rather than a dimer of 17.2 kDa;
.epsilon. migrated just after an ovalbumin standard (43.5 kDa)
consistent with .epsilon. as a 28,5 monomer rather than a 57 kDa
dimer.
[0179] To asses the native molecular masses of .theta., .epsilon.
and the .epsilon..theta. complex, the analysis was extended to
sedimentation in glycerol gradients. The Stokes radius and S values
of .theta., .epsilon. and .epsilon..theta. complex were determined
by comparison to protein standards and their observed mass was
calculated. The observed masses of .theta., .epsilon. and
.epsilon..theta. are 11.6 kDa, 32.7 kDa and 35.5 kDa, respectively,
values most consistent with .theta. as a 8.6 kDa monomer, .epsilon.
as a 28.5 kDa monomer, and the .epsilon..theta. complex having a
composition of .epsilon..sub.1.theta..sub.1 (37.1 kDa);
densitometric analysis of the .epsilon..theta. complex yielded a
molar ration of 1 mol of .epsilon. to 0.8 mol .theta., consistent
with this composition.
[0180] The fourth subunit according to the present invention, that
of .sub..psi., was also identified, purified, cloned and sequenced.
N-terminal analysis of the .sub..psi. peptide yielded a protein
which, when translated to its genetic sequence was found to be
identical to a portion of a much larger sequence described by
Yoshikawa [see Mol. Gen. Genet. 209:481 (1987)]. However,
Yoshikawa's description was for a rimI sequence from E. coli
responsible for encoding an enzyme catalyzing acetylation of the
N-terminal portion of ribosomal protein S-18; his upstream
sequencing from this gene's reading frame was purely accidental and
he does not indicate any appreciation of the gene as a coding
sequence for the .sub..psi. peptide.
[0181] The amino acid sequence obtained from the .sub..psi. peptide
is:
31 Met Thr Ser Arg Arg Asp Trp Gln Leu Gln Gln Leu 5 10 Gly Ile Thr
Gln Trp Ser Leu Arg Arg Pro Gly Ala 15 20 Leu Gln Gly Glu Ile Ala
Ile Ala Ile Pro Ala His 25 30 35 Val Arg Leu Val Met Val Ala Asn
Asp Leu Pro Ala 40 45 Leu Thr Asp Pro Leu Val Ser Asp Val Leu arg
Ala 50 55 60 Leu Thr Val Ser Pro Asp Gln Val Leu Gln Leu Thr 65 70
Pro Glu Lys Ile Ala Met Leu Pro Gln Gly Ser His 75 80 Cys Asn Ser
Trp Arg Leu Gly Thr Asp Glu Pro Leu 85 90 95 Ser Leu Glu Gly Ala
Gln Val Ala Ser Pro Ala Leu 100 105 Thr Asp Leu Arg Ala Asn Pro Thr
Ala Arg Ala Ala 110 115 120 Leu Trp Gln Gln Ile Cys Thr Tyr Glu His
Asp Phe 125 130 Phe Pro Gly Asn Asp 135 137
[0182] Using the information above, the sequence was translated
into the genomic structure which is:
32 ATG ACA TCC CGA CGA GAC TGG CAG TTA CAG CAA CTG GGC 39 ATT ACC
CAG TGG TCG CTG CGT CGC CCT GGC GCG TTG CAG 78 GGC GAG ATT GCC ATT
GCG ATC CCG GCA CAC GTC CGT CTG 117 GTG ATG GTG GCA AAC GAT CTT CCC
GCC CTG ACT GAT CCT 156 TTA GTG AGC GAT GTT CTG CGC GCA TTA ACC GTC
AGC CCC 195 GAC CAG GTG CTG CAA CTG ACG CCA GAA AAA ATC GCG ATG 234
CTG CCG CAA GGC AGT CAC TGC AAC AGT TGG CGG TTG GGT 273 ACT GAC GAA
CCG CTA TCA CTG GAA GGC GCT CAG GTG GCA 312 TCA CCG GCG CTC ACC GAT
TTA CGG GCA AAC CCA ACG GCA 351 CGC GCC GCG TTA TGG CAA CAA ATT TGC
ACA TAT GAA CAC 390 GAT TTC TTC CCT GGA AAC GAC 411
[0183] In addition to the normal sequence for the genomic material,
the gene also contains an internal NdeI site.
[0184] The sequence above is preceded by an upstream sequence
containing two underlined RNA polymerase promoter signals (TTGGCG
and TATATT), and a Shine Dalgarno (AGGAG) sequence. The complete
upstream sequence is:
33 GGCGATTATA GCCATATGTT GGCGCGGTA CGACGAATTT GCTATATTTG 50
CGCCCCTGAC AACAGGAGCG ATTCGCT 77.
[0185] In addition, the open reading frame is followed by a
downstream sequence beginning with a stop codon:
34 TGA TTTACCGGCA GCTTACCACA TTGAACAACG CGCCCACGCC TTTCCGTGGA 53
GTGAAAAAAC GTTTGCCAGC AACCAGGGCG AGCGTTATCT CAACTTTCAG 103.
[0186] The .sub..psi. gene was then produced by PCR using E. coli
genomic DNA and the following (5'->3') primers:
35 primer 1 (Psi-N): GATTCCATAT GACATCCCGA CGAGACT; 27 and primer 2
(Psi-C): GACTGGATCC CTGCAGGCCG GTGAATGAGT 30
[0187] As can be seen, primer 1 contains a NdeI site, and primer 2
contains a BamHI site which have been underlined above.
[0188] The PCR-produced DNA was used to clone the .sub..psi. gene
into pET-3c expression plasmid using a two-step cloning procedure
necessitated by the internal NdeI site in the nucleic acid
sequence. Briefly this procedure involved cutting the PCR product
with NdeI restriction enzyme into two portions of 379 (NdeI to
NdeI) and 543 (from NdeI to BamHI) bp. The 543 bp portion was
ligated into plasmid pET-3c (4638 bp) to form an intermediate
pET-3ca (5217 bp). The pET-3ca was then linearized, and the 379 bp
portion inserted to form the desired pET-3c plasmid containing the
complete PCR product insert.
[0189] The overexpression vector containing the complete insert was
then inserted into E. coli, and induced with IPTG as described
herein, and overexpression (an increase to over 20% of total
bacterial protein) of the .sub..psi. protein was seen.
[0190] The .sub..psi. protein was purified by first dissolving the
cell membrane debris in 6 M urea followed by passing the resulting
solutions through a hydroxylapetite column, which had been
equilibrate previously with a 6 M urea buffer (180 g urea, 12.5 ml
1 M Tris at pH 7.5, 0.5 ml of 0.5 M EDTA, and 1 ml of 1 M DTT),
wherein the .sub..psi. peptide will flow through while almost
everything else in solution will be held within the column. The The
.sub..psi. peptide outflow of the hydroxylapetite column was then
bound to a DEAE columnn, rinsed with buffer, and eluted with a
gradient of NaCl. Fractions containing the .sub..psi. peptide were
pooled, dialyzed twice against 1 liter of buffer, and loaded onto a
hexylamine column for final purification. Fractions from the
hexylamine column containing the .sub..psi. peptide were eluted
with a NaCl gradient (0.0 to 0.5 M), pooled and saved as pure
.sub..psi. subunit peptide.
[0191] Studies were also conducted to determine that the .sub..psi.
gene according to the present invention encodes .sub..psi. subunit
peptide. These studied determined that the N-terminal analysis of
native .sub..psi. peptide is predicted by the .sub..psi. gene
sequence according to the present invention; native .sub..psi.
peptide was obtained and digested with trypsin and a few of the
resulting peptides synthesized--the sequenced peptides were encoded
by the gene sequence according to the present invention; the
cloned/overproduced/pure .sub..psi. peptide made in accordance with
the present invention comigrated with the .sub..psi. subunit
peptide within the naturally occurring holoenzyme; and the
.sub..psi. peptide produced from the sequence according to the
present invention formed a .gamma..sub..chi..psi. complex when
mixed with .gamma. and .sub..chi. as would occur with natural
components.
[0192] The .gamma..sub..chi..psi. complex was purified [see J. Bio
Chem. 265:1179 (1990)] from 1.3 kg of the .gamma./.tau.
overproducing strain (HB101 (pNT203, pSK100). The .sub..psi.
subunit was prepared from .gamma. and .sub..chi. by electrophoresis
in a 15% SDS-polyacrylamide gel, then .sub..psi. was electroblotted
onto PVDF membrane for N-terminal sequencing (220 pmol), and onto
nitrocellulose membrane for tryptic digestion (300 pmol) followed
by sequence analysis of tryptic peptides. Proteins were visualized
by Ponceau S stain. The N-terminal analysis was determined to be:
NH.sub.2TSRRDDQLQQLGIT. Two internal tryptic peptides were
determined to be:
36 .psi.-1: NH.sub.2-Leu Gly Thr Asp Glu Pro Leu Ser Leu Glu Glu 5
10 Ala Gln Val Ala Ser Pro; and 15 .psi.-2: NH.sub.2-Ala Ala Leu
Trp Gln Gln Ile Cys Thr Tyr Glu 5 10 His Asp Phe Phe Pro Ala 15
[0193] A 3.2 kb PstI/BamHI (DNA modification enzymes, New Endland
Biolabs) fragment containing holD was excised from .lambda.5CI and
ligated directionally into the polylinker of Blue Script
(Stratagene). The 1.5 kb AccI fragment (one site is in the vector
and one is in the insert) containing holD was excised, the ends
filled in using Klenow polymerase, then ligated into pUC18 (cut
with AccI and the end filled with Klenow) to yield pUC-.sub..psi..
Both strands of DNA were sequenced by the chain termination method
of Sanger using the sequenase kit [.alpha.-.sup.32P] dATP
(radiochemicals, New England Nuclear), and synthetic DNA 17-mer
(Oligos etc. inc.).
[0194] A 922 bp fragment was amplified from genomic DNA (strain
C600) using Vent DNA polymerase and two synthetic primers, an
upstream 32-mer (CAACAGGAGCGATTCCATATGA-CATCCCGACG), and a
downstream 30-mer (GATTCGGATCCCTGCAGGCCG-GTGAATGAGT). The first two
nucleotides in the NdeI site (underlined) of the upstream 32-mer
and the first 11 nucleotides of the downstream 30-mer (including
the underlined BamHI sequence) are not complimentary to the genomic
DNA. Amplification was performed using a thermocycler in a volume
of 100 .mu.l containing 1 ng genomic DNA, 1 .mu.M each primer, and
2.5 units of Vent polymerase in 10 mM Tris-HCl (pH 8.3), 2 mM
MgSO.sub.4, 200 .mu.M each dATP, dCTP, dGTP and TTP. Twenty five
cycles were performed: 1 minute at 94.degree. C., 1 minute at
42.degree. C., 2 minutes at 72.degree. C. Amplified DNA was phenol
extracted, ethanol precipitated, then digested with 50 units NdeI
in 100 .mu.l 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM DTT
and 50 mM potassium acetate (final pH 7.9), overnight at 37.degree.
C. After confirming the NdeI digestion by agarose gel, 50 units of
BamHI was added and digestion was continued for 2 hours. The
NdeI/NdeI fragment,which contained most of the holD gene, and the
NdeI/BamHI fragment were separated in an agarose gel,
electroeluted, phenol/chloroform extracted, ethanol precipitated
and dissolved in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. The holD gene
was cloned into pET3c in two steps. First the NdeI/BamI fragment
encoding the C-terminus of .sub..psi. was ligated into pET3c
digested with NdeI and BamHI to generate pET.psi.c-ter' (linearized
with NdeI and dephosphorylated) to yield the pET-.sub..psi.
overproducer. DNA sequencing of the pET-.sub..psi. confirmed the
correct orientation of the NdeI/NdeI fragment.
[0195] The 25 .mu.l assay contained 72 ng M13mp18 ssDNA (0.03 pmol
as circles) primed with a synthetic DNA 30-mer, 0.98 .mu.g SSB
(13.6 pmol as tetramer), 82 ng .alpha..epsilon. (0.52 pmol), and 33
ng .beta. (0.29 pmol as dimer) in 20 mM Tris-HCl (pH 7.5), 8 mM
MgCl.sub.2, 40 mM NaCl, 5 mM DTT, 0.1 mM EDTA, 40 .mu.g/ml BSA, 0.5
mM ATP, and 60 .mu.M each dCTP and dGTP. Addition of .sub..chi.,
.sub..psi. and .gamma..delta..delta.' complex to the assay was as
follows. The .gamma..delta..delta.' complex, .sub..chi. and
.sub..psi. (.sub..psi. was initially in 4 M urea) subunits were
preincubated before addition to the assay for 30 minutes at
4.degree. C. at concentrations of 2.4 .mu.g/ml
.gamma..delta..delta.' complex (14.2 nM), 0-0.75 .mu.g/ml
.sub..chi. (45 nM), and 0-0.75 .mu.g/ml .sub..psi. (0-48 nM) in 25
mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM EDTA, 50 .mu.g/ml BSA, 20%
glycerol (buffer B) (the concentration of urea in the preincubation
was 8.5 mM or less). One-half .mu.l of this protein mixture was
added to the assay (urea was 0.17 mM or less in the assay after
addition of .sub..psi.) then the assay was shifted to 37.degree. C.
for 5 minutes to allow polymerase assembly before initiating DNA
synthesis upon addition of dATP and [.alpha.-.sup.32P] dTTP to 60
.mu.M and 20 .mu.M, respectively. After 20 seconds, DNA synthesis
was quenched and quantitated as described in the accompanying
report. Assays to quantitate .theta. in purification were performed
likewise except the protein preincubation contained 2.4 .mu.g/ml
.gamma..delta..delta.' (14.2 nM), 0.75 .mu.g/ml .sub..chi. (45 nM)
and up to 0.25 .mu.g/ml of protein fraction containing .theta..
After the 30 minute preincubation, 0.5 .mu.l was added to the assay
reaction. The SSB, .alpha., .epsilon., .beta., .gamma., and .tau.
subunits used in these studies were purified, and the .sub..chi.,
.delta. and .delta.' subunits were prepared from their respective
overproducing strains. Concentrations of .beta., .delta., .delta.',
.sub..chi. and .sub..psi. were determined from their absorbance at
280 nm using their molar extinction coefficients: .beta., 17,900
M.sup.-1cm.sup.-1; .delta., 46,137 M.sup.-1cm-.sup.1; .delta.',
60,136 M.sup.-1cm.sup.-1;.sub..chi., 29,160 M.sup.-1cm.sup.-1; and
.sub..psi., 24,040 M.sup.-1cm.sup.-1.
[0196] The .mu.l assay contained 140 ng M13mp18 ssDNA in 25 mM
Tris-HCl (pH 7.5), and 8 mM MgCl.sub.2, 50 .mu.M
[.gamma..sup.32-P]ATP, 5.45 pmol .gamma. or .tau. (as dimers), 10.9
pmol .sub..chi. and/ or .sub..psi. (as monomers) (Unless indicated
otherwise) and 1.4 .mu.g SSB (19.4 pmol as tetramer) ( when
present). Mixtures of proteins (.sub..psi. was initially 2 mg/ml
(0.13 mM) in 4 M urea) were preincubated 30 minutes on ice at 3.8
.mu.M of each subunit (as monomer) in 30 .mu.l of 25 mM Tris-HCl
(pH 7.5), 0.5 mM EDTA, 20% glycerol (0.1 M urea final
concentration) before addition to the assay (15 mM urea final
concentration). Assays were incubated at 37.degree. C. for 60
minutes 5 minutes for assays containing .tau.) then quenched upon
spotting 0.5 .mu.l on polyethyleneimine thin layer plates (Brinkman
Instruments Co.). After chromatography in 0.5 M LiCl, 1 M formic
acid, the free phosphate at the solvent front and ATP remaining
hear the origin were quantitated by liquid scintillation,
[0197] Samples of .sub..psi. (45 .mu.g, 3 nmol as monomer
(initially in 4 M urea)), or a mixture of .sub..psi. (45 .mu.g, 3
nmol as monomer) with either .gamma. (65 .mu.g, 0.7 nmol as dimer)
or .tau. (98 .mu.g, 0.7 nmol as dimer) were incubated in 200 .mu.l
25 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 1 mM EDTA, 10% glycerol (0.5 M
urea was present after addition of .sub..psi.) for 30 minutes at
15.degree. C. The 200 .mu.l sample was injected onto a Pharmacia HR
10/30 gel filtration column of either Superdex 75 or Superose 12 at
a flow rate of 0.35 ml/min in 25 mM Tris-HCl (pH 7.5), 0.1 M NaCl,
1 mM EDTA, 10% glycerol. After the first 5.6 ml, fractions of 170
.mu.l were collected and analyzed in a 15% SDS polyacrylamide gel
and the value of Kav was calculated.
[0198] A sample of .sub..psi. (45 .mu.g, 3 nmol as monomer,
initially in 4 M urea) in 200 .mu.l 25 mM Tris-HCl (ph 7.5), 0.1 M
NaCl, 1 mM EDTA, 5% glycerol (0.5 M urea final concentration after
addition of .sub..psi.) was layered onto a 12.3 ml gradient of
10%-30% glycerol in 25 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 1 mM EDTA.
Protein standards in 200 .mu.l of the same buffer were loaded in
another tube and the gradients were centrifuged at 270,000.times.g
for 44 hours at 4.degree. C. Fractions of 150 .mu.l were collected
and analyzed in a 15% SDS polyacrylamide gel stained with Coomassie
Blue.
[0199] The .gamma..delta..delta.' complex was formed upon
incubation of 60 .mu.g .delta. (1.55 nmol as monomer) and 60 .mu.g
.delta.' (1.62 nmol as monomer) with an excess of .gamma. (600
.mu.g, 6.4 nmol as dimer) for 30 minutes at 15.degree. C. In 1 ml
of 25 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM EDTA, 20% glycerol
(buffer A). The mixture was chromatographed on a 1 ml HR 5/5 MonoQ
column, and eluted with 30 ml linear gradient of 0 M-0.4 M NaCl in
buffer A. The .gamma..delta..delta.' complex eluted at an unique
position, after the elution of free .delta.', .delta. and .gamma.
(in that order) and was well resolved from the excess .gamma.. The
pure .gamma..delta..delta.' complex was dialyzed against buffer A
to remove salt. Protein concentration was determined using BSA as a
standard. Molarity of .gamma..delta..delta.' was calculated from
protein concentration assuming the 170 kDa mass of a complex with
subunit composition .gamma..sub.2.delta..sub.1.delta..sub.1, the
composition expected from stoichiomhetry of subunits in the .gamma.
complex.
[0200] The .gamma..sub..chi..psi. complex, was prepared from 1.3 kg
of E. coli and the .psi. subunit was resolved from the .gamma. and
.sub..chi. subunits in a SDS-polyacrylamide gel, then
electroblotted onto PVDF membrane for analysis of the amino acid
sequence of the amino terminus of .sub..psi.. The .sub..psi. was
also electroblotted onto nitrocellulose followed by tryptic
digestion, HPLC purification of peptides and sequence analysis of
two tryptic peptides. Search of the GenBank for DNA sequences
encoding these peptides identified a sequence which was published
in a study of the rimI gene [see Mol Gen Genet 209:481 (1987)]. In
order to define the operon structure of this DNA, the DNA upstream
of rimI was sequenced. All three peptide sequences of .sub..psi.
were in one reading frame located immediately upstream of rimI at
99.3 minutes on the E. coli chromosome which putatively encodes
.sub..psi. and referred to as holD.
[0201] The promoter for holD underlined in the sequence has been
identified previously as the promoter for the rimI gene, encoding
the acetylase of ribosomal protein S18, which initiates 29
nucleotides inside of holD. Hence, holD is in an operon of rimI.
Production of .sub..psi. was inefficient relative to
.rho..eta..mu.l protein as judged by the maxicell technique which
detected rimI protein but not .sub..psi.. The promoter measured by
Northern analysis was strong [see Mol Gen Genet 209:481 (1987)] and
the Shine-Dalgarno sequence is a good match to the consensus
sequence, as is the spacing from the ATG needed for sufficient
translation. Although the cellular abundance of .sub..psi. is not
known, if one assumes all the .sub..psi. sequestered within the
holoenzyme, then it is present in very small amounts, there being
only 10-20 copies of the holoenzyme in a cell. Perhaps the 3-11
fold more frequent use of some rare codons may contribute to
inefficient translation (Leu (UUA), Ser (UCA and AGU), Pro (CCU and
CCC), Thr (ACA), Arg (CGA and CGG)).
[0202] The open reading frame of holD encodes a 137 amino acid
protein of 15,174 Da. Amino terminal analysis of the .sub..psi.
protein within the .gamma..chi..psi. complex showed it was missing
the initiating methionine. The molar extinction coefficient of
.sub..psi. calculated from its 4 Trp and 1 Tyr is 24,040
M.sup.-1cm.sup.-1. There is a potential for a leucine zipper at
amino acid residues 25-53, although three prolines fall within the
possible leucine zipper. There is also a helix-turn-helix motif
(A/GX.sub.3GX.sub.5I/V) at G.sub.22G.sub.26I.sub.3- 3, but again
prolines may preclude helix formation. There is no apparent
nucleotide binding site or zinc finger motif.
[0203] The polymerase chain reaction was used to amplify holD from
genomic DNA. The synthetic DNA oligonucleotides used as primers
were designed such that an NdeI site was formed at the initiating
ATG of holD and a BamHI site was formed downstream of holD. The
amplified holD gene was inserted into the NdeI/BamHI sites of pET3c
in two steps to yield pET-.sub..psi. in which holD is under control
of a strong T7 promoter and is in a favorable context for
translation. The sequence of holD in pET-.sub..psi. was found to be
identical to that depicted in the sequence, and transformation into
BL21(DE)plysS cells and subsequent induction of T7 RNA polymerase
with IPTG, the .sub..psi. protein was expressed to approximately
20% of total cell protein.
[0204] The .sub..psi. protein was completely insoluble and resisted
attempts to obtain even detectable amounts of soluble .sub..psi.
(lower temperature during induction, shorter induction time, and
extraction of the cell lysate with 1 M NaCl were tested); it was
necessary to resort to solubilization of the induced cell debris
with 6 M urea followed by column chromatography in urea. The
.sub..psi. was approximately 40% of total protein in the
solubilized cell debris and was purified to apparent homogeneity
upon flowing it through hydroxyapatite, followed by column
chromatography on DEAE sepharose and heparin agarose. By this
procedure, 22 mg of pure .sub..psi. was obtained from 1 liter of
cell culture in 61% yield. The pure .sub..psi. remained in solution
upon complete removal of the urea by dialysis as described in
greater detail below.
[0205] Four liters of E. coli cells (BL21(DE3)plysS) harboring the
pET-.sub..psi. plasmid were grown at 37.degree. C. In LB media
supplemented with 1% glucose, 10 .mu.g/ml thiamin, 50 .mu.g/ml
thymine, 100 .mu.g/ml ampicillin and 30 .mu.g/ml chloramphenicol.
Upon reaching an OD of 1.0, IPTG was added to 0.4 mM and after an
additional 2 hours of growth at 37.degree. C., the cells were
harvested by centrifugation (20 g wet weight), resuspended in 20 ml
of 50 mM Tris-HCl (pH 7.5) and 10% sucrose (Tris-sucrose) and
frozen at -70.degree. C. The cells lysed upon thawing (due to
lysozyme formed by the plysS plasmid), and the following components
were added on ice to pack the DNA and precipitate the cell debris:
69 ml Tris-sucrose, 1.2 ml unneutralized 2 M Tris base, 0.2 ml 1 M
DTT, and 9 ml of heat lysis buffer (0.3 M spermidine, 1 M NaCl, 50
mM Tris-HCl (pH 7.5), 10% sucrose). After 30 minutes incubation on
ice, the suspension was centrifuged in a GSA rotor at 10,000 rpm
for 1 hour at 4.degree. C. The cell debris pellet was resuspended
in 50 ml buffer B using a dounce homogenizer (B pestle), then
sonicated until the viscosity was greatly reduced (approximately 2
minutes total in 15 second intervals) and centrifuged in two tubes
at 18,000 rpm in a SS34 rotor for 30 minutes at 4.degree. C. The
pellet was resuspended in 50 ml buffer B containing 1 M NaCl using
the dounce homogenizer, then pelleted again. This was repeated, and
followed again by homogenizing the pellet once again in 50 ml
buffer B and pelleted as was done initially. The following
procedures were at 4.degree. C. using only one-fourth of the pellet
(equivalent to 1 liter of the cell culture). The assay for
.sub..psi. is described above, and column fractions were analyzed
in 15% SDS polyacrylamide gels to directly visualized the
.sub..psi. protein and aid the exclusion of contaminants during the
pooling of column fractions. The pellet was solubilized in 25 ml
buffer A containing freshly deionized 6 M urea. The solubilized
pellet fraction (fraction I, 85 mg, 22 ml) was passed over a 10 ml
column of hydroxyapatite and equilibrated in buffer A plus 6 M
urea. The .sub..psi. quantitatively flowed through the
hydroxyapatite column giving substantial purification. The protein
which flowed through the hydroxyapatite column was immediately
loaded onto a 10 ml column of DEAE sephacel, equilibrated in buffer
A containing 6 M freshly deionized urea, and eluted with a 100 ml
gradient of 0-0.5 M NaCl in buffer A containing 6 M freshly
deionized urea over a period of 4 hours. Fractions of 1.25 ml were
collected and analyzed for .sub..psi. as described. Fractions were
pooled and dialyzed overnight against 2 liters of buffer A
containing 3 M freshly deionized urea and then loaded onto a 10 ml
column of hexylamine sepharose. The hexylamine column was eluted
with a 200 ml gradient of 0 M-0.5 M NaCl in buffer B containing 3 M
freshly deionized urea over a period of 4 hours. Eighty fractions
were collected (2.5 ml each) and were analyzed for A, then
fractions containing .sub..psi. were pooled (fraction IV, 21.6 mg)
and urea was removed by extensive dialysis against 25 mM Tris-HCl
(pH 7.5), 0.1 M NaCl, 0.5 MM EDTA (3 changes of 2 liters each).
Protein concentration was determined using BSA as a standard,
except at the last step in which a more accurate assessment of
concentration was performed by absorbance using the value
.epsilon..sub.280 equal 24,040 M.sup.-1cm.sup.-1 calculated from
the sequence of holD. After the absorbance measurement, DTT was
added back to 5 mM and the TV was aliquoted and stored at
-70.degree. C.
37 total specific fold protein total activity purifica- % Fraction
(mg) units.sup.1 (units/mg) tion yield I Solubilized 85.0 104.7
.times. 10.sup.7 12.0 .times. 10.sup.6 1.0 100 pellet II Hydroxy-
42.5 95.9 .times. 10.sup.7 22.6 .times. 10.sup.6 1.8 92 lapatite
III DEAE 30.6 89.7 .times. 10.sup.7 29.3 .times. 10.sup.6 2.4 86
Sepharose IV Hexlyamine 21.6 63.9 .times. 10.sup.7 29.6 .times.
10.sup.6 2.4 61 Sepharose .sup.1One unit is defined as pmol of
nucleotide incorporated in one minute over and above the pmol
incorporated in the assay in the absence of added .psi.
[0206] The pure y protein comigrated with the y subunit of polIII*
(holoenzyme lacking only .beta.) in a 15% SDS-polyacrylamide gel.
Analysis of the N-terminal sequence of the pure cloned .sub..psi.
matched that of the holD sequence and the sequence of the natural
.sub..psi. from within the .gamma..sub..chi..psi. complex
indicating that the purified protein encoded by the gene had been
cloned.
[0207] The pure .sub..psi. appeared fully soluble in the absence of
urea. However, a 2 mg/ml solution of .sub..psi. which appeared
clear, and could not be sedimented in a table top centrifuge, still
behaved as an aggregate in a gel filtration column. Therefore, even
though .sub..psi. appeared soluble it was still an aggregate. The
aggregated .sub..psi. had only weak activity in the replication
assay and was inefficient in binding to other proteins in physical
studies. Therefore before using .sub..psi. in assays or in physical
binding experiments, urea was added to a concentration of 4 M to
disaggregate .sub..psi.. Once disaggregated, the urea could be
quickly removed by gel filtration and .sub..psi. behaved well
during filtration in the absence of urea in the column buffer.
However, upon standing a full day at high concentration (>1
mg/ml) in the absence of urea, it would aggregate again. .sub..psi.
would work in urea provided the preparation was sufficiently
concentrated 2 mg/ml) such that it could be diluted at least 8-fold
(to 0.5 M urea) for protein-protein interaction studies, 300-fold
for AT Pase assays, and 30,000-fold for replication assays. In 0.5
M urea, the .sub..psi. bound to .gamma. and .tau., and also to the
.sub..chi. subunit. .sub..psi. treated in this manner was also
functional in stoichiometric amounts with other proteins in
replication and ATPase assays.
[0208] In a previous study, a .gamma..sub..chi..psi. complex was
purified by resolving the .delta. and .delta.' subunits out of the
.gamma. complex leaving only a complex of .gamma..sub..chi..psi..
Compared to .gamma., this .gamma..sub..chi..psi. complex was
approximately 3-fold more active in reconstituting the processive
polymerase with .delta., .beta., and .alpha..epsilon. at elevated
salt concentration. The simplest explanation for this result is
that at elevated salt, .gamma..sub..chi..psi..delta. is more active
than .gamma..delta. in assembling the .beta. ring around primed
DNA.
[0209] The present invention indicates that a mixture of the
.gamma., .delta. and .delta.' subunits formed a stabile (gel
filterable) .gamma..delta..delta.' complex when the
.alpha..epsilon. complex and .beta. subunit were incubated with the
.gamma..delta..delta.' complex (with or without .sub..chi. and/or
.sub..psi.) in a reaction containing SSB "coated" M13mp18 ssDNA
primed with a synthetic DNA oligonucleotide and in the presence of
40 mM added NaCl, and the reaction was incubated at 37.degree. C.
for 5 minutes to allow the accessory proteins time to assemble the
preinitiation complex clamp and for the .alpha..epsilon. to bind
the preinitiation complex (the preinitiation complex is known to
consist of a .beta. dimer ring clamped onto the DNA). Replication
of the circular DNA was then initiated upon addition of the
remaining dNTPs and was quenched after 20 seconds, sufficient time
for the rapid and processive holoenzyme to complete the circle.
[0210] The results indicated that as .sub..psi. is titrated into
the assay the replication activity increased approximately 3.5-fold
and plateaued at approximately 1 mol .sub..psi. (as monomer) per
mol .gamma..delta..delta.' complex. .sub..psi. (without .sub..chi.)
stimulates .gamma..delta..delta.' and .sub..chi. does not stimulate
the reaction, but the presence of both .sub..chi. and .sub..psi.
yields the most synthesis as though .sub..chi. does exert an
influence on the assay but only when .sub..psi. is also
present.
[0211] Previously .gamma. was observed to contain a low level of
DNA dependent ATPase activity (0.1 mol ATP hydrolyzed/mol
.gamma./minute) compared to the ATPase of the .gamma. complex (6.8
mol ATP/mol .gamma. complex/minute). The .gamma..sub..chi..psi.
complex resolved out of the .gamma. complex appeared to contain
approximately 3-4 fold more DNA dependent ATPase activity than
.gamma. suggesting that .sub..chi. and/or .sub..psi. stimulated the
ATPase activity of .gamma., or that there was an ATPase activity
inherent within .sub..chi. and/or .sub..psi.. Now that the holC and
holD genes have made available pure .sub..chi. and .sub..psi. in
quantity, they have been studied studied them for ATPase activity
and for their effect on the DNA dependent ATPase activity of
.gamma..
[0212] As part of these studies of ATPase activity, all possible
combinations of .sub..chi., .sub..psi. and .sub..psi. have been
tested. These assays were performed in the presence of M13mp18
ssDNA, one of the best DNA effectors in the previous study of the
.gamma. complex ATPase activity. The results showed that .sub..psi.
alone, .sub..chi. alone, and a mixture of .sub..chi. and .sub..psi.
had no detectable ATPase activity and therefore neither .sub..psi.
nor .sub..chi. would appear to have an intrinsic ATPase activity,
although on the basis of negative evidence we can not rule out the
possibility of a cryptic ATPase; the .gamma. subunit has a weak
ATPase activity. The .sub..chi. subunit has no effect on the ATPase
activity of .gamma.. However, addition of .sub..psi. to .gamma.
stimulated the ATPase activity of .gamma. approximately 3-fold.
Titration of .sub..psi. into the ATPase assay showed .sub..psi.
saturated the ATPase assay at approximately 2 mol .sub..psi. (as
monomer) to 1 mol .gamma. (as dimer). Addition of the .sub..chi.
subunit to the .gamma..sub..psi. mixture resulted in a further 30%
increase in ATPase activity.
[0213] In the presence of SSB which "coats" the ssDNA, the ATPase
activity of .gamma., .gamma..sub..psi. and .gamma..sub..chi..psi.
were all greatly reduced (50-fold). However, of the remaining
activity, the .gamma..sub..chi..psi. complex was 4-fold more active
than .gamma..sub..psi. showing that .sub..chi. significantly
stimulates the .gamma..sub..psi. ATPase which the DNA is "coated"
with SSB.
[0214] The ATPase assay of .sub..psi. and .sub..chi. was extended
to the DNA dependent ATPase activity of the .tau. subunit. The
.tau. and .gamma. subunits are encoded by the same gene and, as a
result, .tau. contains the .gamma. sequence plus approximately
another 24 kDa of protein which is responsible for both the ability
of .tau. to bind DNA and to bind the polymerase subunit, .alpha..
In addition, .tau. has a much greater DNA dependent ATPase activity
than .gamma., approximately 6-10 mol ATP hydrolyzed/mol
.tau./minute for a 60-fold greater activity of .tau. relative to
.gamma..
[0215] Neither .sub..psi., .sub..chi., or a mixture of .sub..chi.
and .sub..psi. had a significant influence on the ATPase activity
of .tau.. "Coating" the ssDNA with SSB reduced the ATPase activity
of .tau. 20-fold, and now the .sub..chi. and .sub..psi. subunits
stimulated the .tau. ATPase 10-fold to bring its activity back to
about half of its value in the absence of SSB. In this case, with
SSB present, the .sub..psi. stimulated .tau. approximately 3-fold,
and .sub..chi., which had no effect on .tau. without .sub..psi.,
stimulated the .gamma..sub..psi. ATPase another 3-fold.
[0216] To gain a better understanding of the .sub..psi. molecule
the present invention studied the hydrodynamic properties of
.sub..psi. in gel filtration and glycerol gradient sedimentation to
determine whether .sub..psi. is a monomer or a dimer (or larger).
The Stokes radius of .sub..psi. was 19 .ANG. upon comparing its
position of elution from a gel filtration column with that of
protein standard of know Stokes radius. The .sub..psi. eluted in
the same position as myoglobin (17.5 kDa) indicating .sub..psi. is
a 15 kDa monomer rather than a dimer of 30 kDa. The .sub..psi.
protein sedimented with an S value of 1.95 relative to several
protein standards, and was slightly slower than myoglobin which is
consistent with .sub..psi. as a monomer. If a protein has an
asymmetric shape, its migration will not reflect its true weight in
either of these techniques. However the effect of asymmetric shape
has opposite effects in these techniques and can be eliminated by
the fact that the shape factor cancels when the S value and Stokes
radius are both combined in one mass equation. This calculation
results in a native molecular mass for .sub..psi. of 15.76 kDa,
close to the 15 kDa monomeric mass of .sub..psi. calculated from
its gene sequence. Hence .sub..psi. behaves as a monomer under
these conditions. The frictional coefficient of .sub..psi.
calculated from its Stokes radius and native mass is 1.13, slightly
greater than 1.0 which indicates some asymmetry in the shape of
.sub..psi..
[0217] Although the initial use of 4 M urea would have monomerized
.sub..psi. if it were a native dimer, the .sub..psi. preparation
was diluted such that the concentration of urea was 0.5 M before it
was applied to either the gel filtration column or the glycerol
gradient, and the buffer used in the column and in the gradient
contained no urea. Of course, one should still be concerned that
0.5 M urea is high enough to disaggregate a dimer of .sub..psi. and
that the dimer hasn't time to reassociate during filtration and
sedimentation. Yet under these very conditions it was found that
.sub..psi. forms a protein-protein complex with .gamma., with .tau.
and also with .sub..chi.. Therefore it seems likely that if
.sub..psi. were naturally a dimer, that the dimer could have
reformed under these same conditions under which .sub..psi. can
bind all these other subunits. Further, a monomeric nature of
.sub..psi. is not unusual as most subunits of the holoenzyme are
monomers when isolated (.alpha., .epsilon., .theta., .sub..chi.,
.delta., .delta.', (only .gamma., .tau. and .beta. are dimers).
[0218] A complex of .gamma..sub..chi..psi. can be purified from
cells indicating that .sub..psi. or .sub..chi. (or both) must
directly interact with .gamma..
[0219] Gel filtration of a mixture of .gamma. with a 4-fold molar
excess of .sub..psi. showed that .sub..psi. coeluted with the
.gamma. subunit followed later by the elution of the rest of the
excess .sub..psi.. Hence, the .sub..psi. subunit does in fact bind
directly to .gamma..
[0220] The fifth subunit according to the present invention,
.sub..chi., began with the N-terminal analysis of .sub..chi. which
provided a sequence a portion of which, was found to have been
related, in part, to the sequence of the xerB gene [see EMBO
8(5):1623 (1989)]. Although not included in the 1692 bp sequence in
the publication, a fuller more complete sequence (from 1 to 2035)
of the xerB gene was provided to GenBank. In this submission, the
"front-end" portion of the .sub..chi. gene according to the present
invention was presented. However, in neither the publication nor in
GenBank was the "front-end" portion as coding for a protein. Based
upon the molecular weight of .sub..chi. as determined in a SDS-PAG
gel analysis, the "front-end" portion reported in GenBank predicts
approximately 70% of the expected length of .sub..chi..
[0221] A subsequent literature study located a gene named valS
which was located downstream of the xerB gene. It appeared (and was
confirmed during the research leading to the present invention)
that the .sub..chi., in its entirety, must be located between the
xerB and valS genes.
[0222] Edman degradation amino acid sequencing was performed on an
Applied Biosystems 470A gas phase microsequencing apparatus. The
.gamma..sub..chi..psi. complex of the holoenzyme was purified, and
10 .mu.g was electrophoresed in a 15% polyacrylamide gel [see
Nature 227:680 (1970)] followed by transfer to an Immobilon
membrane PVDF (Millipore) for N-terminal sequence analysis as with
the previous subunits according to, the present invention. Internal
peptide sequences were obtained by electrophoresis of 10 .mu.g of
the .gamma..sub..chi..psi. complex in 15% polyacrylamide gel,
followed by transfer to nitrocellulose membrane, digestion by
trypsin in situ, and analysis by gas phase microsequencing.
[0223] The .sub..chi. or holC gene, according to the present
invention, is located at 96.5 minutes on the, E. coli chromosome
and encodes a 147 amino acid protein of 16.6 kDa.
[0224] The recombinant .lambda. phage 5C4 from the overlapping
.lambda. library of Kohara [see Cell 50:495 (1987)] was used in
determining the DNA sequence of the .sub..chi. gene, The DNA
fragment containing the .sub..chi. gene was identified and cloned
into pUC18 using conventional techniques. The DNA sequence for both
strands of the .sub..chi. gene were performed on the dublex plasmid
by the dideoxy chain termination method of Sanger using the
Sequenase kit; sequencing reactions were analyzed on 6%
polyacrylamide, 50% (w/v) urea gels.
[0225] The sequence of the primers (5'->3') used for PCR
amplification of the .sub..chi. gene during the cloning of the
.sub..chi. gene are as follows:
38 30-mer primer: CCCCACATAT GAAAAACGCG ACGTTCTACC 30; 28-mer
primer: ACCCGGATCC 0AAACTGCCGG TGACATTC 28
[0226] The 30-mer hybridizes over the initiation codon of
.sub..chi., and a two nucleotide mismatch results in a NdeI
restriction site (underlined) at the ATG initiation codon upon
amplification of the gene. The 28-mer anneals within the valS gene
downstream of the c gene; a BamHI restriction site (underlined) is
embedded within the six nucleotides which do not hybridize to
valS.
[0227] Using these codons, subsequent studies isolated the c gene
sequence which is, according to the present invention:
39 ATG AAA AAC GCG ACG TTC TAC CTT CTG GAC AAT GAC ACC 39 ACC GTC
GAT GGC TTA AGC GCC GTT GAG CAC CTG GTG TGT 78 GAA ATT GCC GCA GAA
CGT TGG CGC AGC GGT AAG CGC GTG 117 CTC ATC GCC TGT GAA GAT GAA AAG
CAG GCT TAC GCC CTG 156 GAT GAA GCC CTG TGG GCG CGT CCG GCA GAA AGC
TTT GTT 195 CCG CAT AAT TTA GCG GGA GAA GGA CCG CGC GGC GGT GTA 234
CCG GTG GAG ATC GCC TGG CCG CAA AAG CGT AGC AGC AGC 273 CGG CGC GAT
ATA TTG ATT AGT CTG CGA ACA AGC TTT GCA 312 GAT TTT GCC ACC GCT TTT
ACA GAA GTG GTA GAC TTC GTT 351 CCT CAT GAA GAT TCT CTG AAA CAA CTG
GCG CGC GAA CGC 390 TAT AAA GCC TAC CGC GTG GCT GGT TTC AAC CTG AAT
ACG 429 GCA ACT TGG AAA 441
[0228] The upstream portion of the holC gene is:
40 TAACGGCGAA GAGTAATTGC GTCAGGCAAG GCTGTTATTG CCGGATGCGG 50
CGTGAACGCC TTATCCGACC TACACAGCAC TGAACTCGTA GGCCTGATAA 100
GACACAACAG CGTCGCATCA GGCGCTGCGG TGTATACCTG ATGCGTATTT 150
AAATCCACCA CAAGAAGCCC CATTT 175
[0229] The downstream sequence begins with the stop codon:
41 TAA TGGAAAA GACATATAAC CCACAAGATA TCGAACAGCC 40 GCTTTACGAG
CACTGGGAAA AAAGCCAGGA AAGTTTCTGC 80 ATCATGATCC CGCCGCCGAA 100
[0230] The underlined nucleotide sequences indicate the potential
Shine-Dalgarno sequence (AAGAAG) of holC and the nearest possible
promoter signals (TTGCCG) are highlighted in the first underlined
region. The stop codon of the upstream XerB gene (TAA) and the
start codon of the downstream ValS gene (ATG) are each double
underlined.
[0231] This translated into the correct peptide which is:
42 Met Lys Asp Ala Thr Phe Tyr Leu Leu Asp Asn Asp Thr Thr Val 5 10
15 Asp Gly Leu Ser Ala Val Glu Gln Leu Val Cys Glu Ile Ala Ala 20
25 30 Glu Arg Trp ARg Ser Gly Lys Arg Val Leu Ile Ala Cys Glu Asp
35 40 45 Glu Lys Gln Ala Tyr Arg Leu Asp Glu Ala Leu Trp Ala Arg
Pro 50 55 60 Ala Glu Ser Phe Val Pro His Asn Leu Ala Gly Glu Gly
Pro Arg 65 70 75 Gly Gly ala Pro Val Glu Ile Ala Trp Pro Gln Lys
Arg Ser Ser 80 85 90 Ser Arg Arg Asp Ile Leu Ile Ser Leu Arg Thr
Ser Phe Ala Asp 95 100 105 Phe Ala Thr Ala Phe Thr Glu Val Val Asp
Phe Val Pro Tyr Glu 110 115 120 Asp Ser Leu Lys Gln Leu Ala Arg Glu
Arg Tyr Lys Ala Tyr Arg 125 130 135 Val Ala Gly Phe Asn Leu Asn Thr
Ala Thr Trp Lys 140 145 147
EXAMPLE IV
Molecular Cloning, Cell Growth and Purification
[0232] PCR reactions were performed with both Vent polymerase
(Biolabs) and Taq polymerase. In a 100 .mu.l volume, the PCR
reaction was conducted in a reaction buffer containing 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, and 0.01% (w/v)
gelatin, 1.0 .mu.M of each primer, and 200 .mu.M each dNTP
(Pharmacia-LKB), on 1 ng E. coli genomic DNA (prepared from K12
strain C600) with 2.5 u polymerase. PCR amplification was performed
in a DNA Thermal cycler model 9801 using the following cycle:
melting temperature 94.degree. C. for 1 min, annealing temperature
60.degree. C. for 2 min, and extension temperature 72.degree. C.
for 2 min. After 30 cycles, the amplified DNA was purified by
phenol extraction in 2% SDS followed by sequential digestion of 10
.mu.g DNA with 10 u NdeI, followed by 10 u BamHI. The 600 bp DNA
fragment was purified from a 0.8% agarose gel by electroelution,
and ligated into pET3c previously digested with both NdeI and BamHI
restriction enzymes. The resulting plasmids (pET.sub..chi.-1,
pET.sub..chi.-2 and pET.sub..chi.-3) were ligated into E. coli
strain BL21(DE3)pLysS.
[0233] The freshly transformed BL21(DE3)pLysSpET.sub..chi. cells
were grown in LB media containing 100 .mu.g/ml ampicillin and 30
.mu.g/ml chloramphenicol at 37.degree. C. Upon growth to an
OD.sub.600 of 0.7, isopropyl B-D-galactopyranoside (IPTG) was added
to a final concentration of 0.4 mM. Incubation was continued for 3
hr at 37.degree. C. before harvesting the cells.
[0234] Seven mg of homogeneous .sub..chi. protein was purified from
a 4-liter induced culture in which nearly 30% overproduced
.sub..chi. protein was in soluble form. The 4-liter culture was
grown in an OD.sub.600 of 0.7 before addition of IPTG to 0.4 mM.
After a further 3 hr incubation at 37.degree. C., the cells (25 g)
were harvested, resuspended in 25 ml ice-cold 50 mM tris/10%
sucrose, and lysed by 25 mg lysozyme on ice for 45 min and a
subsequent incubation at 37.degree. C. for 5 min in 5 mM Tris, 1%
sucrose, 30 mM spermidine, and 100 mM NaCl. The cell lysate was
clarified by centrifugation at 12,000 rpm for I hr at 4.degree. C.
All subsequent column chromatography procedures were at 4.degree.
C. All the columns were equilibrated in buffer A (20 mM Tris (pH
7.5), 0.5 mM EDTA, 5 mM DTT, and 20% glycerol). The .sub..chi.
protein was followed through the purification process by SDS-PAGE
gel analysis. Total protein was estimated [see Anal. Biochem
72:248.(1976)] using bovine serum albumin as a standard. The
soluble lysate (120 ml, 520 mg protein) was dialyzed against 4
liter buffer A for 16 hours before being loaded onto a 60 ml
heparin-agarose column. The fractions containing .sub..chi., which
eluted off the column during wash with buffer A, were pooled (360
ml, 365 mg protein), and loaded directly onto a FPLC 26/10 Q
sepharose fast flow column. The Q sepharose fast flow column was
eluted with a 650-ml linear gradient of 0 M to 0.5 M NaCl in buffer
A. The fractions containing .sub..chi., eluted at approximately
0.16 M salt in a volume of 45 ml (60 mg protein), were pooled,
dialyzed overnight against 4 liter buffer A, and loaded onto a 1 ml
N-6 ATP-agarose column. The .gamma. complex
(.gamma..delta..delta.'.sub..chi..psi.) binds to the column tightly
due to the strong ATP binding capacity of .gamma., while .sub..chi.
protein by itself flows through. This column was included to
eliminate any .gamma. complex from the .sub..chi. preparation.
[0235] The flow-through, of the ATP-agarose column was loaded onto
an 8 ml hexylamine column and .sub..chi. was eluted with an 80 ml
linear gradient of 0 to 0.5 M NaCl in buffer A. The .sub..chi.
protein eluted at approximately 0.25 M salt. Fractions containing
the peptide (81 ml, 36 mg protein) were pooled and dialyzed against
buffer A. The .sub..chi. protein was loaded onto an 8 ml FPLC Mono
Q column, and eluted with a 80 ml linear gradient of 0 to 0.5 M
NaCl in buffer A. The fractions containing .sub..chi. (28 ml 8.5 mg
protein) eluted sharply at 0.16 M salt. The .sub..chi. protein was
pooled and dialyzed overnight against 4 liters of buffer A, then
aliquoted and stored at -70.degree. C.
[0236] The concentration of purified .sub..chi. protein was
determined from its absorbance at 280 nm. The molar extinction
coefficient at 280 nm (.epsilon..sub.280) of a protein in its
native state can be calculated from its gene sequence to within
+/-5% by using the equation
.epsilon..sub.280=Trp.sub.m(5690M.sup.-1cm.sup.-1)+Tyr.sub.n(1280M.sup.-1-
cm.sup.-1) [see Analytical Biochemistry 182:319 (1989)] wherein m
and n are the numbers of tryptophan and tyrosine residues,
respectively, in the peptide. The molar extinction coefficients of
tryptophan and tyrosine are known [see Biochemistry 5 6:1948
(1967)]. For .sub..chi. protein where m equals 4 and n equals 5,
.epsilon..sub.280=29,160 M.sup.-1cm.sup.-1. The .sub..chi. protein
was dialyzed against buffer A containing no DTT prior to absorbance
measurement. SDS-PAG was in 15% polyacrylamide 0.1% SDS gel in
Tris/glycine/SDS buffet [see Nature 227:680 (1970)]. Proteins were
visualized by Coomassie Brilliant Blue Stain.
[0237] The .gamma..sub..chi..psi. complex was purifeid from 1.3 kg
of E. coli. The .sub..chi. subunit was resolved from the .gamma.
and .sub..psi. subunits upon electorphoresis in a 15% SDS
polyacrylamide gel followed by transfer of .sub..chi. onto PVDF
membrane for N-terminal sequence analysis (210 pmol), and onto
nitrocellulose membrane for tryptic analysis (300 pmol). Proteins
were visualized by Ponceau S stain. The amino terminus of c was
determined to be:
43 NH.sub.2-Met Lys Asn Ala Thr Phe Tyr Leu Leu Asp Asn Asp Thr Thr
Val 5 10 15 Asp Gly Leu Ser Ala Val Glu Gln Leu Val Xxx Glu Ile Ala
20 25 wherein Xxx is an unidentified residue. Tryptic digestion and
analysis of four internal peptides were determined to be: .chi.-1:
NH.sub.2-Val Leu Ile Ala Xxx Glu Asp Glu Lys 5 .chi.-2:
NH.sub.2-Leu Asp Glu Ala Leu Trp Ala Ala Pro Ala Glu Ser Phe Val
Pro 5 10 15 His Asn Leu Ala Gly Glu 20 .chi.-3: NH.sub.2-Gly Gly
Ala Pro Val Glu Ile Ala Trp Pro 5 10 .chi.-4: NH.sub.2-Gly Phe Asn
Leu Asn Thr Ala Thr 5
[0238] The 3.4 kb BamHI fragment containing holC was excised from
.lambda. 5C4 and ligated into the BamHI site of pUC-.sub..chi..
Both strands of the holC gene were sequenced on the duplex plasmid
by the chain termination method of Sanger, and synthetic 17-mer DNA
oligonucleotides. Sequencing reactions were analyzed on 6% (w/v)
acrylamide, 50% (w/v) urea gels and were performed with both dGTP
and DITP.
[0239] The sequences of the primers used to amplify the holC gene
were:
44 upstream 30-mer: CCCCACATAT GAAAAACGCG ACGTTCTACC 30 Downstream
28-mer: ACCCGGATCC AAACTGCCGG TGACGTTC 28
[0240] The upstream 30-mer hybridizes over the initiation codon of
holC, and a two-nucleotide mismatch results in a NdeI restriction
site (underlined above) at the ATG initiation codon upon
amplification of the gene. The downstream 28-mer sequence within
the valS gene downstream of holC. A BamHI restriction site
(underlined) is embedded in the sequence resulting in three
nucleotides which are not complementary to valS. Amplification
reactions contained 1.0 .mu.M of each primer, 200 .mu.M of each
dNTP, 1 ng E. coli genomic DNA (from strain C600), and 2.5 units of
Taq I DNA polymerase in a final volume of 100 .mu.l 10 mM Tris-HCl
(pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, and 0.01% (w/v) gelatin.
Amplification was performed in a thermal cycler using the following
cycle: 94.degree. C., 1 minute; 60.degree. C., 2 minutes; and
72.degree. C., 2 minutes. After 30 cycles, the amplified 604 bp DNA
was purified by phenol extraction in 2% SDS followed by sequential
digestion of 10 .mu.g DNA in 10 units of NdeI and then 10 units of
BamHI according to manufacturer's specifications. The NdeI-BamHI
fragment was electroeluted from a 0.8% agarose gel and ligated into
gel purified pET3c previously digested with both NdeI and BamHI.
The resulting plasmid, pET-.sub..chi. was sequenced which confirmed
that no errors had been introduced during amplification, and it was
then transformed into strain BL21 (DE3)plysS.
[0241] The .gamma. subunit was purified from an overproducing
strain, and the .delta., .delta.' and .sub..psi. subunits were
purified from their respective overproducing strains as described
above. A mixture of 48 .mu.g .gamma. (0.51 nmol as dimer), 144
.mu.g .delta. (3.7 nmol as monomer}, 144 .mu.g .delta.' (3.9 nmol
as monomer), and 192 .mu.g .sub..psi. (12.7 nmol as monomer) was
incubated at 15.degree. C. for 1 hour and then loaded onto a 1 ml
HR 5/5 Mono Q column. The concentration of .gamma. was determined
using BSA as a standard. Concentrations of .delta., .delta.' and
.sub..psi. were determined from their absorbance at 280 nm using
the molar extinction coefficients 46,137 M.sup.-1cm.sup.-1, 60,136
M.sup.-1cm.sup.-1, and 24040 M.sup.-1cm.sup.-1, respectively. The
column was eluted with a 32 ml gradient of 0 M-0.4 M NaCl in 20 mM
Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM DTT, And 20% glycerol (buffer
A) whereupon the .gamma..delta..delta.'.sub..psi. complex resolved
from uncomplexed subunits by eluting later than all the rest.
Eighty fractions were collected and analyzed by a Coomassie Blue
stained 15% SDS polyacrylamide gel. Fractions containing the
.gamma..delta..delta.'.sub..- psi. complex, were pooled, the
protein concentration was determined using BSA as a standard, and
then the .gamma..delta..delta.'.sub..psi. complex was aliquoted and
stored at -70.degree. C. Molarity of .gamma..delta..delta.' was
calculated from the protein concentration assuming the 185 kDa mass
calculated from gene sequences assuming a stoichiometry of
.gamma..sub.2.delta..sub.1.delta.'.sub.1.psi.1 as expected from the
tentative stoichiometry of subunits in the .gamma. complex.
[0242] The reconstitution assay contained 72 ng M13mp18 ssDNA (0.03
pmol as circles) uniquely primed with a DNA 30-mer, 980 ng SSB
(13.6 pmol as tetramer), 10 ng .beta. (0.13 pmol as dimer), 55 ng
.alpha..epsilon. complex (0.35 pmol) in a final volume (after
addition of proteins) of 25 .mu.l 20 mM Tris-HCl (pH 7.5), 0.1 mM
EDTA, 8 mM MgCl.sub.2, 5 mM DTT, 4% glycerol, 40 .mu.g/ml BSA, 0.5
mM ATP, 40 mM NaCl, 60 .mu.M each dCTP, dGTP, dATP, and 20 .mu.M
[.alpha.-.sup.32P]. Pure .sub..chi. protein or column pool
containing .sub..chi. (1-12 ng) was preincubated on ice for 30
minutes with 37 ng .gamma..delta..delta.'.sub..psi. complex (0.2
pmol) in 20 .mu.l of 20 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM
EDTA, 20% glycerol, and 50 .mu.g/ml BSA before dilution with the
same buffer such that 0.14 ng (0.76 fmol) of the
.gamma..delta..delta.'.sub..psi. complex was added to the assay in
a 1-2 .mu.l volume. The assay was then shifted to 37.degree. C. for
5 minutes. DNA synthesis was quenched by spotting directly onto
DE81 filter paper and quantitated. The .alpha..epsilon. complex,
.beta. and SSB proteins used in the reconstitution assay were
purified and their concentrations determined using BSA as a
standard except for .beta. which was determined by absorbance using
an .epsilon.280 value of 17,900 M.sup.-1cm.sup.-1.
[0243] Gel filtration analysis was performed using the Pharmacia HR
10/30 fast protein liquid chromatography columns, Superdex 75 and
Superose 12. Proteins were incubated together for 1 hour at
15.degree. C. In a final volume of 200 .mu.l buffer B (25 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 10% glycerol, and 100 mnM NaCl). The
.sub..psi. protein was first brought to 4 M in urea to disaggregate
it, and when present with other proteins the final concentration of
urea in buffer B was 0.5 M. The entire sample was injected, the
column was developed with buffer B, and after collecting the first
6 ml, fractions of 170 .mu.l were collected. The .sub..chi. protein
was located in column fractions by analysis in 15%
SDS-polyacrylamide gels. Densitometry of Coomassie Blue-stained
gels was performed using a laser densitometer (Ultrascan XL).
[0244] Individual samples of .sub..chi. (46 ng, 2.8 nmol as
monomer) and of .sub..psi. (45 ng, 3 nmol as monomer), and a
mixture of .sub..chi. (218 ng, 13 nmol as monomer) and .sub..psi.
(45 ng, 3 nmol as monomer) were incubated 30 minutes at 4.degree.
C. In 200 .mu.l buffet B with 5% glycerol (samples containing
.sub..psi. contained a final concentration of 0.5 M urea in the 200
.mu.l as explained above). Samples were layered onto 12.3 ml
gradients of 10%-30% glycerol in 25 mM Tris-HCl (pH 7.5), 0.1 M
NaCl and 1 mM EDTA. Protein standards in 200 .mu.l of buffer B with
5% glycerol were layered onto another gradient and the gradients
were centrifuged at 270,000.times.g for 44 hours at 4.degree. C.
Fractions were collected and analyzed.
[0245] The polymerase chain reaction was used to precisely clone
the holC gene into the T7 based pET expression system [see Methods
in Enzymology 185:60 (1990)]. Primers upstream and downstream of
holC were synthesized to amplify a 604 bp fragment containing the
holC gene from E. coli genomic DNA. The upstream primer hybridized
over the start codon of holC and included two mismatched
nucleotides in order to create an NdeI restriction site at the
initiating ATG. The primer downstream of holC included three
mismatched nucleotides to create a BamHI restriction site. The
amplified 604 bp fragment was digested with NdeI and BamHI and
cloned into the NdeI-BamHI site of the T7 based expression vector
pET3c to yield PET-.sub..chi.. In the PET-.sub..chi. plasmid, the
holC gene is under the control of a strong T7 RNA polymerase
promoter and an efficient Shine-Dalgarno sequence in favorable
context for translation initiation. DNA sequencing of the
pET-.sub..chi. plasmid showed its sequence was identical to that of
pUC-.sub..chi., and therefore no errors were incurred during
amplification.
[0246] The pET-.sub..chi. expression plasmid was introduced into
strain BL21(DE)plyS which is a lysogen carrying the T7 RNA
polymerase gene under the control of the IPTG-inducible lac UV5
promoter. Upon induction with IPTG and continued growth for 3
hours, the .sub..chi. protein was expressed to a level of 27% total
cell protein. Upon cell lysis, only about 30% of the .sub..chi.
protein was in the soluble fraction, the rest being found in the
cell debris. Induction at lower temperature (20.degree. C.) or for
shorter times did not appear to increase the proportion of
.sub..chi. in the soluble fraction.
[0247] Four liters of induced cells were lysed and 38 mg of pure
.sub..chi. was obtained in 38% overall yield upon fractionation
with ammonium sulfate precipitation, followed by column
chromatography using Q sepharose and heparin agarose. The
.sub..chi. protein was well behaved throughout the purification and
showed no tendency to aggregate. The N-terminal sequence analysis
of the pure cloned .sub..chi. matched that of the holC gene
indicating that the protein had been successfully cloned and
purified. The expressed c protein also comigrated with the
authentic .sub..chi. subunit contained within polIII*.
[0248] In summary, as a result of the present invention, the
location and sequence of .sub..chi. was determined. The .sub..chi.
subunit (400 pmol) was separated from .gamma. and .sub..chi.
subunits of the .gamma..sub..chi..psi. complex by SDS denaturation
and resolution on a 15% polyacrylamide gel, and 100 pmol
transferred to a PVDF immobilon membrane for amino terminal
sequence analysis; the remainder was transferred to nitrocellulose
for sequence analysis of internal peptides following trypsin
digestion. After transfer, the protein was visualized by Ponceau S
stain and excised from the gel. The sequence of the N-terminal
amino acids and four internal peptides were determined as described
above, and these sequences were used to search the GenBank
database. One single exact match was found at about 96.5 minutes on
the E. coli chromosome between the xerB and valS genes.
[0249] The recombinant Kohara .lambda. clone 5C4, contains the DNA
fragment encompassing the xerB and partial vals genes, and the
.sub..chi. gene was subcloned by ligation of the BamHI fragment of
.lambda. 5C4 into pUC18. Sequence analysis was performed directly
on the plasmid. As shown above, the open reading frame of the
.sub..chi. gene was 441 nucleotides long. Its initiation codon is
160 nucleotides downstream of the stop codon of the xerB gene,
while its termination codon, TAA, has one base overlapping with the
start codon of the valS gene. Since the xerB and .sub..chi. genes
were transcribed in the same direction, and that no promoter
consensus sequences were found for the .sub..chi. gene alone,
suggests that these two genes are in the same operon.
[0250] When PCR was applied to clone the .sub..chi. gene into the
T7 based expression system, PCR primers based upon the known
sequences of the xerB and valS genes were made to amplify the
fragment between the two genes. As described, E coli genomic DNA
was used as the PCR template, and a fragment of approximately 600
base pairs was amplified. The PCR fragment, after being digested
with NdeI and BamHI, was cloned into the NdeI-BamHI site of the
expression vector pET3c in similar manner to what was done with the
preceding gene sequences. Thus, the putative .sub..chi. gene was
put under the control of a strong T7 RNA polymerase promoter gene
as well as the efficient translation initiation signal, and
transcription termination sequence downstream of the BamHI site.
Direct DNA sequencing of the plasmids formed showed that they were
all identical to the sequence of the .lambda. clone.
[0251] The resulting plasmids were transformed into E. coli
BL21(DE)pLysS that contained a lysogen carrying the T7 RNA
polymerase gene under the control of the IPTG-inducible lac UV5
promoter [see Methods in Enzymology,185:60 (1990)]. Transformants
were selected by ampicillin and chloramphenicol resistance, and
subsequently subjected to IPTG induction as described above. A
protein of about 17 kDa was overproduced in all three PCR clones.
The .gamma. complex was run in parallel with the three clones on
SDS-PAG gel, and when the overproduced and the .chi. subunit were
at similar amounts, they showed the same gel mobility. This
observation supported the identity of the overproduced protein as
the .chi. subunit.
[0252] In addition to the specific sequences provided above for the
individual genes according to the present invention, the present
invention also extends to mutations, deletions and additions to
these sequences provided such changes do not substantially affect
the present properties of the listed sequences.
[0253] As described, the naturally occurring holoenzyme consists of
10 protein subunits and is capable of extending DNA faster than
polymerase t, and producing a product many times larger then the
polymerase 1 enzyme. Thus, these unique properties of the 5,
preferably 6, active subunits of the present invention are likely
to find wide application in, for example, long chain PCR--using the
active sequence according to the present invention PCR can be
performed over several tens of kb; PCR performed at room
temperature--the active sequence according to the present invention
is uniquely adapted to be a polymerase of choice for PCR at room
temperature due to its high fidelity; extension of site mutated
primers without catalyzing strand displacement; and for sequencing
operations wherein other polymerases find difficulty. Other uses
will become more apparent to those skilled in the art as the
science of molecular genetics continues to progress.
[0254] The sequence listing for the nucleic acid sequences and
peptide sequences which are contained in the present description is
as follows:
[0255] Thus, while I have illustrated and desribed the preferred
embodiment of my invention, it is to be understood that this
invention is capable of variation and modification, and I therefore
do not wish to be limited to the precise terms set forth, but
desire to avail myself of such changes and alterations which may be
made for adapting the invention to various usages and conditions.
Accordingly, such changes and alterations are properly intended to
be within the full range of equivalents, and therefore within the
purview of the following claims.
[0256] Having thus described my invention and the manner and a
process of making and using it in such full, clear, concise and
exact terms so as to enable any person skilled in the art to which
it pertains, or with which it is most nearly connected, to make and
use the same;
Sequence CWU 1
1
60 1 28 PRT Escherichia coli 1 Met Leu Arg Leu Tyr Pro Glu Gln Leu
Arg Ala Gln Leu Asn Glu Gly 1 5 10 15 Leu Arg Ala Ala Tyr Leu Leu
Leu Gly Asn Asp Pro 20 25 2 21 PRT Escherichia coli 2 Ala Ala Tyr
Leu Leu Leu Gly Asn Asp Pro Leu Leu Leu Gln Glu Ser 1 5 10 15 Gln
Asp Ala Val Arg 20 3 14 PRT Escherichia coli 3 Ala Gln Glu Asn Ala
Ala Trp Phe Thr Ala Leu Ala Asn Arg 1 5 10 4 24 PRT Escherichia
coli 4 Val Glu Gln Ala Val Asn Asp Ala Ala His Phe Thr Pro Phe His
Trp 1 5 10 15 Val Asp Ala Leu Leu Met Gly Lys 20 5 33 DNA
Escherichia coli 5 gtacaaccga atcatatgtt acccagcgag ctc 33 6 1032
DNA Escherichia coli 6 atgattcggt tgtacccgga acaactccgc gcgcagctca
atgaagggct gcgcgcggcg 60 tatcttttac ttggtaacga tcctctgtta
ttgcaggaaa gccaggacgc tgttcgtcag 120 gtagctgcgg cacaaggatt
cgaagaacac cacacttttt ccattgatcc caacactgac 180 tggaatgcga
tcttttcgtt atgccaggct atgagtctgt ttgccagtcg acaaacgcta 240
ttgctgttgt taccagaaaa cggaccgaat gcggcgatca atgagcaact tctcacactc
300 accggacttc tgcatgacga cctgctgttg atcgtccgcg gtaataaatt
aagcaaagcg 360 caagaaaatg ccgcctggtt tactgcgctt gcgaatcgca
gcgtgcaggt gacctgtcag 420 acaccggagc aggctcagct tccccgctgg
gttgctgcgc gcgcaaaaca gctcaactta 480 gaactggatg acgcggcaaa
tcaggtgctc tgctactgtt atgaaggtaa cctgctggcg 540 ctggctcagg
cactggagcg tttatcgctg ctctggccag acggcaaatt gacattaccg 600
cgcgttgaac aggcggtgaa tgatgccgcg catttcaccc cttttcattg ggttgatgct
660 ttgttgatgg gaaaaagtaa gcgcgcattg catattcttc agcaactgcg
tctggaaggc 720 agcgaaccgg ttattttgtt gcgcacatta caacgtgaac
tgttgttact ggttaacctg 780 aaacgccagt ctgcccatac gccactgcgt
gcgttgtttg ataagcatcg ggtatggcag 840 aaccgccggg gcatgatggg
cgaggcgtta aatcgcttaa gtcagacgca gttacgtcag 900 gccgtgcaac
tcctgacacg aacggaactc accctcaaac aagattacgg tcagtcagtg 960
tgggcagagc tggaagggtt atctcttctg ttgtgccata aacccctggc ggacgtattt
1020 atcgacggtt ga 1032 7 127 DNA Escherichia coli 7 ccgaacagct
gattcgtaag ctgccaagca tccgtgctgc ggatattcgt tccgacgaag 60
aacagacgtc gaccacaacg gatactccgg caacgcctgc acgcgtctcc accacgctgg
120 gtaactg 127 8 104 DNA Escherichia coli 8 tgaatgaaat ctttacaggc
tctgtttggc ggcacctttg atccggtgca ctatggtcat 60 ctaaaacccg
ttggaagcgt ggccgaagtt ttgattggtc tgac 104 9 343 PRT Escherichia
coli 9 Met Ile Arg Leu Tyr Pro Glu Gln Leu Arg Ala Gln Leu Asn Glu
Gly 1 5 10 15 Leu Arg Ala Ala Tyr Leu Leu Leu Gly Asn Asp Pro Leu
Leu Leu Gln 20 25 30 Glu Ser Gln Asp Ala Val Arg Gln Val Ala Ala
Ala Gln Gly Phe Glu 35 40 45 Glu His His Thr Phe Ser Ile Asp Pro
Asn Thr Asp Trp Asn Ala Ile 50 55 60 Phe Ser Leu Cys Gln Ala Met
Ser Leu Phe Ala Ser Arg Gln Thr Leu 65 70 75 80 Leu Leu Leu Leu Pro
Glu Asn Gly Pro Asn Ala Ala Ile Asn Glu Gln 85 90 95 Leu Leu Thr
Leu Thr Gly Leu Leu His Asp Asp Leu Leu Leu Ile Val 100 105 110 Arg
Gly Asn Lys Leu Ser Lys Ala Gln Glu Asn Ala Ala Trp Phe Thr 115 120
125 Ala Leu Ala Asn Arg Ser Val Gln Val Thr Cys Gln Thr Pro Glu Gln
130 135 140 Ala Gln Leu Pro Arg Trp Val Ala Ala Arg Ala Lys Gln Leu
Asn Leu 145 150 155 160 Glu Leu Asp Asp Ala Ala Asn Gln Val Leu Cys
Tyr Cys Tyr Glu Gly 165 170 175 Asn Leu Leu Asn Leu Ala Gln Ala Leu
Glu Arg Leu Ser Leu Leu Trp 180 185 190 Pro Asp Gly Lys Leu Thr Leu
Pro Arg Val Glu Gln Ala Val Asn Asp 195 200 205 Ala Ala His Phe Thr
Pro Phe His Trp Val Asp Ala Leu Leu Met Gly 210 215 220 Lys Ser Lys
Arg Ala Leu His Ile Leu Gln Gln Leu Arg Leu Gly Gly 225 230 235 240
Ser Glu Pro Val Ile Leu Leu Arg Thr Leu Gln Arg Glu Leu Leu Leu 245
250 255 Leu Val Asn Leu Lys Arg Gln Ser Ala His Thr Pro Leu Arg Ala
Leu 260 265 270 Phe Asp Lys His Arg Val Trp Gln Asn Arg Arg Gly Met
Met Gly Glu 275 280 285 Ala Leu Asn Arg Leu Ser Gln Thr Gln Leu Arg
Gln Ala Val Gln Leu 290 295 300 Leu Thr Arg Thr Glu Leu Thr Leu Lys
Gln Asp Tyr Gly Gln Ser Val 305 310 315 320 Trp Ala Glu Leu Glu Gly
Leu Ser Leu Leu Leu Cys His Lys Pro Leu 325 330 335 Ala Asp Val Phe
Ile Asp Gly 340 10 334 PRT Escherichia coli 10 Met Arg Trp Tyr Pro
Trp Leu Arg Pro Asp Phe Glu Lys Leu Val Ala 1 5 10 15 Ser Tyr Gln
Ala Gly Arg Gly His His Ala Leu Leu Ile Gln Ala Leu 20 25 30 Pro
Gly Met Gly Asp Asp Ala Leu Ile Tyr Ala Leu Ser Arg Tyr Leu 35 40
45 Leu Cys Gln Gln Pro Gln Gly His Lys Ser Cys Gly His Cys Arg Gly
50 55 60 Cys Gln Leu Met Gln Ala Gly Thr His Pro Asp Tyr Tyr Thr
Leu Ala 65 70 75 80 Pro Glu Lys Gly Lys Asn Thr Leu Gly Val Asp Ala
Val Arg Glu Val 85 90 95 Thr Glu Lys Leu Asn Glu His Ala Arg Leu
Gly Gly Ala Lys Val Val 100 105 110 Trp Val Thr Asp Ala Ala Leu Leu
Thr Asp Ala Ala Ala Asn Ala Leu 115 120 125 Leu Lys Thr Leu Glu Glu
Pro Pro Ala Glu Thr Trp Phe Phe Leu Ala 130 135 140 Thr Arg Glu Pro
Glu Arg Leu Leu Ala Thr Leu Arg Ser Arg Cys Arg 145 150 155 160 Leu
His Tyr Leu Ala Pro Pro Pro Glu Gln Tyr Ala Val Thr Trp Leu 165 170
175 Ser Arg Glu Val Thr Met Ser Gln Asp Ala Leu Leu Ala Ala Leu Arg
180 185 190 Leu Ser Ala Gly Ser Pro Gly Ala Ala Leu Ala Leu Phe Gln
Gly Asp 195 200 205 Asn Trp Gln Ala Arg Glu Thr Leu Cys Gln Ala Leu
Ala Tyr Ser Val 210 215 220 Pro Ser Gly Asp Trp Tyr Ser Leu Leu Ala
Ala Leu Asn His Glu Gln 225 230 235 240 Ala Pro Ala Arg Leu His Trp
Leu Ala Thr Leu Leu Met Asp Ala Leu 245 250 255 Lys Arg His His Gly
Ala Ala Gln Val Thr Asn Val Asp Val Pro Gly 260 265 270 Leu Val Ala
Glu Leu Ala Asn His Leu Ser Pro Ser Arg Leu Gln Ala 275 280 285 Ile
Leu Gly Asp Val Cys His Ile Arg Glu Gln Leu Met Ser Val Thr 290 295
300 Gly Ile Asn Arg Glu Leu Leu Ile Thr Asp Leu Leu Leu Arg Ile Glu
305 310 315 320 His Tyr Leu Gln Pro Gly Val Val Leu Pro Val Pro His
Leu 325 330 11 57 DNA Escherichia coli 11 actctggaag aaccgccggc
tgaaacttgg ttttttctgg ctactcgtga accggaa 57 12 54 DNA Escherichia
coli 12 gctggttctc cgggtgctgc tctggctctg tttcagggtg atgactggca ggct
54 13 1002 DNA Escherichia coli 13 atgagatggt atccatggtt acgacctgat
ttcgaaaaac tggtagccag ctatcaggcc 60 ggaagaggtc accatgcgct
actcattcag gcgttaccgg gcatgggcga tgatgcttta 120 atctacgccc
tgagccgtta tttactctgc caacaaccgc agggccacaa aagttgcggt 180
cactgtcgtg gatgtcagtt gatgcaggct ggcacgcatc ccgattacta caccctggct
240 cccgaaaaag gaaaaaatac gctgggcgtt gatgcggtac gtgaggtcac
cgaaaagctg 300 aatgagcacg cacgcttagg tggtgcgaaa gtcgtttggg
taaccgatgc tgccttacta 360 accgacgccg cggctaacgc attgctgaaa
acgcttgaag agccaccagc agaaacttgg 420 tttttcctgg ctacccgcga
gcctgaacgt ttactggcaa cattacgtag tcgttgtcgg 480 ttacattacc
ttgcgccgcc gccggaacag tacgccgtga cctggctttc acgcgaagtg 540
acaatgtcac aggatgcatt acttgccgca ttgcgcttaa gcgccggttc gcctggcgcg
600 gcactggcgt tgtttcaggg agataactgg caggctcgtg aaacattgtg
tcaggcgttg 660 gcatatagcg tgccatcggg cgattggtat tcgctgctag
cggcccttaa tcatgaacaa 720 gtcccggcgc gtttacactg gctggcaacg
ttgctgatgg atgcgctaaa acgccatcat 780 ggtgctgcgc aggtgaccaa
tgttgatgtg ccgggcctgg tcgccgaact ggcaaaccat 840 ctttctccct
cgcgcctgca ggctatactg ggggatgttt gccacattcg tgaacagtta 900
atgtctgtta caggcatcaa ccgcgagctt ctcatcaccg atcttttact gcgtattgag
960 cattacctgc aaccgggcgt tgtgctaccg gttcctcatc tt 1002 14 157 DNA
Escherichia coli 14 aagaatcttt cgatttcttt aatcgcaccc gcgcccgcta
tctggaactg gcagcacaag 60 ataaaagcat tcataccatt gatgccaccc
agccgctgga ggccgtgatg gatgcaatcc 120 gcactaccgt gacccactgg
gtgaaggagt tggacgc 157 15 143 DNA Escherichia coli 15 ttagagagac
atcatgtttt tagtggactc acactgccat ctcgatggtc tggattatga 60
atctttgcat aaggacgtgg atgacgttct ggcgaaagcc gccgcacgcg atgtgaaatt
120 ttgtctggca gtcgccacaa cat 143 16 16 PRT Escherichia coli 16 Met
Arg Trp Tyr Pro Pro Leu Arg Pro Asp Phe Glu Lys Leu Val Ala 1 5 10
15 17 11 PRT Escherichia coli 17 Glu Val Thr Glu Lys Leu Asn Glu
His Ala Arg 1 5 10 18 20 PRT Escherichia coli 18 Val Val Trp Val
Thr Asp Ala Ala Leu Leu Thr Asp Ala Ala Ala Asn 1 5 10 15 Ala Leu
Leu Lys 20 19 25 PRT Escherichia coli 19 Thr Leu Glu Glu Pro Pro
Ala Glu Thr Trp Phe Phe Leu Ala Thr Arg 1 5 10 15 Glu Pro Glu Arg
Leu Leu Ala Thr Leu 20 25 20 18 PRT Escherichia coli 20 Leu His Tyr
Leu Ala Pro Pro Pro Glu Gln Tyr Ala Val Thr Trp Leu 1 5 10 15 Ser
Arg 21 21 PRT Escherichia coli 21 Leu Ser Ala Gly Ser Pro Gly Ala
Ala Leu Ala Leu Phe Gln Gly Asp 1 5 10 15 Asn Trp Gln Ala Arg 20 22
5 PRT Escherichia coli 22 Leu Gly Gly Ala Lys 1 5 23 58 PRT
Escherichia coli 23 Ala Cys Thr Cys Thr Gly Gly Ala Ala Gly Ala Ala
Cys Cys Gly Cys 1 5 10 15 Cys Gly Gly Cys Thr Thr Gly Ala Ala Ala
Cys Thr Thr Gly Gly Thr 20 25 30 Thr Thr Thr Thr Thr Cys Thr Gly
Gly Cys Thr Ala Cys Thr Cys Gly 35 40 45 Thr Gly Ala Ala Cys Cys
Gly Gly Ala Ala 50 55 24 54 PRT Escherichia coli 24 Gly Cys Thr Gly
Gly Thr Thr Cys Thr Cys Cys Gly Gly Gly Thr Gly 1 5 10 15 Cys Thr
Gly Cys Thr Cys Thr Gly Gly Cys Thr Cys Thr Gly Thr Thr 20 25 30
Thr Cys Ala Gly Gly Gly Thr Gly Ala Thr Ala Ala Cys Thr Gly Gly 35
40 45 Cys Ala Gly Gly Cys Thr 50 25 33 PRT Escherichia coli 25 Gly
Gly Thr Gly Ala Ala Gly Gly Ala Gly Thr Thr Gly Gly Ala Cys 1 5 10
15 Ala Thr Ala Thr Gly Ala Gly Ala Thr Gly Gly Thr Ala Thr Cys Cys
20 25 30 Ala 26 40 PRT Escherichia coli 26 Met Leu Lys Asn Leu Ala
Lys Leu Asp Gln Thr Glu Met Asp Lys Val 1 5 10 15 Asn Val Asp Leu
Ala Ala Ala Gly Val Ala Phe Lys Glu Arg Tyr Asn 20 25 30 Met Pro
Val Ile Ala Glu Ala Val 35 40 27 57 DNA Escherichia coli 27
atgctgaaaa acctggctaa actggatcag actgaaatgg ataaagttaa cgttgat 57
28 57 DNA Escherichia coli 28 ctggctgctg ctggtgttgc ttttaaggaa
cgttataaca tgccggttat tgctgaa 57 29 228 DNA Escherichia coli 29
atgctgaaga atctggctaa actggatcaa acagaaatgg ataaagtgaa tgtcgatttg
60 gcggcggccg gggtggcatt taaagaacgc tacaatatgc cggtgatcgc
tgaagcggtt 120 gaacgtgaac agcctgaaca tttgcgcagc tggtttcgcg
agcggcttat tgcccaccgt 180 ttggcttcgg tcaatctgtc acgtttacct
tacgagccca aacttaaa 228 30 172 DNA Escherichia coli 30 aggcgtagcg
aagggagcgt gcagttgaag ccatattatc tattcctttt tgtaataact 60
tttttacaga cgataacctt gtctaatgtc tgagtcgagg atcatcaatt ccggcttgcc
120 atcctggctc actcttagta acttttgccc gcgaatgatg aggagattaa ga 172
31 107 DNA Escherichia coli 31 taaaacttat acagagttac actttcttac
ataacgcctg ctaaattatg agtattttct 60 aaaccgcact cataatttgc
agtcattttg aaaaggaagt cattatg 107 32 76 PRT Escherichia coli 32 Met
Leu Lys Asn Leu Ala Lys Leu Asp Gln Thr Glu Met Asp Lys Val 1 5 10
15 Asn Val Asp Leu Ala Ala Ala Gly Val Ala Phe Lys Glu Arg Tyr Asn
20 25 30 Met Pro Val Ile Ala Glu Ala Val Glu Arg Glu Gln Pro Glu
His Leu 35 40 45 Arg Ser Trp Phe Arg Glu Arg Leu Ile Ala His Arg
Leu Ala Ser Val 50 55 60 Asn Leu Ser Arg Leu Pro Tyr Glu Pro Lys
Leu Lys 65 70 75 33 40 PRT Escherichia coli 33 Met Leu Lys Asn Leu
Ala Lys Leu Asp Gln Thr Glu Met Asp Lys Val 1 5 10 15 Asn Val Asp
Leu Ala Ala Ala Gly Val Ala Phe Lys Glu Ala Tyr Asn 20 25 30 Met
Pro Val Ile Ala Glu Ala Val 35 40 34 57 DNA Escherichia coli 34
atgctgaaaa acctggctaa actggatcag actgaaatgg ataaagttaa cgttgat 57
35 57 DNA Escherichia coli 35 ctggctgctg ctggtgttgc ttttaaagaa
cgttataaca tgccggttat tgctgaa 57 36 33 DNA Escherichia coli 36
atgatgagga gattacatat gctgaagaat ctg 33 37 51 DNA Escherichia coli
37 gaggaattcg gcttttttgc cgaattcctc ggcccctagg agatctcagc t 51 38
137 PRT Escherichia coli 38 Met Thr Ser Arg Arg Asp Trp Gln Leu Gln
Gln Leu Gly Ile Thr Gln 1 5 10 15 Trp Ser Leu Arg Arg Pro Gly Ala
Leu Gln Gly Glu Ile Ala Ile Ala 20 25 30 Ile Pro Ala His Val Arg
Leu Val Met Val Ala Asn Asp Leu Pro Ala 35 40 45 Leu Thr Asp Pro
Leu Val Ser Asp Val Leu Arg Ala Leu Thr Val Ser 50 55 60 Pro Asp
Gln Val Leu Gln Leu Thr Pro Glu Lys Ile Ala Met Leu Pro 65 70 75 80
Gln Gly Ser His Cys Asn Ser Trp Arg Leu Gly Thr Asp Glu Pro Leu 85
90 95 Ser Leu Glu Gly Ala Gln Val Ala Ser Pro Ala Leu Thr Asp Leu
Arg 100 105 110 Ala Asn Pro Thr Ala Arg Ala Ala Leu Trp Gln Gln Ile
Cys Thr Tyr 115 120 125 Glu His Asp Phe Phe Pro Gly Asn Asp 130 135
39 411 DNA Escherichia coli 39 atgacatccc gacgagactg gcagttacag
caactgggca ttacccagtg gtcgctgcgt 60 cgccctggcg cgttgcaggg
cgagattgcc attgcgatcc cggcacacgt ccgtctggtg 120 atggtggcaa
acgatcttcc cgccctgact gatcctttag tgagcgatgt tctgcgcgca 180
ttaaccgtca gccccgacca ggtgctgcaa ctgacgccag aaaaaatcgc gatgctgccg
240 caaggcagtc actgcaacag ttggcggttg ggtactgacg aaccgctatc
actggaaggc 300 gctcaggtgg catcaccggc gctcaccgat ttacgggcaa
acccaacggc acgcgccgcg 360 ttatggcaac aaatttgcac atatgaacac
gatttcttcc ctggaaacga c 411 40 77 DNA Escherichia coli 40
ggcgattata gccatatgtt ggcgcggtat cgacgaattt gctatatttg cgcccctgac
60 aacaggagcg attcgct 77 41 103 DNA Escherichia coli 41 tgatttaccg
gcagcttacc acattgaaca acgcgcccac gcctttccgt ggagtgaaaa 60
aacgtttgcc agcaaccagg gcgagcgtta tctcaacttt cag 103 42 27 DNA
Escherichia coli 42 gattccatat gacatcccga cgagact 27 43 30 DNA
Escherichia coli 43 gactggatcc ctgcaggccg gtgaatgagt 30 44 17 PRT
Escherichia coli 44 Leu Gly Thr Asp Glu Pro Leu Ser Leu Glu Glu Ala
Gln Val Ala Ser 1 5 10 15 Pro 45 17 PRT Escherichia coli 45 Ala Ala
Leu Trp Gln Gln Ile Cys Thr Tyr Glu His Asp Phe Phe Pro 1 5 10 15
Ala 46 32 DNA Escherichia coli 46 caacaggagc gattccatat gacatcccga
cg 32 47 31 DNA Escherichia coli 47 gattcggatc cctgcaggcc
ggtgaatgag t 31 48 30 DNA Escherichia coli 48 ccccacatat gaaaaacgcg
acgttctacc 30 49 28 DNA Escherichia coli 49 acccggatcc aaactgccgg
tgacattc 28 50 441 DNA Escherichia coli 50 atgaaaaacg cgacgttcta
ccttctggac aatgacacca ccgtcgatgg cttaagcgcc 60 gttgagcacc
tggtgtgtga aattgccgca gaacgttggc gcagcggtaa gcgcgtgctc 120
atcgcctgtg aagatgaaaa gcaggcttac gccctggatg aagccctgtg ggcgcgtccg
180 gcagaaagct ttgttccgca taatttagcg ggagaaggac cgcgcggcgg
tgtaccggtg 240 gagatcgcct ggccgcaaaa gcgtagcagc agccggcgcg
atatattgat tagtctgcga 300 acaagctttg cagattttgc caccgctttt
acagaagtgg tagacttcgt tcctcatgaa 360 gattctctga aacaactggc
gcgcgaacgc tataaagcct accgcgtggc tggtttcaac 420 ctgaatacgg
caacttggaa a 441 51 175 DNA Escherichia coli 51 taacggcgaa
gagtaattgc gtcaggcaag
gctgttattg ccggatgcgg cgtgaacgcc 60 ttatccgacc tacacagcac
tgaactcgta ggcctgataa gacacaacag cgtcgcatca 120 ggcgctgcgg
tgtatacctg atgcgtattt aaatccacca caagaagccc cattt 175 52 100 DNA
Escherichia coli 52 taatggaaaa gacatataac ccacaagata tcgaacagcc
gctttacgag cactgggaaa 60 aaagccagga aagtttctgc atcatgatcc
cgccgccgaa 100 53 147 PRT Escherichia coli 53 Met Lys Asp Ala Thr
Phe Tyr Leu Leu Asp Asn Asp Thr Thr Val Asp 1 5 10 15 Gly Leu Ser
Ala Val Glu Gln Leu Val Cys Glu Ile Ala Ala Glu Arg 20 25 30 Trp
Arg Ser Gly Lys Arg Val Leu Ile Ala Cys Glu Asp Glu Lys Gln 35 40
45 Ala Tyr Arg Leu Asp Glu Ala Leu Trp Ala Arg Pro Ala Glu Ser Phe
50 55 60 Val Pro His Asn Leu Ala Gly Glu Gly Pro Arg Gly Gly Ala
Pro Val 65 70 75 80 Glu Ile Ala Trp Pro Gln Lys Arg Ser Ser Ser Arg
Arg Asp Ile Leu 85 90 95 Ile Ser Leu Arg Thr Ser Phe Ala Asp Phe
Ala Thr Ala Phe Thr Glu 100 105 110 Val Val Asp Phe Val Pro Tyr Glu
Asp Ser Leu Lys Gln Leu Ala Arg 115 120 125 Glu Arg Tyr Lys Ala Tyr
Arg Val Ala Gly Phe Asn Leu Asn Thr Ala 130 135 140 Thr Trp Lys 145
54 29 PRT Escherichia coli UNSURE (26) Xaa at position 26 is
unidentified 54 Met Lys Asn Ala Thr Phe Tyr Leu Leu Asp Asn Asp Thr
Thr Val Asp 1 5 10 15 Gly Leu Ser Ala Val Glu Gln Leu Val Xaa Glu
Ile Ala 20 25 55 9 PRT Escherichia coli UNSURE (5) Xaa at position
5 is unidentified 55 Val Leu Ile Ala Xaa Glu Asp Glu Lys 1 5 56 21
PRT Escherichia coli 56 Leu Asp Glu Ala Leu Trp Ala Ala Pro Ala Glu
Ser Phe Val Pro His 1 5 10 15 Asn Leu Ala Gly Glu 20 57 10 PRT
Escherichia coli 57 Gly Gly Ala Pro Val Glu Ile Ala Trp Pro 1 5 10
58 8 PRT Escherichia coli 58 Gly Phe Asn Leu Asn Thr Ala Thr 1 5 59
30 DNA Escherichia coli 59 ccccacatat gaaaaacgcg acgttctacc 30 60
28 DNA Escherichia coli 60 acccggatcc aaactgccgg tgacgttc 28
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