U.S. patent application number 10/839456 was filed with the patent office on 2005-07-21 for compositions and methods utilizing dna polymerases.
This patent application is currently assigned to Stratagene California. Invention is credited to Hansen, Connie Jo, Hogrefe, Holly Hurlbut, Sorge, Joseph A..
Application Number | 20050158730 10/839456 |
Document ID | / |
Family ID | 22586340 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158730 |
Kind Code |
A1 |
Sorge, Joseph A. ; et
al. |
July 21, 2005 |
Compositions and methods utilizing DNA polymerases
Abstract
The invention features a novel isolated Family B DNA polymerase,
a Thermococcus polymerase JDF-3, and mutant recombinant forms
thereof. Mutant polymerases of the invention are deficient in 3' to
5' exonuclease activity and/or exhibit reduced discrimination
against non-conventional nucleotides relative to the wild-type form
of the polymerase.
Inventors: |
Sorge, Joseph A.; (Wilson,
WY) ; Hogrefe, Holly Hurlbut; (San Diego, CA)
; Hansen, Connie Jo; (San Diego, CA) |
Correspondence
Address: |
PALMER & DODGE, LLP
KATHLEEN M. WILLIAMS / STR
111 HUNTINGTON AVENUE
BOSTON
MA
02199
US
|
Assignee: |
Stratagene California
|
Family ID: |
22586340 |
Appl. No.: |
10/839456 |
Filed: |
May 5, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10839456 |
May 5, 2004 |
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09698341 |
Oct 27, 2000 |
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60162600 |
Oct 29, 1999 |
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Current U.S.
Class: |
435/6.11 ;
435/199; 435/320.1; 435/325; 435/6.12; 435/69.1; 435/91.2;
536/23.2 |
Current CPC
Class: |
C12N 9/1252 20130101;
B82Y 10/00 20130101; B82Y 5/00 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/091.2; 435/199; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 019/34; C12N 009/22 |
Claims
1. An isolated recombinant family B DNA polymerase comprising a
sequence selected from the sequences as indicated by accession
numbers listed in Table II, and further comprising a mutation at a
position corresponding to D141 and/or E143 of SEQ ID NO. 2.
2. The isolated recombinant family B DNA polymerase of claim 1,
wherein said said mutation at D141 is an aspartic acid (D) to
threonine (T) or alanine (A) mutation, and said mutation at E143 is
a glutamic acid (E) to alanine (A) mutation.
3. The isolated recombinant family B DNA polymerase of claim 1,
further comprising a mutation at a position corresponding to L408
and/or P410 of SEQ ID NO. 2.
4. The isolated recombinant family B DNA polymerase of claim 3,
wherein said mutation at L408 is a leucine (L) to histidine (H) or
phenylalanine (F) mutation and said mutation at P410 is a proline
(P) to leucine (P) mutation.
5. The isolated recombinant family B DNA polymerase of claim 1,
further comprising a mutation at one or more additional amino acids
selected from the group of positions corresponding to A485, S345,
T604, Y497, I630, E645, E578, R465, V401, N424, P569, E617, V640,
S651, L396, E459, L456, E658, V437, L478, Y496, Y409 and A490 of
SEQ ID NO: 2.
6. The isolated recombinant family B DNA polymerase of claim 5,
wherein said said mutation at S345 is serine (S) to proline (P),
said mutation at A485 is alanine (A) to threonine (T), cysteine
(C), serine (S), leucine (L), isoleucine (I), phenylalanine (F) or
valine (V), said mutation at Y497 is tyrosine (Y) to cysteine (C),
said mutation at I630 is isoleucine (I) to valine (V), said
mutation at E645 is glutamic acid (E) to lysine (L), said mutation
at E578 is glutamic acid (E) to lysine (L), said mutation at R465
is arginine (R) to methionine (M), said mutation at L396 is leucine
(L) to glutamine (Q) or to proline (P), said mutation at S651 is
serine (S) to asparagine (B), said mutation at L456 is leucine (L)
to histidine (H), said mutation at Y496 is tyrosine (Y) to
asparagine (B) or leucine (L), said mutation at Y409 is tyrosine
(Y) to valine (V), said mutation at A490 is alanine (A) to tyrosine
(Y).
7. The isolated recombinant family B DNA polymerase of claim 3,
further comprising a mutation at one or more additional amino acids
selected from the group of positions corresponding to A485, S345,
T604, Y497, I630, E645, E578, R465, V401, N424, P569, E617, V640,
S651, L396, E459, L456, E658, V437, L478, Y496, Y409 and A490 of
SEQ ID NO: 2.
8. The isolated recombinant family B DNA polymerase of claim 7,
wherein said said mutation at S345 is serine (S) to proline (P),
said mutation at A485 is alanine (A) to threonine (T), cysteine
(C), serine (S), leucine (L), isoleucine (I), phenylalanine (F) or
valine (V), said mutation at Y497 is tyrosine (Y) to cysteine (C),
said mutation at I630 is isoleucine (I) to valine (V), said
mutation at E645 is glutamic acid (E) to lysine (L), said mutation
at E578 is glutamic acid (E) to lysine (L), said mutation at R465
is arginine (R) to methionine (M), said mutation at L396 is leucine
(L) to glutamine (Q) or to proline (P), said mutation at S651 is
serine (S) to asparagine (B), said mutation at L456 is leucine (L)
to histidine (H), said mutation at Y496 is tyrosine (Y) to
asparagine (B) or leucine (L), said mutation at Y409 is tyrosine
(Y) to valine (V), said mutation at A490 is alanine (A) to tyrosine
(Y).
9. The isolated recombinant family B DNA polymerase of claim 1,
further comprising reduced discrimination against a
non-conventional nucleotide selected from the group consisting of:
dideoxynucleotides, ribonucleotides and conjugated nucleotides.
10. The isolated recombinant family B DNA polymerase of claim 9,
wherein said conjugated nucleotide is selected from the group
consisting of radiolabeled nucleotides, fluorescently labeled
nucleotides, biotin labeled nucleotides, chemiluminescently labeled
nucleotides and quantum dot labeled nucleotides.
11. The isolated recombinant family B DNA polymerase of claim 3,
further comprising reduced discrimination against a
non-conventional nucleotide selected from the group consisting of:
dideoxynucleotides, ribonucleotides and conjugated nucleotides.
12. The isolated recombinant family B DNA polymerase of claim 11,
wherein said conjugated nucleotide is selected from the group
consisting of radiolabeled nucleotides, fluorescently labeled
nucleotides, biotin labeled nucleotides, chemiluminescently labeled
nucleotides and quantum dot labeled nucleotides.
13. An isolated recombinant family B DNA polymerase comprising at
least two mutations selected from the group consisting of: an
aspartic acid (D) to threonine (T) or alanine (A) mutation at
position corresponding to D141 of SEQ ID NO. 2, a glutamic acid (E)
to alanine (A) mutation at E143 of SEQ ID NO. 2, a proline (P) to
leucine (L) mutation at P410 of SEQ ID NO. 2, a leucine (L) to
Histidine (H) or phenylalanine (F) mutation at L408 of SEQ ID NO.
2, an alanine (A) to threonine (T) mutation at A485 of SEQ ID NO:
2, and a serine (S) to proline (P) mutation of SEQ ID NO. 2.
14. A kit comprising an isolated recombinant family B DNA
polymerase of claim 1 and packaging material therefor.
15. A kit comprising an isolated recombinant family B DNA
polymerase of claim 3 and packaging material therefor.
16. A method of synthesizing a complementary strand of DNA, said
method comprising: contacting a template DNA molecule with a
non-conventional nucleotide and a recombinant family B DNA
polymerase of claim 1; and incorporating said non-conventional
nucleotide to synthesize a complementary DNA strand.
17. A method of sequencing DNA comprising the steps of: contacting
a template DNA strand with a sequencing primer, a chain-terminating
nucleotide analog, and a recombinant family B DNA polymerase of
claim 1; and incorporating said non-conventional nucleotide to
synthesize a complementary DNA strand, wherein incorporation of
said chain-terminating nucleotide analog results in the termination
of chain elongation, such that the nucleotide sequence of said DNA
strand is determined.
18. The method of claim 17, wherein said chain-terminating
nucleotide analog is a dideoxynucleotide.
19. The method of claim 18 wherein said dideoxynucleotide is
detectably labeled.
20. The method of claim 19, wherein said dideoxynucleotide is
fluorescently labeled.
21. The method of claim 20, wherein said dideoxynucleotide is
labeled with a moiety selected from the group consisting of
fluorescein and rhodamine.
22. A method of synthesizing a complementary strand of DNA, said
method comprising: contacting a template DNA molecule with a
non-conventional nucleotide and a recombinant family B DNA
polymerase of claim 3; and incorporating said non-conventional
nucleotide to synthesize a complementary DNA strand.
23. A method of sequencing DNA comprising the steps of: contacting
a template DNA strand with a sequencing primer, a chain-terminating
nucleotide analog, and a recombinant family B DNA polymerase of
claim 3; and incorporating said non-conventional nucleotide to
synthesize a complementary DNA strand, wherein incorporation of
said chain-terminating nucleotide analog results in the termination
of chain elongation, such that the nucleotide sequence of said DNA
strand is determined.
24. The method of claim 23, wherein said chain-terminating
nucleotide analog is a dideoxynucleotide.
25. The method of claim 24 wherein said dideoxynucleotide is
detectably labeled.
26. The method of claim 25, wherein said dideoxynucleotide is
fluorescently labeled.
27. The method of claim 26, wherein said dideoxynucleotide is
labeled with a moiety selected from the group consisting of
fluorescein and rhodamine.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions and methods
utilizing DNA polymerase enzymes with reduced discrimination for
non-conventional nucleotides. The enzymes of the invention are
useful in many applications calling for the detectable labeling of
nucleic acids and are particularly useful in DNA sequencing
applications.
BACKGROUND OF THE INVENTION
[0002] Detectable labeling of nucleic acids is required for many
applications in molecular biology, including applications for
research as well as clinical diagnostic techniques. A commonly used
method of labeling nucleic acids uses one or more unconventional
nucleotides and a polymerase enzyme that catalyzes the
template-dependent incorporation of the unconventional
nucleotide(s) into the newly synthesized complementary strand.
[0003] The ability of a DNA polymerase to incorporate the correct
deoxynucleotide is the basis for high fidelity DNA replication in
vivo. Amino acids within the active site of polymerases form a
specific binding pocket that favors the placement of the correct
complementary nucleotide opposite the template nucleotide. If a
mismatched nucleotide, ribonucleotide, or nucleotide analog fills
that position, the precise alignment of the amino acids contacting
the incoming nucleotide may be distorted into a position
unfavorable for DNA polymerization. Because of this, the
unconventional nucleotides or nucleotide analogs used to label DNA
tend to be incorporated into the elongated strand less efficiently
than do the standard deoxynucleotide triphosphates (dNTPs; the
so-called "standard" dNTPs include deoxyadenosine triphosphate
(dATP), deoxycytosine triphosphate (dCTP), deoxyguanosine
triphosphate (dGTP), and thymidine triphosphate (TTP)).
[0004] The reduced efficiency with which unconventional nucleotides
are incorporated by the polymerase increases the amount of the
unconventional nucleotide necessary for DNA labeling. The reduced
efficiency of incorporation of a particular nucleotide can also
adversely affect the performance of techniques or assays, such as
DNA sequencing, that depend upon unbiased incorporation of
unconventional nucleotides for homogeneous signal strength.
[0005] The identity and exact arrangement of the amino acids of a
DNA polymerase that contact an incoming nucleotide triphosphate
determine the nature of the nucleotides, both conventional and
unconventional, that may be incorporated by that polymerase enzyme.
Changes in the exact placement of the amino acids that contact the
incoming nucleotide triphosphate at any stage of binding or chain
elongation can dramatically alter the polymerase's capacity for
utilization of unusual or unconventional nucleotides. Sometimes
changes in distant amino acids can influence the incorporation of
nucleotide analogs due to indirect global or structural effects.
Polymerases with increased capacity to incorporate nucleotide
analogs are useful for labeling DNA or RNA strands with nucleotides
modified with signal moieties such as dyes, reactive groups or
unstable isotopes.
[0006] In addition to labeled nucleotides, an extremely important
class of modified nucleotides is the dideoxynucleotides. The
so-called "Sanger" or "dideoxy" DNA sequencing method (Sanger et
al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463, which is
incorporated herein by reference) relies upon the template-directed
incorporation of nucleotides onto an annealed primer by a DNA
polymerase from a mixture containing deoxy- and dideoxynucleotides.
The incorporation of a dideoxynucleotide results in chain
termination, the inability of the enzyme to catalyze further
extension of that strand. Electrophoretic separation of reaction
products results in a "ladder" of extension products wherein each
extension product ends in a particular dideoxynucleotide
complementary to the nucleotide opposite it in the template. The
distance of the dideoxynucleotide analog from the primer is
indicated by the length of the extension product. When four
reactions, each containing one of the four dideoxynucleotide
analogs ddA, ddC, ddG, or ddT (ddNTPs) are separated on the same
gel, the sequence of the template may be read directly from the
ladder patterns. Extension products may be detected in several
ways, including for example, the inclusion of isotopically- or
fluorescently-labeled primers, deoxynucleotide triphosphates or
dideoxynucleotide triphosphates in the reaction.
[0007] Fluorescent labeling has the advantages of faster data
collection, since detection may be performed while the gel is
running, and longer reads of sequence data from a single reaction
and gel. Further, fluorescent sequence detection has allowed
sequencing to be performed in a single reaction tube containing
four differentially-labeled fluorescent dye terminators (the
so-called dye-terminator method, Lee et al., 1992, Nucleic Acids
Res. 20: 2471, incorporated herein by reference).
[0008] A desirable quality of a polymerase useful for DNA
sequencing is improved incorporation of dideoxynucleotides.
Improved incorporation of dideoxynucleotides can make processes
such as DNA sequencing more cost effective by reducing the
requirement for expensive radioactive or fluorescent dye-labeled
dideoxynucleotides. Moreover, unbiased dideoxynucleotide
incorporation provides improved signal uniformity, leading to
increased accuracy of base determination. The even signal output
further allows subtle sequence differences caused by factors like
allelic variation to be detected. Allelic variation, which produces
two different half strength signals at the position of relevance,
can easily be concealed by the varied signal strengths caused by
polymerases with non-uniform ddNTP utilization.
[0009] Incorporation of ribonucleotides by the native form of DNA
polymerase is a rare event. Mutants that incorporate higher levels
of ribonucleotides can be used for application s such as sequencing
by partial ribosubstitution. In this system, a mixture of
ribonucleotides and deoxynucleotides corresponding to the same base
are incorporated by the mutant polymerase (Barnes, 1978 J. Mol.
Biol. 119: 83-99). When the ribosequencing reactions are exposed to
alkaline conditions and heat, fragmentation of the extended strand
occurs. If the reactions for all four bases are separated on a
denaturing acrylamide gel, they produce a sequencing ladder there
is a need in the art for polymerase mutants with higher utilization
of ribonucleotides for this alternative method of sequencing.
[0010] Alternatively, the incorporation of ribonucleotides followed
by alkaline hydrolysis could be utilized in a system that requires
random cleavage of DNA molecules such as DNA shuffling ((Stemmer,
1994, Nature, 370: 389-391) which has also been called molecular
breeding, sexual PCR and directed evolution).
[0011] Another desirable quality in a DNA labeling enzyme is
thermal stability. DNA polymerases exhibiting thermal stability
have revolutionized many aspects of molecular biology and clinical
diagnostics since the development of the polymerase chain reaction
(PCR), which uses cycles of thermal denaturation, primer annealing,
and enzymatic primer extension to amplify DNA templates. The
prototype thermostable DNA polymerase is Taq polymerase, originally
isolated from the thermophilic eubacterium Thermus aquaticus.
So-called "cycle sequencing" reactions using thermostable DNA
polymerases have the advantage of requiring smaller amounts of
starting template relative to conventional (i.e., non-cycle)
sequencing reactions.
[0012] There are three major families of DNA polymerases, termed
families A, B and C. The classification of a polymerase into one of
these three families is based on structural similarity of a given
polymerase to E. coli DNA polymerase I (Family A), II (Family B) or
III (family C). As examples, Family A DNA polymerases include, but
are not limited to Klenow DNA polymerase, Thermus aquaticus DNA
polymerase I (Taq polymerase) and bacteriophage T7 DNA polymerase;
Family B DNA polymerases, formerly known as .alpha.-family
polymerases (Braithwaite and Ito, 1991, Nuc. Acids Res. 19: 4045),
include, but are not limited to human .alpha., .delta. and
.epsilon. DNA polymerases, T4, RB69 and .phi.29 bacteriophage DNA
polymerases, and Pyrococcus furiosus DNA polymerase (Pfu
polymerase); and family C DNA polymerases include, but are not
limited to Bacillus subtilis DNA polymerase III, and E. coli DNA
polymerase III .alpha. and .epsilon. subunits (listed as products
of the dnaE and dnaQ genes, respectively, by Brathwaite and Ito,
1993, Nucleic Acids Res. 21: 787). An alignment of DNA polymerase
protein sequences of each family across a broad spectrum of
archaeal, bacterial, viral and eukaryotic organisms is presented in
Braithwaite and Ito (1993, supra), which is incorporated herein by
reference.
[0013] The term used to describe the tendency of DNA polymerases to
not to carry the incorporation of unnatural nucleotides into the
nascent DNA polymer is "discrimination". In Family A DNA
polymerases, the effective discrimination against incorporation of
dideoxynucleotide analogs is largely associated with a single amino
acid residue. The majority of enzymes from the Family A DNA
polymerases have a phenylalanine (phe or F) residue at the position
equivalent to F762 in E. coli Klenow fragment of DNA polymerase and
demonstrate a strong discrimination against dideoxynucleotides. A
few polymerases (e.g. T7 DNA polymerase) have a tyrosine (tyr or Y)
residue at the corresponding position and exhibit relatively weak
discrimination against dideoxynucleotides. Family A polymerases
with tyrosine at this position readily incorporate
dideoxynucleotides at levels equal to or only slightly different
from the levels at which they incorporate deoxynucleotides.
Conversion of the tyrosine or phenylalanine residues in the site
responsible for discrimination reverses the dideoxynucleotide
discrimination profile of the Family A enzymes (Tabor and
Richardson, 1995, Proc. Natl. Acad. Sci. USA 92: 6449).
[0014] Among the thermostable DNA polymerases, a mutant form of the
Family A DNA polymerase from Thermus aqaticus, known as AmpliTaq
FS.RTM. (Perkin Elmer), contains a F667Y mutation at the position
equivalent to F762 of Klenow DNA polymerase and exhibits increased
dideoxynucleotide uptake (i.e., reduced discrimination against
ddNTPs) relative to the wild-type enzyme. The reduced
discrimination for dideoxynucleotide uptake makes it more useful
for fluorescent and labeled dideoxynucleotide sequencing than the
wild-type enzyme.
[0015] The F667Y mutant of Taq DNA polymerase is not suited,
however, for use with fluorescein-labeled dideoxynucleotides,
necessitating the use of rhodamine dye terminators. Rhodamine dye
terminators that are currently utilized with Taq sequencing
reactions, however, stabilize DNA secondary structure, causing
compression of signal. Efforts to eliminate compression problems
have resulted in systems that use high amounts of the nucleotide
analog deoxyinosine triphosphate (dITP) in place of deoxyguanosine
triphosphate. While incorporation of (dITP) reduces the compression
of the signal, the presence of dITP in the reaction produces
additional complications including lowered reaction temperatures
and increased reaction times. Additionally, the use of rhodamine
dyes in sequencing requires undesirable post-reaction purification
(Brandis, 1999 Nuc. Acid Res. 27: 1912).
[0016] Family B DNA polymerases exhibit substantially different
structure compared to Family A DNA polymerases, with the exception
of the position of acidic residues involved in catalysis in the
so-called palm domain (Wang et al., 1997, Cell 89: 1087; Hopfner et
al., 1999, Proc. Natl. Acad. Sci. USA 96: 3600). The unique
structure of Family B DNA polymerases may permit a completely
different spectrum of interactions with nucleotide analogs, perhaps
allowing utilization of analogs which are unsuitable for use with
Family A DNA polymerases due to structural constraints.
Thermostable Family B DNA polymerases have been identified in
hyperthermophilic archaea. These organisms grow at temperatures
higher than 901C and their enzymes demonstrate greater
themostability (Mathur et al., 1992, Stratagies 5: 11) than the
thermophilic eubacterial Family A DNA polymerases. Family B
polymerases from hyperthermophilic archaea may be well suited
starting substrates for modification(s) to reduce discrimination
against non-conventional nucleotides.
[0017] Although the crystal structures of three Family B DNA
polymerases have been solved (Wang et al., 1997, supra; Hopfner,
K.-P. et al., 1999, Proc. Natl. Acad. Sci. 96: 3600; Zhao, 1999,
Structure Fold Des., 7: 1189), the structures of DNA-polymerase or
dNTP-polymerase co-complexes have not yet been reported. At
present, identification of amino acid residues contributing to
nucleotide analog discrimination can only be inferred from
extrapolation to Family A-dNTP structures or from mutagenesis
studies carried out with related Family B DNA polymerases (e.g.,
human pola, phage T4, phage .phi.29, T. litoralis DNA
polymerase).
[0018] Sequence comparison of the Family B DNA polymerases indicate
six conserved regions numbered I-VI (Braithwaite and Ito, 1993,
supra). The crystal structure of bacteriophage RB69 DNA polymerase
(Family B) proposed by Wang et al. (Wang et al., 1997, supra) shows
that Y416 in region II (which corresponds to Y409 in the Family B
DNA polymerase of Thermococcus species JDF-3) has the same position
as Y115 in HIV reverse transcriptase (RT) and E710 in the Klenow
fragment (Family A polymerases). Modeling of the dNTP and primer
template complex in RB69 was carried out using the atomic
coordinates of the reverse transcriptase-DNA cocrystal. This model
predicts the RB69 Y416 packs under the deoxyribose portion of the
dNTP. Tyrosine at this position has been implicated in ribose
selectivity, contributing to polymerase discrimination between
ribonucleotides and deoxribonucleotides in mammalian reverse
transcriptases (Y115) (Gao et al., 1997, Proc. Natl. Acad. Sci. USA
94: 407; Joyce, 1994, Proc. Natl. Acad. Sci. USA 94: 1619) and in
Family A DNA polymerases where modification of the corresponding
invariable glutamate residue (E710) reduces discrimination against
ribonucleotides (Gelfand et al., 1998, Pat. No. EPO823479; Astatke
et al., 1998, Proc. Natl. Acad. Sci. USA 96: 3402).
[0019] Mutagenesis studies done in Family B DNA polymerases also
implicate the region containing the analogous Y in region II in
dNTP incorporation and ribose selectivity. Mutations at the
corresponding Y865 in human DNA polymerase .alpha. affect
polymerase fidelity and sensitivity to dNTP nucleotide inhibitors
such as AZT-TP, which has a bulky 3'-azido group in place of the
3'-OH group, BuPdGTP, which contains a butylphenyl group attached
to the amino group at the C-2 position in the guanine base of dGTP
(resulting in a bulkier and more hydrophobic purine base
nucleotide) and aphidicolin, a competitive inhibitor of pyrimidine
deoxynucleotide triphosphate. Interestingly, the mutants showed no
difference in their uptake of ddCTP (Dong et al., 1993, J. Biol.
Chem. 268: 26143). Additionally, mutants of bacteriophage T4 DNA
polymerase, which have converted L412 to methionine (M) or
isoleucine (I) just one amino acid before the analogous Y (Y411),
show extreme and mild sensitivity, respectively, to the inorganic
pyrophosphate analog phosphonoacetic acid (PAA). Alterations in PAA
sensitivity have been shown to predict polymerase interactions with
nucleotide analogs. L412 in T4 DNA polymerase corresponds to L410
in Thermococcus species JDF-3 DNA polymerase. The L412M T4 DNA
polymerase mutant was inhibited with 50-fold less ddGTP than
wild-type polymerase while the K.sub.ms for dGTP was similar. As
stated by the authors in that study, "[d]espite the sensitivity of
the L412M DNA polymerase to ddGTP, there was no difference found in
the incorporation of ddNTPs by wild-type and L412M DNA polymerase."
(Reha-Krantz et al., 1993, J. Virol. 67: 60). In bacteriophage
.phi.29, mutations in region II (LYP where Y is analogous to
Thermococcus species JDF3 DNA polymerase Y409) produce mixed
results when challenged with PAA; P255S was hypersensitive to PAA
while L253V was shown to be less sensitive than the wild-type
enzyme (Blasco et al., 1993, J. Biol. Chem. 268: 24106). These data
support the role of the LYP region (region II) in
polymerase-nucleotide interactions, but improved incorporation of
ddNTPs was not achieved in these references.
[0020] In another study, extensive mutation of region II in the
archaeal Family B DNA polymerase from Thermococcus litoralis DNA
polymerase (VENT.TM. polymerase, New England Biolabs) was
performed. In that study, 26 different site-directed mutants were
made for the sole intent of examining nucleotide analog
discrimination (Gardner and Jack, 1999, Nucleic Acids Res. 27:
2545). Site-directed mutagenesis of VENT.TM. DNA polymerase
demonstrated that three mutations at Y412 (which corresponds to
JDF-3 DNA polymerase Y409) could alter nucleotide binding (Gardner
and Jack, 1999, supra). Y412V was most significant with a 2 fold
increase in dideoxynucleotide incorporation and a 200 fold increase
in the incorporation of ribonucleotide ATP. The mutation Y412F
showed no change in analog incorporation.
[0021] Region III of the Family B polymerases (also referred to as
motif B) has also been demonstrated to play a role in nucleotide
recognition. This region, which corresponds to AA 487 to 495 of
JDF-3 Family B DNA polymerase, has a consensus sequence
KX.sub.3NSXYG (Jung et al., 1990, supra; Blasco et al., 1992,
supra; Dong et al., 1993, J. Biol. Chem. 268: 21163; Zhu et al.,
1994, Biochem. Biophys. Acta 1219: 260; Dong and Wang, 1995, J.
Biol. Chem. 270: 21563), and is functionally, but not structurally
(Wang et al., 1997, supra), analogous to KX.sub.3(F/Y)GX.sub.2YG in
helix 0 of the Family A DNA polymerases. In Family A DNA
polymerases, such as the Klenow fragment and Taq DNA polymerases,
the O helix contains amino acids that play a major role in dNTP
binding (Astatke et al., 1998, J. Mol. Biol. 278: 147; Astatke et
al., 1995, J. Biol. Chem. 270: 1945; Polesky et al., 1992, J. Biol.
Chem 267: 8417; Polesky et al., 1990, J. Biol. Chem. 265: 14579;
Pandey et al., 1994, J. Biol. Chem. 269: 13259; Kaushik et al.,
1996, Biochem. 35: 7256). Specifically, helix 0 contains the F
(F763 in the Klenow fragment; F667 in Taq) which confers ddNTP
discrimination in Family A DNA polymerases
(KX.sub.3(F/Y)GX.sub.2YG) (Tabor and Richardson, 1995, supra).
[0022] Directed mutagenesis studies in region III of VENT.TM. DNA
polymerase also targeted an alanine analogous to A485 of the
Thermococcus species JDF-3 DNA polymerase (Jung et al., 1990,
supra). These mutants (A C, A S, A L, A I, A F and A V) exhibited a
range of specific activities from 0.12 to 1.2 times the polymerase
activity of the progenitor enzyme (Gardner and Jack, 1999, Nucl.
Acids Res. 27: 2545). The dideoxynucleotide incorporation ranged
from 4 to 15 times the unmutated enzyme. Interestingly, the mutant
with the highest dideoxynucleotide incorporation (15.times.) had a
specific activity of only 0.12.times. of the original enzyme.
[0023] Site-directed mutagenesis studies on the Family B DNA
polymerase from Thermococcus barossii modified each residue
independently in the sequence ILANSF, which corresponds to AA
residues 488-493 of the JDF-3 DNA polymerase, to tyrosine (Reidl et
al., U.S. Pat. No. 5,882,904). That study indicated that an L489Y
mutant exhibits approximately 3 times greater incorporation of
dideoxynucleotides relative to an enzyme bearing the wild-type
leucine residue at this site.
[0024] One area of active research involves the use of nucleic acid
arrays, often referred to as nucleic acid or DNA "chips", in the
simultaneous analyses of multiple different nucleic acid sequences.
Many of these applications, such as those described in U.S. Pat.
No. 5,882,904 (Reidl et al., issued Mar. 16, 1999) will benefit
from DNA polymerases exhibiting reduced discrimination against
non-conventional nucleotides, particularly fluorescently-labeled
non-conventional nucleotides. Applications being addressed in the
chip format include DNA sequencing and mutation detection, among
others. For example, the "mini-sequencing" methods (e.g., Pastinen
et al., 1997, Genome Res. 7: 606; Syvanen, 1999, Human Mutation 13:
1-10) and the arrayed primer extension (APEX) mutation detection
method (Shumaker et al., 1996, Hum. Mutat. 7: 346) and methods like
them can benefit from DNA polymerases with reduced discrimination
against fluorescently-labeled or other non-conventional
nucleotides. There is a need in the art for a non-discriminating
DNA polymerase for use in chip or gel based mini-sequencing
systems. Such a system would advantageously permit detection of
multiplexed single nucleotide polymorphisms (SNPs) and allow for
quantitative genotyping. Identification of sequence variation
permits the diagnosis and treatment of genetic disorders,
predisposition to multifactorial diseases, and sensitivity to new
or existing pharmaceutical products.
[0025] There is a need in the art for DNA polymerases with reduced
discrimination against unconventional nucleotides. There is
particularly a need in the art for thermostable DNA polymerases
exhibiting reduced discrimination against dideoxynucleotides, and
further, for DNA polymerases exhibiting reduced discrimination
against fluorescently labeled dideoxynucleotides.
SUMMARY OF THE INVENTION
[0026] The present invention relates to compositions and methods
utilizing DNA polymerase enzymes exhibiting reduced discrimination
against non-conventional nucleotides. Enzymes with this quality are
useful in many applications calling for the detectable labeling of
nucleic acids and are particularly useful in DNA sequencing
applications.
[0027] The invention further relates to a Family B DNA polymerase
having one or more mutations at a site or sites corresponding to
L408, P410, S345, and/or A485 of SEQ ID NO: 2, or a fragment
thereof which retains the ability to direct the template-dependent
polymerization of nucleic acid. The invention also encompasses
mutants and modified versions (e.g., reversibly inactivated
versions of a Family B polymerase prepared, for example, by
chemical modification or antibody complexing) of a Family B
polymerase mutated at sites corresponding to L408, P410 and or A485
of SEQ ID NO: 2.
[0028] In one embodiment, the DNA polymerase has a dual mutation
comprising comprising a serine to proline mutation at a site
corresponding to S345 of SEQ ID NO: 2; and a proline to leucine
mutation at a site corresponding to P410 of SEQ ID NO: 2.
[0029] The invention encompasses purified thermostable DNA
polymerase having an amino acid sequence presented in SEQ ID NO: 2
from residue 1 to 776.
[0030] In one embodiment, the thermostable DNA polymerase is
isolated from Thermococcus species JDF-3.
[0031] In another embodiment, the thermostable polymerase is
isolated from a recombinant organism transformed with a vector that
codes for the expression of Thermococcus species JDF-3 DNA
polymerase.
[0032] The invention further encompasses a recombinant vector
comprising the nucleotide sequence presented in SEQ ID NO: 1.
[0033] The invention further encompasses an isolated recombinant
polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a
functional fragment thereof.
[0034] The invention further encompasses an isolated recombinant
DNA polymerase from Thermococcus species JDF-3 that is 3' to 5'
exonuclease deficient.
[0035] In one embodiment, the isolated recombinant DNA polymerase
of has an aspartic acid to threonine or alanine mutation at the
amino acid corresponding to D141 of SEQ ID NO: 2 or a glutamic acid
to alanine mutation at the amino acid corresponding to E143 of SEQ
ID NO: 2.
[0036] In another embodiment, the isolated recombinant DNA
polymerase has an aspartic acid to threonine or alanine mutation at
the amino acid corresponding to D141 of SEQ ID NO: 2 and a glutamic
acid to alanine mutation at the amino acid corresponding to E143 of
SEQ ID NO: 2.
[0037] The invention further encompasses an isolated recombinant
DNA polymerase having reduced discrimination against
non-conventional nucleotides.
[0038] In one embodiment, the DNA polymerase is a Family B DNA
polymerase.
[0039] In another embodiment, the DNA polymerase further comprises
a mutation selected from the group consisting of: a leucine to
histidine mutation at a site corresponding to L408 of SEQ ID NO: 2;
a leucine to phenylalanine mutation at a site corresponding to L408
of SEQ ID NO: 2; a proline to leucine mutation at a site
corresponding to P410 of SEQ ID NO: 2; and an alanine to threonine
mutation at a site corresponding to A485 of SEQ ID NO: 2.
[0040] The invention further encompasses an isolated recombinant
DNA polymerase having the alanine to threonine mutation at the site
corresponding to A485 of SEQ ID NO:2 further comprising a mutation
selected from the group consisting of: a leucine to histidine
mutation at a site corresponding to L408 of SEQ ID NO: 2; a leucine
to phenylalanine mutation at a site corresponding to L408 of SEQ ID
NO: 2; and a proline to leucine mutation at a site corresponding to
P410 of SEQ ID NO: 2.
[0041] The invention further encompasses an isolated recombinant
DNA polymerase having the a proline to leucine mutation at a site
corresponding to P410 of SEQ ID NO: 2, further comprising of serine
to proline mutation at a site corresponding to S345 of SEQ ID NO:
2.
[0042] In another embodiment, the DNA polymerase has reduced
discrimination against a non-conventional nucleotide selected from
the group consisting of: dideoxynucleotides, ribonucleotides and
conjugated nucleotides.
[0043] In another embodiment, conjugated nucleotide is selected
from the group consisting of radiolabeled nucleotides,
fluorescently labeled nucleotides, biotin labeled nucleotides,
chemiluminescently labeled nucleotides and quantum dot labeled
nucleotides.
[0044] The invention further encompasses an isolated recombinant
Family B DNA polymerase comprising an alanine to threonine mutation
at the site corresponding to A485 of SEQ ID NO: 2 or a mutation at
a site corresponding to L408 or P410 of SEQ ID NO: 2, wherein the
DNA polymerase has reduced discrimination against non-conventional
nucleotides relative to the wild-type form of that polymerase.
[0045] In one embodiment, the Family B DNA polymerase is 3' to 5'
exonuclease deficient.
[0046] In another embodiment, the Family B DNA polymerase has a
mutation at an amino acid corresponding to D141 or E143 of SEQ ID
NO: 2.
[0047] In another embodiment, the Family B DNA polymerase has an
aspartic acid to threonine or alanine mutation at a site
corresponding to D141 of SEQ ID NO: 2.
[0048] In another embodiment, the Family B DNA polymerase has a
glutamic acid to alanine mutation at a site corresponding to E143
of SEQ ID NO: 2.
[0049] In another embodiment, the Family B DNA polymerase has a
glutamic acid to alanine mutation at a site corresponding to E143
of SEQ ID NO: 2 and has an aspartic acid to threonine or alanine
mutation at the amino acid corresponding to D141 of SEQ ID NO:
2.
[0050] In another embodiment, the Family B DNA polymerase is
thermostable.
[0051] In another embodiment, the Family B DNA polymerase is
archaeal.
[0052] In another embodiment, the Family B DNA polymerase comprises
a leucine to histidine mutation at a site corresponding to L408 of
SEQ ID NO: 2.
[0053] In another embodiment, the Family B DNA polymerase comprises
a leucine to phenylalanine mutation at a site corresponding to L408
of SEQ ID NO: 2.
[0054] In another embodiment, the Family B DNA polymerase comprises
a proline to leucine mutation at a site corresponding to P410 of
SEQ ID NO: 2.
[0055] In another embodiment, the Family B DNA polymerase comprises
an alanine to threonine mutation at a site corresponding to A485 of
SEQ ID NO: 2.
[0056] In another embodiment, the Family B DNA polymerase
comprising an alanine to threonine mutation at a site corresponding
to A485 of SEQ ID NO: 2 comprises a leucine to histidine mutation
at a site corresponding to L408 of SEQ ID NO: 2.
[0057] In another embodiment, the Family B DNA polymerase
comprising an alanine to threonine mutation at a site corresponding
to A485 of SEQ ID NO: 2 comprises a leucine to phenylalanine
mutation at a site corresponding to L408 of SEQ ID NO: 2.
[0058] In another embodiment, the Family B DNA polymerase
comprising an alanine to threonine mutation at a site corresponding
to A485 of SEQ ID NO: 2 comprises a proline to leucine mutation at
a site corresponding to P410 of SEQ ID NO: 2.
[0059] In another embodiment, the Family B DNA polymerase
comprising a proline to leucine mutation at a site corresponding to
P410 of SEQ ID NO: 2, further having a serine to proline mutation
at a site corresponding to S345 of SEQ ID NO: 2.
[0060] In another embodiment, the Family B DNA polymerase comprises
a serine to proline mutation at a site corresponding to S345 of SEQ
ID NO: 2, and may further comprise a mutation at a site
corresponding to T604 of SEQ ID NO: 2.
[0061] In another embodiment, the Family B DNA polymerase comprises
a tyrosine to cysteine mutation at a site corresponding to Y497 of
SEQ ID NO: 2, and may further comprise an isoleucine to valine
mutation at a site corresponding to 1630 of SEQ ID NO:2.
[0062] In another embodiment, the Family B DNA polymerase comprises
a glutamic acid to lysine mutation at a site corresponding to E645
of SEQ ID NO: 2.
[0063] In another embodiment, the Family B DNA polymerase comprises
a glutamic acid to lysine mutation at a site corresponding to E578
of SEQ ID NO: 2, and may further comprise an arginine to methionine
mutation at a site corresponding to R465 of SEQ ID NO: 2.
[0064] In another embodiment, the Family B DNA polymerase comprises
a leucine to glutamine mutation at a site corresponding to L396 of
SEQ ID NO: 2, and may further comprise a mutation at a site
corresponding to V401, N424, P569, E617, or V640 of SEQ ID NO:
2.
[0065] In another embodiment, the Family B DNA polymerase comprises
a serine to asparagene mutation at a site corresponding to S651 of
SEQ ID NO: 2.
[0066] In another embodiment, the Family B DNA polymerase comprises
a leucine to proline mutation at a site corresponding to L396 of
SEQ ID NO: 2, and may further comprise a mutation at a site
corresponding to E459 of SEQ ID NO: 2.
[0067] In another embodiment, the Family B DNA polymerase comprises
a leucine to proline mutation at a site corresponding to L456 of
SEQ ID NO: 2, and may further comprise a mutation at a site
corresponding to E658 of SEQ ID NO: 2.
[0068] In another embodiment, the Family B DNA polymerase comprises
a leucine to histidine mutation at a site corresponding to L408 of
SEQ ID NO: 2, and may further comprise a mutation at a site
corresponding to V437, or L478 of SEQ ID NO: 2. The L408H mutation
was isolated both in the dideoxynucleotide and the
dye-dideoxynucleotide screens described herein.
[0069] In another embodiment, the Family B DNA polymerase comprises
an tyrosine to asparagine mutation at a site corresponding to Y496
of SEQ ID NO: 2.
[0070] In another embodiment, the Family B DNA polymerase has
reduced discrimination against a non-conventional nucleotide
selected from the group consisting of: dideoxynucleotides,
ribonucleotides and conjugated nucleotides.
[0071] In another embodiment, the conjugated nucleotide is selected
from the group consisting of radiolabeled nucleotides,
fluorescently labeled nucleotides, biotin labeled nucleotides,
chemiluminescently labeled nucleotides and quantum dot labeled
nucleotides.
[0072] In another embodiment, an isolated recombinant DNA
polymerase having reduced discrimination against non-conventional
nucleotides or an isolated recombinant Family B DNA polymerase
comprising an alanine to threonine mutation at the site
corresponding to A485 of SEQ ID NO: 2 or a mutation at a site
corresponding to L408 or P410 of SEQ ID NO: 2, wherein the DNA
polymerase has reduced discrimination against non-conventional
nucleotides relative to the wild-type form of that polymerase
further comprises a mutation at an amino acid residue in the
polymerase that corresponds to a mutation selected from the group
consisting of: a Y to V mutation at amino acid 409 of SEQ ID NO:2;
an A to C, S, L, I, F, or V mutation at amino acid 485 of SEQ ID
NO: 2; a Y to S mutation at amino acid 494 of SEQ ID NO: 2; a Y to
L mutation at amino acid 496 of SEQ ID NO: 2; and an A to Y
mutation at amino acid 490 of SEQ ID NO: 2.
[0073] In another embodiment, an isolated recombinant DNA
polymerase having reduced discrimination against non-conventional
nucleotides or an isolated recombinant Family B DNA polymerase
comprising an alanine to threonine mutation at the site
corresponding to A485 of SEQ ID NO: 2 or a mutation at a site
corresponding to L408 or P410 of SEQ ID NO: 2, wherein the DNA
polymerase has reduced discrimination against non-conventional
nucleotides relative to the wild-type form of that polymerase
further comprises a mutation at an amino acid of the polymerase
corresponding to one of amino acids 483 to 496, inclusive, of SEQ
ID NO: 2.
[0074] In one embodiment, the mutation is at an amino acid of the
polymerase corresponding to one of amino acids 485, 490, 494, or
496 of SEQ ID NO: 2.
[0075] The invention further encompasses an isolated recombinant
Family B DNA polymerase comprising an alanine to threonine mutation
at an amino acid corresponding to A485T of SEQ ID NO: 2 and at
least one substitution in the polymerase of an amino acid
corresponding to L408, Y409, or P410, respectively, of SEQ ID NO:
2.
[0076] The invention further encompasses an isolated recombinant
Family B DNA polymerase comprising an amino acid other than A at an
amino acid of the polymerase corresponding to A485 of SEQ ID NO: 2,
and at least one substitution in the polymerase of an amino acid
corresponding to L408, Y409, or P410, respectively, of SEQ ID NO:
2.
[0077] The invention further encompasses a recombinant vector
comprising a nucleic acid sequence encoding the Family B DNA
polymerase.
[0078] The invention further encompasses a method of labeling a
complementary strand of DNA, the method comprising the step of
contacting a template DNA molecule with a recombinant Family B DNA
polymerase from Thermococcus species JDF-3, wherein the DNA
polymerase has reduced discrimination against non-conventional
nucleotides, and a non-conventional nucleotide, under conditions
and for a time sufficient to permit the DNA polymerase to
synthesize a complementary DNA strand and to incorporate the
non-conventional nucleotide into the synthesized complementary DNA
strand. The invention further encompasses a method of labeling a
complementary strand of DNA, the method comprising the step of
contacting a template DNA molecule with a recombinant Family B DNA
polymerase comprising an alanine to threonine mutation at a site
corresponding to A485 of SEQ ID NO: 2 or a mutation at a site
corresponding to L408 or P410 of SEQ ID NO: 2, wherein the DNA
polymerase has reduced discrimination against non-conventional
nucleotides, and a non-conventional nucleotide, under conditions
and for a time sufficient to permit the DNA polymerase to
synthesize a complementary DNA strand and to incorporate the
non-conventional nucleotide into the synthesized complementary DNA
strand.
[0079] In one embodiment, the recombinant Family B DNA polymerase
is 3' to 5' exonuclease deficient.
[0080] In another embodiment, the recombinant Family B polymerase
comprises a leucine to histidine mutation at a site corresponding
to amino acid L408 of SEQ ID NO: 2.
[0081] In another embodiment, the recombinant Family B polymerase
comprises a leucine to phenylalanine mutation at a site
corresponding to amino acid L408 of SEQ ID NO: 2.
[0082] In another embodiment, the recombinant Family B polymerase
comprises a proline to leucine mutation at a site corresponding to
amino acid P410 of SEQ ID NO: 2.
[0083] In another embodiment, the recombinant Family B polymerase
comprises an alanine to threonine mutation at a site corresponding
to amino acid A485 of SEQ ID NO: 2.
[0084] In another embodiment, the recombinant Family B polymerase
comprising an alanine to threonine mutation at a site corresponding
to amino acid A485 of SEQ ID NO: 2 comprises a leucine to histidine
mutation at an amino acid corresponding to L408 of SEQ ID NO:
2.
[0085] In another embodiment, the recombinant Family B polymerase
comprising an alanine to threonine mutation at a site corresponding
to amino acid A485 of SEQ ID NO: 2 comprises a leucine to
phenylalanine mutation at an amino acid corresponding to L408 of
SEQ ID NO: 2.
[0086] In another embodiment, the recombinant Family B polymerase
comprising an alanine to threonine mutation at a site corresponding
to amino acid A485 of SEQ ID NO: 2 comprises a proline to leucine
mutation at an amino acid corresponding to P410 of SEQ ID NO:
2.
[0087] In another embodiment, the recombinant Family B polymerase
has reduced discrimination against a non-conventional nucleotide
selected from the group consisting of: dideoxynucleotides,
ribonucleotides, and conjugated nucleotides.
[0088] In another embodiment, the conjugated nucleotide is selected
from the group consisting of radiolabeled nucleotides,
fluorescently labeled nucleotides, biotin labeled nucleotides,
chemiluminescently labeled nucleotides and quantum dot labeled
nucleotides.
[0089] The invention further encompasses a method of sequencing DNA
comprising the steps of contacting a DNA strand to be sequenced
with a sequencing primer, a recombinant Family B DNA polymerase
from Thermococcus species JDF-3, wherein the DNA polymerase has
reduced discrimination against non-conventional nucleotides, and a
chain-terminating nucleotide analog, under conditions that permit
the DNA polymerase to synthesize a complementary DNA strand, and to
incorporate nucleotides into the synthesized complementary DNA
strand, wherein incorporation of a chain-terminating nucleotide
analog results in the termination of chain elongation, such that
the nucleotide sequence of the template DNA strand is
determined.
[0090] The invention further encompasses a method of sequencing DNA
comprising the steps of contacting a DNA strand to be sequenced
with a sequencing primer, a recombinant Family B DNA polymerase
comprising an alanine to threonine mutation at a site corresponding
to A485 of SEQ ID NO: 2 or a mutation at a site corresponding to
L408, S345 or P410 of SEQ ID NO: 2, where the DNA polymerase has
reduced discrimination against non-conventional nucleotides, and a
chain-terminating nucleotide analog, under conditions that permit
the DNA polymerase to synthesize a complementary DNA strand, and to
incorporate nucleotides into the synthesized complementary DNA
strand, wherein incorporation of a chain-terminating nucleotide
analog results in the termination of chain elongation, such that
the nucleotide sequence of the template DNA strand is
determined.
[0091] In one embodiment, the recombinant DNA polymerase is
deficient in 3' to 5' exonuclease activity.
[0092] In another embodiment, the recombinant Family B polymerase
has a leucine to histidine mutation at a site corresponding to
amino acid L408 of SEQ ID NO: 2.
[0093] In another embodiment, the recombinant Family B polymerase
has a leucine to phenylalanine mutation at a site corresponding to
amino acid L408 of SEQ ID NO: 2.
[0094] In another embodiment, the recombinant Family B polymerase
has a proline to leucine mutation at a site corresponding to amino
acid P410 of SEQ ID NO: 2.
[0095] In another embodiment, the Family B DNA polymerase
comprising a proline to leucine mutation at a site corresponding to
P410 of SEQ ID NO: 2, further having a serine to proline mutation
at a site corresponding to S345 of SEQ ID NO: 2.
[0096] In another embodiment, the recombinant Family B polymerase
has an alanine to threonine mutation at a site corresponding to
amino acid A485 of SEQ ID NO: 2.
[0097] In another embodiment, the recombinant Family B polymerase
having an alanine to threonine mutation at a site corresponding to
amino acid A485 of SEQ ID NO: 2 has a leucine to histidine mutation
at a site corresponding to L408 of SEQ ID NO: 2.
[0098] In another embodiment, the recombinant Family B polymerase
having an alanine to threonine mutation at a site corresponding to
amino acid A485 of SEQ ID NO: 2 has a leucine to phenylalanine
mutation at a site corresponding to L408 of SEQ ID NO: 2.
[0099] In another embodiment, the recombinant Family B polymerase
having an alanine to threonine mutation at a site corresponding to
amino acid A485 of SEQ ID NO: 2 has a proline to leucine mutation
at a site corresponding to P410 of SEQ ID NO: 2.
[0100] In another embodiment, the chain-terminating nucleotide
analog is a dideoxynucleotide.
[0101] In another embodiment, the dideoxynucleotide is detectably
labeled.
[0102] In another embodiment, the dideoxynucleotide is
fluorescently labeled.
[0103] In another embodiment, the dideoxynucleotide is labeled with
a moiety selected from the group consisting of fluorescein and
rhodamine.
[0104] The invention also encompasses a kit for performing the
methods disclosed herein.
[0105] The invention also encompasses methods of making a
recombinant DNA polymerase as disclosed here, comprising culturing
a host cell containing a nucleic acid sequence encoding said
polymerase under conditions which permit production of said DNA
polymerase.
[0106] The invention encompasses a mixture of a mutant DNA
polymerase described herein and another DNA polymerase such as Taq
DNA polymerase (preferably the mutant form, F667Y). Such a mixture
is useful in that it may increase signal uniformity generated from
polymerization of a labeled nucleotide into a synthetic
nucleotide.
[0107] As used herein, "discrimination" refers to the tendency of
DNA polymerase to not incorporate non-conventional nucleotides into
a nascent DNA polymer. DNA polymerase has the ability to sense
nucleotide structure, including but not limited to nucleotide base
complementarity, and structural features of the sugar and
heterocyclic base, thereby allowing DNA polymerase to
preferentially utilize conventional deoxynucleotides rather than
non-conventional nucleotides for incorporation into a nascent
polymer. DNA polymerase strongly prefers to incorporate the
conventional deoxynucleotides dATP, dCTP, dGTP and TTP into DNA
polymers; the polymerase is unlikely to progress with an
unconventional nucleotide in its binding pocket.
[0108] As used herein, "reduced discrimination" refers to a
reduction of at least 50% in the tendency of a DNA polymerase to
exclude a non-conventional nucleotide from (that is, to not
incorporate non-conventional nucleotides into) a nascent DNA
polymer, relative to a parental or wild type DNA polymerase which
does not exhibit reduced discrimination. The preference of DNA
polymerase to incorporate the conventional deoxynucleotides dATP,
dCTP, dGTP and TTP rather than non-conventional nucleotides into
DNA polymers is thereby reduced compared to the natural level of
preference, such that non-conventional nucleotides are more readily
incorporated into DNA polymers by DNA polymerase. According to the
invention, a polymerase exhibiting reduced discrimination will
exhibit reduced discrimination against at least one
non-conventional nucleotides, but may not exhibit reduced
discrimination against all non-conventional nucleotides.
[0109] According to the invention, discrimination is quantitated by
measuring the concentration of a non-conventional nucleotide
required to inhibit the incorporation of the corresponding
conventional nucleotide by 50%. This concentration is referred to
herein as the "I.sub.50%" for a non-conventional nucleotide.
Discrimination against a given non-conventional nucleotide is
"reduced" if the I.sub.50% for that non-conventional nucleotide is
reduced by at least two fold (50%) relative to an identical assay
containing, in place of the mutant DNA polymerase, a parental DNA
polymerase.
[0110] Alternatively, reduced discrimination may be quantitated by
determining the amount of a non-conventional nucleotide (for
example, a dideoxynucleotide, ribonucleotide, or cordycepin)
required in a reaction with a mutant polymerase having reduced
discrimination to generate a sequencing ladder identical to a
sequencing ladder produced using the wild-type or parental enzyme.
The sequencing ladder can be examined, for example, in the range of
1 to 400 bases from the primer terminus, and the ladders will be
identical in the number of extension products generated as well as
the lengths of extension products generated in the sequencing
reaction. For this type of assay, a constant amount of dNTPs and
varying amounts of non-conventional nucleotides are used to
generate a sequencing ladder with both the wild-type (or parental)
enzyme and the mutant poplymerase (for ribonucleotides, a
sequencing ladder is generated by alkali cleavage of the
polymerization products). See Gardner & Jack, 1999, supra. A
mutant exhibits reduced discrimination if it requires at least
two-fold (50%) less, five-fold (80%) less, ten-fold (100%) less,
etc. of the amount of the non-conventional nucleotide used by the
wild-type or parental polymerase to produce a sequencing ladder
identical (with respect to the number and length of extension
products generated) to that generated by the wild-type or parental
enzyme.
[0111] As used herein, the term "parental" or "progenitor" refers
to a polymerase used as the starting material in generating a
mutant polymerase having reduced discrimination. The term
"parental" is meant to encompass not only a so-called "wild-type"
enzyme as it occurs in nature, but also intermediate forms, for
example, an exonuclease deficient enzyme that is used as the
starting material for generating an enzyme with reduced
discrimination against non-conventional nucleotides.
[0112] As used herein, "non-conventional nucleotide" refers to a) a
nucleotide structure that is not one of the four conventional
deoxynucleotides dATP, dCTP, dGTP, and TTP recognized by and
incorporated by a DNA polymerase, b) a synthetic nucleotide that is
not one of the four conventional deoxynucleotides in (a), c) a
modified conventional nucleotide, or d) a ribonucleotide (since
they are not normally recognized or incorporated by DNA
polymerases) and modified forms of a ribonucleotide.
Non-conventional nucleotides include but are not limited to those
listed in Table III, which are commercially available, for example,
from New England Nuclear. Any one of the above non-conventional
nucleotides may be a "conjugated nucleotide", which as used herein
refers to nucleotides bearing a detectable label, including but not
limited to a fluorescent label, isotope, chemiluminescent label,
quantum dot label, antigen, or affinity moiety.
[0113] As used herein, the term "cell", "cell line" and "cell
culture" can be used interchangeably and all such designations
include progeny. Thus, the words "transformants" or "transformed
cells" includes the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same functionality as screened for in the originally
transformed cell are included.
[0114] As used herein, the term "organism transformed with a
vector" refers to an organism carrying a recombinant gene
construct.
[0115] As used herein, "thermostable" refers to a property of a DNA
polymerase, such that the enzyme active at elevated temperatures
and is resistant to DNA duplex-denaturing temperatures in the range
of about 93.degree. C. to about 97.degree. C. "Active" means the
enzyme retains the ability to effect primer extension reactions
when subjected to elevated or denaturing temperatures for the time
necessary to effect denaturation of double-stranded nucleic acids.
Elevated temperatures as used herein refer to the range of about
70.degree. C. to about 75.degree. C., whereas non-elevated
temperatures as used herein refer to the range of about 35.degree.
C. to about 50.degree. C.
[0116] As used herein, "archaeal" refers to an organism or to a DNA
polymerase from an organism of the kingdom Archaea.
[0117] As used herein, "sequencing primer" refers to an
oligonucleotide, whether natural or synthetic, which serves as a
point of initiation of nucleic acid synthesis by a polymerase
following annealing to a DNA strand to be sequenced. A primer is
typically a single-stranded oligodeoxyribonucleotide. The
appropriate length of a primer depends on the intended use of the
primer, but for DNA sequencing applications typically ranges from
about 15 to about 40 nucleotides in length.
[0118] As used herein, "Family B DNA polymerase" refers to any DNA
polymerase that is classified as a member of the Family B DNA
polymerases, where the Family B classification is based on
structural similarity to E. coli DNA polymerase II. The Family B
DNA polymerases, formerly known as .alpha.-family polymerases,
include, but are not limited to those listed as such in Table
I.
[0119] As used herein, "Family A DNA polymerase" refers to any DNA
polymerase that is classified as a member of the Family A DNA
polymerases, where the Family A classification is based on
structural similarity to E. coli DNA polymerase I. Family A DNA
polymerases include, but are not limited to those listed as such in
Table I.
[0120] As used herein, "3' to 5' exonuclease deficient" or "3' to
5' exo.sup.-" refers to an enzyme that substantially lacks the
ability to remove incorporated nucleotides from the 3' end of a DNA
polymer. DNA polymerase exonuclease activities, such as the 3' to
5' exonuclease activity exemplified by members of the Family B
polymerases, can be lost through mutation, yielding an
exonuclease-deficient polymerase. As used herein, a DNA polymerase
that is deficient in 3' to 5' exonuclease activity substantially
lacks 3' to 5' exonuclease activity. "Substantially lacks"
encompasses a complete lack of activity, or a "substantial" lack of
activity. "Substantial" lack of activity means that the 3'
exonuclease activity of the mutant polymerase relative to the
parental polymerase is 0.03%, and also may be 0.05%, 0.1%, 1%, 5%,
10%, or 20%, but is not higher than 50% of the 3' exonuclease
activity of the parental or wild type polymerase.
[0121] As used herein, "mutation" refers to a change introduced
into a starting parental DNA sequence that changes the amino acid
sequence encoded by the DNA. The consequences of a mutation include
but are not limited to the creation of a new character, property,
function, or trait not found in the protein encoded by the parental
DNA.
[0122] As used herein, "wild-type" refers to the typical state of
an organism, strain, gene, protein or characteristic as it occurs
in nature. The wild-type is therefore the natural state that is
distinguished from a mutant, which was derived from the wild type
by introduction of change(s) to the wild-type.
[0123] As used herein, "corresponding" refers to sequence
similarity in a comparison of two or more nucleic acids or
polypeptides, where functionally equivalent domains or
sub-sequences are identified; such functionally equivalent domains
or sub-sequences or amino acids within such a domain or
sub-sequence are said to "correspond". That is, two or more
sequences are compared through a comparative alignment analysis in
which an entire sequence is examined for regions of sequence that
are similar or identical, and thus regions likely to be
functionally equivalent to regions from the other sequence(s) are
identified.
[0124] As used herein in reference to comparisons of an amino acid,
amino acid sequence, or protein domain, the term "similar" refers
to amino acids or domains that although not identical, represent
"conservative" differences. By "conservative" is meant that the
differing amino acid has like characteristics with the amino acid
in the corresponding or reference sequence. Typical conservative
substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;
among the acidic residues Asp and Glu; among Asn and Gln; and among
the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
In calculating the degree (most often as a percentage) of
similarity between two polypeptide sequences, one considers the
number of positions at which identity or similarity is observed
between corresponding amino acid residues in the two polypeptide
sequences in relation to the entire lengths of the two molecules
being compared.
[0125] As used herein, the term "functionally equivalent" means
that a given motif, region, or amino acid within a motif or region
performs the same function with regard to the overall function of
the enzyme as a motif, region or amino acid within a motif or
region performs in another enzyme.
[0126] As used herein, "chain terminating nucleotide analog" refers
to a nucleotide analog that once incorporated cannot serve as a
substrate for subsequent extension by a DNA polymerase, thereby
terminating the elongation of a DNA polymer by a DNA polymerase.
Such a nucleotide analog typically lacks a hydroxyl group on its
sugar moiety to which DNA polymerase can synthesize a
phosphodiester bond with an incoming nucleotide. Chain terminating
nucleotide analogs are a subset of non-conventional nucleotides,
and include but are not limited to dideoxynucleotides.
[0127] As used herein, "detectably labeled" refers to a structural
modification that incorporates a functional group (label) that can
be readily detected by various means. Compounds that can be
detectably labeled include but are not limited to nucleotide
analogs. Detectable nucleotide analog labels include but are not
limited to fluorescent compounds, isotopic compounds,
chemiluminescent compound, quantum dot labels, biotin, enzymes,
electron-dense reagents, and haptens or proteins for which antisera
or monoclonal antibodies are available. The various means of
detection include but are not limited to spectroscopic,
photochemical, biochemical, immunochemical, or chemical means.
[0128] As used herein in reference to a polynucleotide or
polypeptide, the term "isolated" means that a naturally occurring
sequence has been removed from its normal cellular environment or
is synthesized in a non-natural environment (e.g., artificially
synthesized). Thus, the sequence may be in a cell-free solution or
placed in a different cellular environment. The term does not imply
that the sequence is the only nucleotide or polypeptide chain
present, but that it is essentially free (about 90-95% pure at
least) of non-nucleotide or non-polypeptide material, respectively,
naturally associated with it.
[0129] As used herein, the term "recombinant" refers to a
polynucleotide or polypeptide that is altered by genetic
engineering (i.e., by modification or manipulation of the genetic
material encoding that polynucleotide or polypeptide).
[0130] The invention encompasses full length mutant DNA
polymerases, as described herein, as well as a functional fragment
of a mutant polymerase, that is, a fragment of a DNA polymerase
that is less than the entire amino acid sequence of the mutant
polymerase and retains the ability, under at least one set of
conditions, to catalyze the polymerization of a polynucleotide.
Such a functional fragment may exist as a separate entity, or it
may be a constituent of a larger polypeptide, such as a fusion
protein.
[0131] As used herein, the term "complementary DNA strand" refers
to that DNA molecule synthesized from a template DNA molecule by a
DNA polymerase in a primer extension reaction.
[0132] As used herein, the term "template DNA molecule" refers to
that strand of a nucleic acid from which a complementary nucleic
acid strand is synthesized by a DNA polymerase, for example, in a
primer extension reaction.
[0133] Further features and advantages of the invention will become
more fully apparent in the following description of the embodiments
and drawings thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0134] FIG. 1 shows the DNA sequence encoding Thermococcus species
JDF-3 DNA polymerase (intein removed) (SEQ ID NO: 1).
[0135] FIG. 2 shows the amino sequence of Thermococcus species
JDF-3 DNA polymerase (intein removed) (SEQ ID NO: 2).
[0136] FIG. 3 shows the amino acid sequence of the genomic clone
encoding Thermococcus species JDF-3 DNA polymerase (SEQ ID NO: 3).
The position of an intein, removed by post-translational
processing, is shown.
[0137] FIG. 4 shows the DNA sequence of the genomic clone encoding
Thermococcus species JDF-3 DNA polymerase (SEQ ID NO: 4). DNA
sequences are shown which correspond to 5' and 3' untranslated
regions, polymerase-coding regions (exteins), and an intein-coding
region.
[0138] FIG. 5 shows nucleotide incorporation by JDF-3 mutants.
Lambda phage clones which incorporated .sup.33P-labeled ddNTPs in
the primary library screen were rescreened to assess .sup.33P-ddNTP
incorporation in the presence of: (panel 1) 0.5 mM MnCl.sub.2 or
(panel 2) 1.5 mM MgCl.sub.2. Polymerase activity was measured using
.sup.33P-dNTPs in the presence of 1.5 mM MgCl.sub.2 (panel 3).
Nucleotide utilization is shown for clones 1-18 and for the
parental #550 clone.
[0139] FIG. 6 shows .sup.33P-ddNTP cycle sequencing reactions
performed using JDF-3 polymerase mutants. Purified JDF-3 mutants
were substituted into the Thermo Sequenase radiolabeled terminator
cycle sequencing kit. DNA sequencing ladders were generated as per
the kit's instructions using the following polymerases: (A) Thermo
Sequenase (B) JDF-3 #550 clone (parental) (C) JDF-3 A485T mutant
(clone p12) (D) JDF-3 P410L mutant (clone p11) (E) JDF-3 P410L
mutant (clone p8). The top of the original sequencing gel is shown
on the side. The lanes are: (bottom) ddGTP, ddATP, ddTTP, ddCTP
(top). Clones p8, p11, and p12 contain ancillary mutations and an
amino-terminal tag.
[0140] FIG. 7 shows cycle sequencing reactions performed using
dye-labeled ddNTPs and JDF-3 polymerase mutants. DNA sequencing
ladders were generated using (1) 2.14 .mu.M dNTP: 0.0214 .mu.M
ddNTP; (2) 2.14 .mu.M dNTP: 0.214 .mu.M ddNTP; or (3) 2.14 .mu.M
dNTP: 2.14 .mu.M ddNTP. The following purified DNA polymerases were
used: (A) JDF-3 #550 clone (parental) (B) Thermo Sequenase (C)
JDF-3 P410L mutant (clone p8, contains ancillary mutations and an
amino tag) (E) JDF-3 L408H mutant (clone 1-1). The top of the
original sequencing gel is shown on the right hand side.
[0141] FIG. 8 shows cycle sequencing reactions performed using the
JDF-3 P410L/A485T double mutant and .alpha.-.sup.33P
Dideoxynucleotides. DNA sequencing ladders were generated using the
JDF-3 P410L/A485T double mutant at (A) 2 .mu.l (B) 1 .mu.l (C) 0.5
.mu.l, the JDF-3 P410L mutant (clone p8, contains ancillary
mutations and an amino-terminal tag)(D), or Thermo Sequenase (E).
The top of the original sequencing gel is shown on the left side.
The lanes are: (bottom) ddGTP, ddATP, ddTTP, ddCTP (top).
[0142] FIG. 9 shows the result of ribonucleotide incorporation
assays using exo JDF-3 (550) and mutants of this progenitor clone.
The ratios of ribonucleotide versus deoxynucleotide incorporation
are plotted for JDF-3 550, JDF-3 L408H, JDF-3 L408F and JDF-3
A485T.
[0143] FIG. 10 shows the traces of the sequence generated by four
versions of JDF-3 DNA polymerase and FAM ddCTP. Panel A shows the
minimal trace produced by the progenitor polymerase JDF-3 550,
Panel B demonstrates the slightly improved trace made by JDF-3
P410L, Panel C shows the sequence generated by the double mutant
S345P and P410L, and Panel D shows the trace created by JDF-3
S345P.
[0144] FIG. 11 shows the difference in peak uniformity demonstrated
by Thermo Sequenase in Panel A and the double mutant JDF-3
S345P+P410L in Panel B.
[0145] FIG. 12 shows the separated products of 3' extension of a
labeled oligonucleotide with the dideoxynucleotide thymidine
triphosphate of ROX-ddUTP (New England Nuclear (NEN) NEL476) or
Fluorescein-12-ddUTP (NEN NEL401). Mutant 4 is JDF-3 S345P, Mutant
2 is JDF-3 P410L, Mutant 3 is JDF-3 A485T and Mutant 5 is Y496N. F
indicates FLU ddUTP and R indicates ROX ddUTP.
[0146] FIG. 13 shows a graphic representation of the relative band
intensities form FIG. 12. The numerical values are generated by
dividing the intensity value of the ddTTP band into the intensity
value for the Fluroescein-12-ddUTP bands.
[0147] FIG. 14 shows the sequence alignment of
dye-dideoxynucleotide selected JDF-3 mutants (amino acids 301-480).
Nucleic acid residues highlighted by white boxes indication the
location of a mutation. The mutation S345P is one of two mutations
present in mutant 28.
[0148] FIG. 15 shows the sequence alignment of
dye-dideoxynucleotide selected JDF-3 (amino acids 481-660). Nucleic
acid residues highlighted by white boxes indication the location of
a mutation.
DESCRIPTION
[0149] The invention is based on the discovery of Family B DNA
polymerases that bear one or more genetic alterations resulting in
reduced discrimination against non-conventional nucleotides
relative to their unmodified wild-type forms. All references
described herein are incorporated by reference herein in their
entirety.
[0150] Family B DNA Polymerase Exhibiting Reduced Discrimination
Against Non-Conventional Nucleotides:
[0151] A. DNA Polymerases Useful According to the Invention
[0152] According to the invention, DNA polymerases of Family B may
be mutated to generate enzymes exhibiting reduced discrimination
against non-conventional nucleotides. Table I includes a
non-limiting list of known DNA polymerases categorized by
family.
1TABLE I DNA POLYMERASES BY FAMILY Reference FAMILY A DNA
POLYMERASES Bacterial DNA Polymerases a) E. coli DNA polymerase I
(1) b) Streptococcus pneumoniae DNA polymerase I (2) c) Thermus
aquaticus DNA polymerase I (3) d) Thermus flavus DNA polymerase I
(4) e) Thermotoga maritima DNA polymerase I Bacteriophage DNA
Polymerases a) T5 DNA polymerase (5) b) T7 DNA polymerase (6) c)
Spo1 DNA polymerase (7) d) Spo2 DNA polymerase (8) Mitochondrial
DNA polymerase Yeast Mitochondrial DNA polymerase II (9, 10, 11)
FAMILY B DNA POLYMERASES Bacterial DNA polymerase E. coli DNA
polymerase II (15) Bacteriophage DNA polymerase a) PRD1 DNA
polymerase (16, 17) b) .phi.29 DNA polymerase (18) c) M2 DNA
polymerase (19) d) T4 DNA polymerase (20) Archaeal DNA polymerase
a) Thermococcus litoralis DNA polymerase (Vent) (21) b) Pyrococcus
furiosus DNA polymerase (22) c) Sulfolobus solfataricus DNA
polymerase (23) d) Thermococcus gorgonarius DNA polymerase (64) e)
Thermococcus species TY (65) f) Pyrococcus species strain KODI (66)
g) Sulfolobus acidocaldarius (67) h) Thermococcus species 9.degree.
N-7 (68) i) Pyrodictium occultum (69) j) Methanococcus voltae (70)
k) Desulfurococcus strain TOK (D. Tok Pol) (71) Eukaryotic Cell DNA
polymerase (1) DNA polymerase alpha a) Human DNA polymerase (alpha)
(24) b) S. cerevisiae DNA polymerase (alpha) (25) c) S. pombe DNA
polymerase I (alpha) (26) d) Drosophila melanogaster DNA polymerase
(alpha) (27) e) Trypanosoma brucei DNA polymerase (alpha) (28) (2)
DNA polymerase delta a) Human DNA polymerase (delta) (29, 30) b)
Bovine DNA polymerase (delta) (31) c) S. cerevisiae DNA polymerase
III (delta) (32) d) S. pombe DNA polymerase III (delta) (33) e)
Plasmodiun falciparum DNA polymerase (delta) (34) (3) DNA
polymerase epsilon S. cerevisiae DNA polymerase II (epsilon) (35)
(4) Other eukaryotic DNA polymerase S. cerevisiae DNA polymerase
Rev3 (36) Viral DNA polymerases a) Herpes Simplex virus type 1 DNA
polymerase (37) b) Equine herpes virus type 1 DNA polymerase (38)
c) Varicella-Zoster virus DNA polymerase (39) d) Epstein-Barr virus
DNA polymerase (40) e) Herpes virus saimiri DNA polymerase (41) f)
Human cytomegalovirus DNA polymerase (42) g) Murine cytomegalovirus
DNA polymerase (43) h) Human herpes virus type 6 DNA polymerase
(44) i) Channel Catfish virus DNA polymerase (45) j) Chlorella
virus DNA polymerase (46) k) Fowlpox virus DNA polymerase (47) l)
Vaccinia virus DNA polymerase (48) m) Choristoneura biennis DNA
polymerase (49) n) Autographa california nuclear polymerase virus
(50) (AcMNPV) DNA polymerase o) Lymantria dispar nuclear
polyhedrosis virus DNA (51) polymerase p) Adenovirus-2 DNA
polymerase (52) q) Adenovirus-7 DNA polymerase (53) r)
Adenovirus-12 DNA polymerase (54) Eukaryotic linear DNA plasmid
encoded DNA polymerases a) S-1 Maize DNA polymerase (55) b) kalilo
neurospora intermedia DNA polymerase (56) c) pA12 ascobolus
immersus DNA polymerase (57) d) pCLK1 Claviceps purpurea DNA
polymerase (58) e) maranhar neurospora crassa DNA polymerase (59)
f) pEM Agaricus bitorquis DNA polymerase (60) g) pGKL1
Kluyveromyces lactis DNA polymerase (61) h) pGKL2 Kluyveromyces
lactis DNA polymerase (62) i) pSKL Saccharomyces kluyveri DNA
polymerase (63)
[0153] B. Plasmids
[0154] The starting sequences for the generation of Family B DNA
polymerases according to the invention may be contained in a
plasmid vector. A non-limiting list of cloned Family B DNA
polymerases and their GenBank Accession numbers are listed in Table
II.
2TABLE II Accession Information for Cloned Family B Polymerases
Vent Thermococcus litoralis ACCESSION AAA72101 PID g348689 VERSION
AAA72101.1 GI: 348689 DBSOURCE locus THCVDPE accession M74198.1
THEST THERMOCOCCUS SP. (STRAIN TY) ACCESSION O33845 PID g3913524
VERSION O33845 GI: 3913524 DBSOURCE swissprot: locus DPOL_THEST,
accession O33845 Pab Pyrococcus abyssi ACCESSION P77916 PID
g3913529 VERSION P77916 GI: 3913529 DBSOURCE swissprot: locus
DPOL_PYRAB, accession P77916 PYRHO Pyrococcus horikoshii ACCESSION
O59610 PID g3913526 VERSION O59610 GI: 3913526 DBSOURCE swissprot:
locus DPOL_PYRHO, accession O59610 PYRSE PYROCOCCUS SP. (STRAIN
GE23) ACCESSION P77932 PID g3913530 VERSION P77932 GI: 3913530
DBSOURCE swissprot: locus DPOL_PYRSE, accession P77932 DeepVent
Pyrococcus sp. ACCESSION AAA67131 PID g436495 VERSION AAA67131.1
GI: 436495 DBSOURCE locus PSU00707 accession U00707.1 Pfu
Pyrococcus furiosus ACCESSION P80061 PID g399403 VERSION P80061 GI:
399403 DBSOURCE swissprot: locus DPOL_PYRFU, accession P80061 JDF-3
Thermococcus sp. Unpublished Baross
gi.vertline.2097756.vertline.pat.vertline.US.ve-
rtline.5602011.vertline.12 Sequence 12 from patent U.S. Pat. No.
5602011 9degN THERMOCOCCUS SP. (STRAIN 9ON-7). ACCESSION Q56366 PID
g3913540 VERSION Q56366 GI: 3913540 DBSOURCE swissprot: locus
DPOL_THES9, accession Q56366 KOD Pyrococcus sp. ACCESSION BAA06142
PID g1620911 VERSION BAA06142.1 GI: 1620911 DBSOURCE locus
PYWKODPOL accession D29671.1 Tgo Thermococcus gorgonarius.
ACCESSION 4699806 PID g4699806 VERSION GI: 4699806 DBSOURCE pdb:
chain 65, release Feb. 23, 1999 THEFM Thermococcus fumicolans
ACCESSION P74918 PID g3913528 VERSION P74918 GI: 3913528 DBSOURCE
swissprot: locus DPOL_THEFM, accession P74918 METTH
Methanobacterium thermoautotrophicum ACCESSION O27276 PID g3913522
VERSION O27276 GI: 3913522 DBSOURCE swissprot: locus DPOL_METTH,
accession O27276 Metja Methanococcus jannaschii ACCESSION Q58295
PID g3915679 VERSION Q58295 GI: 3915679 DBSOURCE swissprot: locus
DPOL_METJA, accession Q58295 POC Pyrodictium occultum ACCESSION
B56277 PID g1363344 VERSION B56277 GI: 1363344 DBSOURCE pir: locus
B56277 ApeI Aeropyrum pernix ACCESSION BAA81109 PID g5105797
VERSION BAA81109.1 GI: 5105797 DBSOURCE locus AP000063 accession
AP000063.1 ARCFU Archaeoglobus fulgidus ACCESSION O29753 PID
g3122019 VERSION O29753 GI: 3122019 DBSOURCE swissprot: locus
DPOL_ARCFU, accession O29753 Desulfurococcus sp. Tok. ACCESSION
6435708 PID g64357089 VERSION GT: 6435708 DBSOURCE pdb. chain 65,
release Jun. 2, 1999
[0155] Plasmids acceptable for the expression of modified forms of
Family B DNA polymerases may be selected from a large number known
in the art by one of skill in the art. A plasmid vector for
expression of a modified DNA polymerase according to the invention
will preferably comprise sequences directing high level expression
of a DNA polymerase, and will more preferably comprise sequences
directing inducible, high level expression of a DNA polymerase. As
one example of an inducible high level expression system, plasmids
placing a modified DNA polymerase coding sequence according to the
invention under the control of a bacteriophage T7 promoter may be
introduced to bacteria containing an inducible T7 RNA polymerase
gene within their chromosome. Induction of the T7 RNA polymerase
gene subsequently induces high level expression of the T7
promoter-driven modified DNA polymerase gene (see for example,
Gardner & Jack, Nucleic Acids Res. 27: 2545).
[0156] C. Mutagenesis
[0157] The cloned wild-type form of a Family B DNA polymerase may
be mutated to generate forms exhibiting reduced discrimination
against non-conventional nucleotides by a number of methods.
[0158] First, methods of random mutagenesis which will result in a
panel of mutants bearing one or more randomly-situated mutations
exist in the art. Such a panel of mutants may then be screened for
those exhibiting reduced discrimination relative to the wild-type
polymerase (see "Methods of Evaluating Mutants for Reduced
Discrimination", below). An example of a method for random
mutagenesis is the so-called "error-prone PCR method". As the name
implies, the method amplifies a given sequence under conditions in
which the DNA polymerase does not support high fidelity
incorporation. The conditions encouraging error-prone incorporation
for different DNA polymerases vary, however one skilled in the art
may determine such conditions for a given enzyme. A key variable
for many DNA polymerases in the fidelity of amplification is, for
example, the type and concentration of divalent metal ion in the
buffer. The use of manganese ion and/or variation of the magnesium
or manganese ion concentration may therefore be applied to
influence the error rate of the polymerase.
[0159] Second, there are a number of site-directed mutagenesis
methods known in the art which allow one to mutate a particular
site or region in a straightforward manner. There are a number of
kits available commercially for the performance of site-directed
mutagenesis, including both conventional and PCR-based methods.
Examples include the EXSITE.TM. PCR-Based Site-directed Mutagenesis
Kit available from Stratagene (Catalog No. 200502; PCR based) and
the QUIKCHANGE.TM. Site-directed mutagenesis Kit from Stratagene
(Catalog No. 200518; non-PCR-based), and the CHAMELEON.RTM.
double-stranded Site-directed mutagenesis kit, also from Stratagene
(Catalog No. 200509).
[0160] Older methods of site-directed mutagenesis known in the art
relied upon sub-cloning of the sequence to be mutated into a
vector, such as an M13 bacteriophage vector, that allows the
isolation of single-stranded DNA template. In these methods one
annealed a mutagenic primer (i.e., a primer capable of annealing to
the site to be mutated but bearing one or mismatched nucleotides at
the site to be mutated) to the single-stranded template and then
polymerized the complement of the template starting from the 3' end
of the mutagenic primer. The resulting duplexes were then
transformed into host bacteria and plaques were screened for the
desired mutation.
[0161] More recently, site-directed mutagenesis has employed PCR
methodologies, which have the advantage of not requiring a
single-stranded template. In addition, methods have been developed
that do not require sub-cloning. Several issues must be considered
when PCR-based site-directed mutagenesis is performed. First, in
these methods it is desirable to reduce the number of PCR cycles to
prevent expansion of undesired mutations introduced by the
polymerase. Second, a selection must be employed in order to reduce
the number of non-mutated parental molecules persisting in the
reaction. Third, an extended-length PCR method is preferred in
order to allow the use of a single PCR primer set. And fourth,
because of the non-template-dependent terminal extension activity
of some thermostable polymerases it is often necessary to
incorporate an end-polishing step into the procedure prior to
blunt-end ligation of the PCR-generated mutant product.
[0162] The protocol described below accomodates these
considerations through the following steps. First, the template
concentration used is approximately 1000-fold higher than that used
in conventional PCR reactions, allowing a reduction in the number
of cycles from 25-30 down to 5-10 without dramatically reducing
product yield. Second, the restriction endonuclease DpnI
(recognition target sequence: 5-Gm6ATC-3, where the A residue is
methylated) is used to select against parental DNA, since most
common strains of E. coli Dam methylate their DNA at the sequence
5-GATC-3. Third, Taq Extender is used in the PCR mix in order to
increase the proportion of long (i.e., full plasmid length) PCR
products. Finally, Pfu DNA polymerase is used to polish the ends of
the PCR product prior to intramolecular ligation using T4 DNA
ligase.
[0163] The method is described in detail as follows:
[0164] PCR-Based Site Directed Mutagenesis of the 3'-5' Exonuclease
Domain
[0165] Plasmid template DNA (approximately 0.5 pmole) is added to a
PCR cocktail containing: 1.times.mutagenesis buffer (20 mM Tris
HCl, pH 7.5; 8 mM MgCl2; 40 ug/ml BSA); 12-20 pmole of each primer
(one of skill in the art may design a mutagenic primer as
necessary, giving consideration to those factors such as base
composition, primer length and intended buffer salt concentrations
that affect the annealing characteristics of oligonucleotide
primers; one primer must contain the desired mutation, and one (the
same or the other) must contain a 5' phosphate to facilitate later
ligation), 250 uM each dNTP, 2.5 U Taq DNA polymerase, and 2.5 U of
Taq Extender (Available from Stratagene; See Nielson et al. (1994)
Strategies 7: 27, and U.S. Pat. No. 5,556,772). The PCR cycling is
performed as follows: 1 cycle of 4 min at 94.degree. C., 2 min at
50.degree. C. and 2 min at 72.degree. C.; followed by 5-10 cycles
of 1 min at 94.degree. C., 2 min at 54.degree. C. and 1 min at
72.degree. C. The parental template DNA and the linear,
PCR-generated DNA incorporating the mutagenic primer are treated
with DpnI (10 U) and Pfu DNA polymerase (2.5U). This results in the
DpnI digestion of the in vivo methylated parental template and
hybrid DNA and the removal, by Pfu DNA polymerase, of the
non-template-directed Taq DNA polymerase-extended base(s) on the
linear PCR product. The reaction is incubated at 37.degree. C. for
30 min and then transferred to 72.degree. C. for an additional 30
min. Mutagenesis buffer (115 ul of 1.times.) containing 0.5 mM ATP
is added to the DpnI-digested, Pfu DNA polymerase-polished PCR
products. The solution is mixed and 10 ul are removed to a new
microfuge tube and T4 DNA ligase (2-4 U) is added. The ligation is
incubated for greater than 60 min at 37.degree. C. Finally, the
treated solution is transformed into competent E. coli according to
standard methods.
[0166] D. Non-Conventional Nucleotides Useful According to the
Invention.
[0167] There is a wide variety of non-conventional nucleotides
available in the art. Any or all of them are contemplated for use
with a DNA polymerase of the invention. A non-limiting list of such
non-conventional nucleotides is presented in Table III.
3TABLE III Non-Conventional Nucleotides DIDEOXYNUCLEOTIDE ANALOGS
Fluorescein Labeled Fluorophore Labeled Fluorescein-12-ddCTP
Eosin-6-ddCTP Fluorescein-12-ddUTP Coumarin-5-ddUTP
Fluorescein-12-ddATP Tetramethylrhodamine- 6-ddUTP
Fluorescein-12-ddGTP Texas Red-5-ddATP Fluorescein-N6-ddATP
LISSAMINE .TM. -rhodamine- 5-ddGTP FAM Labeled TAMRA Labeled
FAM-ddUTP TAMRA-ddUTP FAM-ddCTP TAMRA-ddCTP FAM-ddATP TAMRA-ddATP
FAM-ddGTP TAMRA-ddGTP ROX Labeled JOE Labeled ROX-ddUTP JOE-ddUTP
ROX-ddCTP JOE-ddCTP ROX-ddATP JOE-ddATP ROX-ddGTP JOE-ddGTP R6G
Labeled R110 Labeled R6G-ddUTP R110-ddUTP R6G-ddCTP R110-ddCTP
R6G-ddATP R110-ddATP R6G-ddGTP R110-ddGTP BIOTIN Labeled DNP
Labeled Biotin-N6-ATP DNP-N6-ddATP DEOXYNUCLEOTIDE ANALOGS TTP
Analogs dATP-Analogs Fluorescein-12-dUTP Coumarin-5-dATP
Coumarin-5-dUTP Diethylaminocoumarin- 5-dATP
Tetramethylrhodamine-6-dUTP Fluorescein-12-dATP
Tetraethylrhodamine-6-dUTP Fluorescein Chlorotria- zinyl-4-dATP
Texas Red-5-dUTP LISSAMINE .TM.-rhodamine- 5-dATP LISSAMINE
.TM.-rhodamine-5-dUTP Naphthofluorescein- 5-dATP
Naphthofluorescein-5-dUTP Pyrene-8-dATP Fluorescein
Chlorotriazinyl-4-dUTP Tetramethylrhodamine- 6-dATP Pyrene-8-dUTP
Texas Red-5-dATP Diethylaminocoumarin-5-dUTP DNA-N6-dATP
Biotin-N6-dATP dCTP Analogs dGTP Analogs Coumarin-5-dCTP
Coumarin-5-dGTP Fluorescein-12-dCTP Fluorescein-12-dGTP
Tetramethylrhodamine-6-dCTP Tetramethylrhodamine- 6-dGTP Texas
Red-5-dCTP Texas Red-5-dGTP LISSAMINE .TM.-rhodamine-5-dCTP
LISSAMINE .TM.-rhodamine- 5-dGTP Naphthofluorescein-5-dCTP
Fluorescein Chlorotriazinyl-4-dCTP Pyrene-8-dCTP
Diethylaminocoumarin- 5-dCTP Fluorescein-N4-dCTP Biotin-N4-dCTP
DNP-N4-dCTP RIBONUCLEOTIDE ANALOGS CTP Analogs UTP Analogs
Coumarin-5-CTP Fluorescein-12-UTP Fluorescein-12-CTP Coumarin-5-UTP
Tetrainethylrhodainine-6-CTP Tetramethylrhodamine- 6-UTP Texas
Red-5-CTP Texas Red-5-UTP LISSAMINE .TM.-rhodamine-5-CTP LISSAMINE
.TM.-5-UTP Naphthofluorescein-5-CTP Naphthofluorescein- 5-UTP
Fluorescein Chlorotriazinyl-4-CTP Fluorescein Chlorotria-
zinyl-4-UTP Pyrene-8-CTP Pyrene-8-UTP Fluorescein-N4-CTP
Biotin-N4-CTP ATP Analogs Coumarin-5-ATP Fluorescein-12-ATP
Tetramethylrhodamine-6-ATP Texas Red-5-ATP LISSAMINE
.TM.-rhodamine-5-ATP Fluorescein-N6-ATP Biotin-N6-ATP
[0168] DNP-N-6-ATP
[0169] Additional non-conventional nucleotides useful according to
the invention include, but are not limited to 7-deaza-dATP,
7-deaza-dGTP, 5'-methyl-2'-deoxycytidine-5'-triphosphate. Further
non-conventional nucleotides or variations on those listed above
are discussed by Wright & Brown, 1990, Pharmacol. Ther. 47:
447. It is specifically noted that ribonucleotides qualify as
non-conventional nucleotides, since ribonucleotides are not
generally incorporated by DNA polymerases. Modifications of Family
B DNA polymerases that result in the ability, or enhanced ability,
of the polymerase to incorporate labeled or unlabeled
ribonucleotides are specifically contemplated herein.
[0170] E. Methods of Evaluating Mutants for Reduced
Discrimination
[0171] Random or site-directed mutants generated as known in the
art or as described herein and expressed in bacteria may be
screened for reduced discrimination against non-conventional
nucleotides by several different assays. In one method, Family B
DNA polymerase proteins expressed in lytic lambda phage plaques
generated by infection of host bacteria with expression vectors
based on, for example, Lambda ZapII.RTM., are transferred to a
membrane support. The immobilized proteins are then assayed for
polymerase activity on the membrane by immersing the membranes in a
buffer containing a DNA template and the unconventional nucleotides
to be monitored for incorporation.
[0172] Mutant polymerase libraries may be screened using a
variation of the technique used by Sagner et al (Sagner, G., Ruger,
R., and Kessler, C. (1991) Gene 97: 119-123). For this approach,
lambda phage clones are plated at a density of 10-20 plaques per
square centimeter. Proteins present in the plaques are transferred
to filters and moistened with polymerase screening buffer (50 mM
Tris (pH 8.0), 7 mM MgCl.sub.2, 3 mM -ME). The filters are kept
between layers of plastic wrap and glass while the host cell
proteins are heat-inactivated by incubation at 65.degree. C. for 30
minutes. The heat-treated filters are then transferred to fresh
plastic wrap and approximately 35 .mu.l of polymerase assay
cocktail are added for every square centimeter of filter. The assay
cocktail consists of 1.times. cloned Pfu (cPfu) magnesium free
buffer (1.times. buffer is 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10
mM (NH4).sub.2SO.sub.4, 100 ug/ml bovine serum albumin (BSA), and
0.1% Triton X-100; Pfu Magnesium-free buffer may be obtained from
Stratagene (Catalog No. 200534)), 125 ng/ml activated calf thymus
or salmon sperm DNA, 1.29 .mu.Ci/ml .alpha.-.sup.33P ddNTP or
dideoxynucleotides (at a dNTP:dye-ddNTP ratio of 1: 15). Initial
screening was done in the presence of MnCl.sub.2, but the preferred
method was to screen in 1.times.Taq Polymerase buffer (1.5 mM
MgCl.sub.2) The filters are placed between plastic wrap and a glass
plate and then incubated at 65.degree. C. for one hour, and then at
70.degree. C. for one hour and fifteen minutes. Filters are then
washed three times in 2.times.SSC for five minutes per wash before
rinsing twice in 100% ethanol and vacuum drying. Filters are then
exposed to X-ray film (approximately 16 hours), and plaques that
incorporate label are identified by aligning the filters with the
original plate bearing the phage clones. Plaques identified in this
way are re-plated at more dilute concentrations and assayed under
similar conditions to allow the isolation of purified plaques.
[0173] In assays such as the one described above, the signal
generated by the label is a direct measure of the activity of the
polymerase with regard to that particular unconventional nucleotide
or combination of unconventional nucleotides used in the assay.
Unconventional nucleotides corresponding to all four conventional
nucleotides may be included in the reactions, or, alternatively,
only one unconventional nucleotide may be included to assess the
effect of the mutation(s) on utilization of a given unconventional
nucleotide. One approach is to use unconventional nucleotides
corresponding to all four nucleotides in a first screen to identify
clones that incorporate more than a reference wild-type clone, and
then to monitor the incorporation of individual unconventional
nucleotides in a subsequent screen. In the preferred screening
mode, only the dideoxynucleotides and dideoxynucleotide analogs of
ddATP, ddCTP, and ddTTP would be used since ddGTP is not
discriminated against by some DNA polymerases and increases the
background signal of any screen.
[0174] In order to screen for clones with enhanced ability to
incorporate dideoxynucleotides, clones identified in first screens
utilizing only dideoxynucleotides may then be characterized by
their sensitivity to low levels of each of the four
dideoxynucleotides in a DNA polymerase nucleotide incorporation
assay employing all four dNTPs, a ;H-TTP tracer, and a low level of
each ddNTP. Since incorporation of dideoxynucleotides stops DNA
chain elongation, superior ability to incorporate
dideoxynucleotides diminishes the incorporation of tritium labeled
deoxynucleotides relative to wild-type DNA polymerase. Comparisons
of ddNTP concentrations that bring about 50% inhibition of
nucleotide incorporation (I.sub.50%) can be used to compare ddNTP
incorporation efficiency of different polymerases or polymerase
mutants. Comparisons of I.sub.50% values for ddATP, ddCTP, ddGTP,
and ddTTP can be used to identify mutants with reduced selectivity
for particular bases. Such mutants would be expected to produce
more uniform DNA sequencing ladders.
[0175] In order to measure incorporation of individual ddNTPs,
cocktails are prepared which consist of varying concentrations of
the ddNTP of interest, and a total of 200 .mu.M of each nucleotide
triphosphate. For example, the incorporation of ddATP by wild type
JDF-3 polymerase may be measured at 0, 40, 80, 120 and 160 .mu.M
ddATP. In these reactions, dATP concentrations would be adjusted to
200, 160, 120, 80, and 40 .mu.M, respectively, so that the total
amount of adenine nucleotide triphosphate is 200 .mu.M. In
comparison, mutants may be assayed using ddATP concentrations of 0,
5, 10, and 20 .mu.M ddATP, and adjusted dATP concentrations of 200,
195, 190, and 180 .mu.M, respectively (dATP+ddATP=200 .mu.M).
Additional cocktails are prepared to similarly measure ddCTP,
ddGTP, and ddTTP incorporation.
[0176] Incorporation of nucleotides under the concentration
parameters described above may be measured in extension reactions
by adding, for example, 1 .mu.l of appropriately diluted bacterial
extract (i.e., heat-treated and clarified extract of bacterial
cells (see Example 1, part M) expressing a cloned polymerase or
mutated cloned polymerase) to 10 ul of each nucleotide cocktail,
followed by incubation at 72.degree. C. for 30 minutes. Extension
reactions are quenched on ice, and then 5 .mu.l aliquots are
spotted immediately onto DE81 ion-exchange filters (2.3 cm; Whatman
#3658323). Unincorporated label is removed by 6 washes with
2.times.SCC (0.3M NaCl, 30 mM sodium citrate, pH 7.0), followed by
a brief wash with 100% ethanol. Incorporated radioactivity is then
measured by scintillation counting. Reactions that lack enzyme are
also set up along with sample incubations to determine "total cpms"
(omit filter wash steps) and "minimum cpms" (wash filters as
above).
[0177] Cpms bound is proportional to the amount of polymerase
activity present per volume of bacterial extract. The volume of
bacterial extract (generally about 0.25-1 .mu.l) which brings about
incorporation of approximately 10,000 cpms is determined for use in
subsequent nucleotide analog incorporation testing.
[0178] Genes for mutant DNA polymerases generated by random
mutagenesis may be sequenced to identify the sites and number of
mutations. For those mutants comprising more than one mutation, the
effect of a given mutation may be evaluated by introduction of the
identified mutation to the exo.sup.- progenitor gene by
site-directed mutagenesis in isolation from the other mutations
borne by the particular mutant. Screening assays of the single
mutant thus produced will then allow the determination of the
effect of that mutation alone.
[0179] F. Expression of Mutated Family B DNA Polymerase According
to the Invention
[0180] Methods known in the art may be applied to express and
isolate the mutated forms of Family B DNA polymerase according to
the invention. Many bacterial expression vectors contain sequence
elements or combinations of sequence elements allowing high level
inducible expression of the protein encoded by a foreign sequence.
For example, as mentioned above, bacteria expressing an integrated
inducible form of the T7 RNA polymerase gene may be transformed
with an expression vector bearing a mutated DNA polymerase gene
linked to the T7 promoter. Induction of the T7 RNA polymerase by
addition of an appropriate inducer, for example,
isopropyl--D-thiogalacto- pyranoside (IPTG) for a lac-inducible
promoter, induces the high level expression of the mutated gene
from the T7 promoter (see Gardner & Jack, 1999, supra).
[0181] Appropriate host strains of bacteria may be selected from
those available in the art by one of skill in the art. As a
non-limiting example, E. coli strain BL-21 is commonly used for
expression of exogenous proteins since it is protease deficient
relative to other strains of E. coli. BL-21 strains bearing an
inducible T7 RNA polymerase gene include WJ56 and ER2566 (Gardner
& Jack, 1999, supra). For situations in which codon usage for
the particular polymerase gene differs from that normally seen in
E. coli genes, there are strains of BL-21 that are modified to
carry tRNA genes encoding tRNAs with rarer anticodons (for example,
argu, ileY, leuW, and proL tRNA genes), allowing high efficiency
expression of cloned protein genes, for example, cloned archaeal
enzyme genes (several BL21-CODON PLUS.TM. cell strains carrying
rare-codon tRNAs are available from Stratagene, for example).
[0182] There are many methods known to those of skill in the art
that are suitable for the purification of a modified DNA polymerase
of the invention. For example, the method of Lawyer et al. (1993,
PCR Meth. & App. 2: 275) is well suited for the isolation of
thermostable DNA polymerases expressed in E. coli, as it was
designed originally for the isolation of Taq polymerase.
Alternatively, the method of Kong et al. (1993, J. Biol. Chem. 268:
1965, incorporated herein by reference) may be used, which employs
a heat denaturation step to destroy host proteins, and two column
purification steps (over DEAE-Sepharose and heparin-Sepharose
columns) to isolate highly active and approximately 80% pure
thermostable DNA polymerase. Further, as detailed in Example 1,
part N, below, DNA polymerase mutants may be isolated by an
ammonium sulfate fractionation, followed by Q Sepharose and DNA
cellulose columns, or by adsorption of contaminants on a HiTrap Q
column, followed by gradient elution from a HiTrap heparin
column.
[0183] G. Preparation of Thermococcus species JDF-3 Thermostable
DNA Polymerase with Reduced Discrimination
[0184] To prepare thermostable Family B polymerases which exhibit
reduced discrimination for dideoxynucleotide triphosphates
(ddNTPs), the DNA sequence encoding a 3' to 5'
exonuclease-deficient (D141A) Family B polymerase from the
hyperthermophilic archaeon Thermococcus species JDF-3 was subjected
to random mutagenesis using "error-prone PCR" as described herein,
and cloned into the bacteriophage lambda Zap.RTM.II. The polymerase
from JDF-3 was chosen due to superior processivity, polymerization
rate and ddNTP incorporation relative to the Family B DNA
polymerase from Pyrococcus furiosus (Pfu) (see Table IV, below).
The library of mutants was plated on E. coli hosts and the proteins
present in the lytic plaques were transferred to a solid support
that was then immersed in a buffer containing DNA template and all
four .alpha.-.sup.33P labeled dideoxynucleotides. Mutants that
incorporated the labeled dideoxynucleotide produced signals that
corresponded to their ability to incorporate the .alpha.-.sup.33P
ddNTPs. Isolated clones were then characterized by their
sensitivity to low levels of each of the four dideoxynucleotides in
a DNA polymerase nucleotide incorporation assay employing all four
dNTPs and a ;H-TTP tracer. Since incorporation of
dideoxynucleotides stops DNA chain elongation, superior ability to
incorporate dideoxynucleotides diminishes the incorporation of
tritium labeled deoxynucleotides. The unmutated progenitor DNA
polymerase rarely incorporates dideoxynucleotides and is only 50%
inhibited at high ddNTP levels (100-160 micromolar each ddNTP). The
mutant enzymes show 50% inhibition at 5 to 40 micromolar
concentrations of ddNTP and improved incorporation was observed for
all four ddNTPs (ddATP, ddCTP, ddTTP and ddGTP; see Tables V and VI
in Example 1, below).
[0185] The incorporation of non-conventional nucleotides was also
evaluated through use of purified mutant polymerases in cycle
sequencing, with .alpha.-.sup.33P labeled ddNTPs present at 0.021 M
and dNTPs present at 2.1 M each. The mutants readily utilized all
four dideoxynucleotides and produced sequencing ladders that
compared favorably to Thermo Sequenase.RTM., which uses an F667Y
Taq DNA polymerase mutant (VanderHorn et al., 1997, BioTechniques
22: 758).
[0186] The domains of relevance in 17 of the 40 purified mutants
were sequenced. Most randomly mutated clones contained more than
one mutation in the regions sequenced but all mutants contained
mutations at one of three sites. Mutations predicted to confer an
enhanced ddNTP uptake phenotype were introduced into the progenitor
exonuclease deficient DNA polymerase sequence by site-directed
mutagenesis to eliminate ancillary mutations which were not
expected to contribute to the improved dideoxynucleotide uptake
phenotype.
[0187] Sixteen of the seventeen JDF-3 DNA polymerase mutations were
found in region II (motif A) on either side of the tyrosine in the
consensus sequence 404 DxxSLYPSII 413. These mutations consisted of
DFRSLYLSII (P410L), DFRSHYPSII (L408H) and DFRSFYPSII (L408F).
Therefore, the LYP motif of region II appears to be important in
ddNTP discrimination in the JDF-3 Family B polymerase.
[0188] The prior art modification of the tyrosine corresponding to
Y409 in JDF3 Family B DNA polymerase is recognized for its
positioning in the nucleotide binding pocket. As shown herein,
however, modification of the residues neighboring Y409 (L408H or
L408F or P410L) had the unexpected effect of profoundly altering
nucleotide binding, particularly with respect to ddNTP
incorporation.
[0189] The only JDF-3 DNA polymerase mutation leading to enhanced
incorporation of non-conventional nucleotides occurring outside of
region II is an alanine (ala or A) to threonine (thr or T)
conversion at position 485 in region III (A485T). This site is two
residues upstream of KX.sub.3NSXYG (Jung et al., 1990, supra;
Blasco et al., 1992, supra; Dong et al., 1993, J. Biol. Chem. 268:
21163; Zhu et al., 1994, Biochem. Biophys. Acta 1219: 260; Dong and
Wang, 1995, J. Biol. Chem. 270: 21563) (referred to as region III
or motif B) which is functionally, but not structurally (Wang et
al., 1997, supra), analogous to KX.sub.3(F/Y)GX.sub.2YG in helix O
of the Family A DNA polymerases. In Family A DNA polymerases, such
as the Klenow fragment and Taq DNA polymerases, the O helix
contains amino acids that play a major role in dNTP binding
(Astatke et al., 1998, J. Mol. Biol. 278: 147; Astatke et al.,
1995, J. Biol. Chem. 270: 1945; Polesky et al., 1992, J. Biol. Chem
267: 8417; Polesky et al., 1990, J. Biol. Chem. 265: 14579; Pandey
et al., 1994, J. Biol. Chem. 269: 13259; Kaushik et al., 1996,
Biochem. 35: 7256). Specifically, helix O contains the F (F762 in
the Klenow fragment; F667 in Taq) which confers ddNTP
discrimination in Family A DNA polymerases
(KX.sub.3(F/Y)GX.sub.2YG) (Tabor and Richardson, 1995, supra).
[0190] The effect of the A485T mutation on ddNTP incorporation in
the JDF-3 DNA polymerase is surprising since the RB69 and
Thermococcus gorgonarius crystal structures (Hopfner et al., 1999,
supra) show it facing away from the proposed active site of the
nucleotide binding surface. Moreover, the type of side chain
conferring ribose selectivity in archaeal Family B DNA polymerases
(A: small, non-polar) is different from that of the bulky, aromatic
Y and F residues that dictate ddNTP discrimination in Family A DNA
polymerases (Tabor and Richardson, 1995, supra). Additionally, this
position (A485) is not well conserved among either DNA polymerase
family and is not included in the consensus sequence for this
domain (Braithwaite and Ito, 1993, supra), implying a lack of
critical importance in dNTP recognition.
[0191] A JDF-3 double mutant was constructed that contains
mutations P410L and A485T. In dideoxynucleotide cycle sequencing,
the banding pattern intensity demonstrated by the double mutant was
extremely uniform, suggesting little if any preference for any dNTP
over its corresponding ddNTP (See FIG. 8 and Example 1Q). This
polymerase characteristic improves the accuracy of base calling in
automated sequencing. We presume that combinations of P410L and
A485 mutations, L408H and A485 mutations, and L408F and A485
mutations would result in enzymes that exhibit improved ddNTP
incorporation. The efficiency of dideoxynucleotide incorporation by
such double mutant enzymes may also be characterized or quantitated
by measurement of the I.sub.50% as described herein to determine
the relative degree of improvement in incorporation.
EXAMPLES
[0192] The following examples are offered by way of illustration
only and are by no means intended to limit the scope of the claimed
invention.
Example 1
[0193] A. Cloning a DNA Polymerase Gene from Thermococcus species
JDF-3 DNA Polymerase.
[0194] A Thermococcus species was cultured from submarine samples
taken from the Juan de Fuca ridge. Genomic DNA was isolated and
used to prepare a genomic DNA library in ZAP II (Stratagene) using
standard procedures. The lambda library was plated on XL1-Blue MRF'
E. coli and screened for clones with DNA polymerase activity using
a variation of the method described by Sagner et al. (Sagner, G.,
Ruger, R., and Kessler, C. (1991) Gene 97: 119-123). Plaques
containing active polymerase were cored and stored in SM buffer.
Positive primary plaques were re-plated and re-assayed to allow
purification of isolated clones. Secondary clones were excised
according to the instructions provided with the ZAP II system
(Stratagene), and the DNA sequence of the insert determined (FIG.
1).
[0195] The translated amino acid sequence of the JDF-3 DNA
polymerase is shown in FIG. 2. Amino acid sequence alignments show
that JDF-3 DNA polymerase exhibits homology to the class of DNA
polymerases referred to as Family B.
[0196] Recombinant JDF-3 DNA polymerase was purified as described
below (see "Purification of JDF-3" (method 1)). The biochemical
properties of JDF-3 DNA polymerase have been compared to those of
other commercially available archaeal DNA polymerases. The results
shown in Table IV and V indicated that, compared to other enzymes,
JDF-3 exhibits higher processivity, a faster polymerization rate
(K.sub.cat), and a greater tendency to utilize ddNTPs. JDF-3 DNA
polymerase was therefore chosen for development of a DNA sequencing
enzyme.
4TABLE IV Polymerase Activities of Archaeal Family B DNA
Polymerases Specific Activity (U/mg) .times. 10.sup.4 DNA dNTP
Polymerase Activated DNA Primed M13 (nM) (M each) Pfu 2.6 .+-. .07
2.0 .+-. .02 0.7 16 .+-. 2 exo.sup.- Pfu 4.1 .+-. .07 2.3 0.5 12
JDF-3 1.2 .+-. .07 5.2 2.0 16 .+-. 2 Vent .sup. 1.8.sup.a .sup.
0.7.sup.a .sup. 0.1.sup.a .sup. 57.sup.a .sup.aH. Kong, R. B.
Kucera, and W. E Jack, J. Biol. Chem. 268. 1965 (1993).
[0197] B. Intein Removal From the Gene Encoding JDF-3 DNA
Polymerase.
[0198] By alignment to Family B DNA polymerase sequences, the JDF-3
DNA polymerase clone was found to contain an intein sequence (FIGS.
3 and 4). To improve expression of recombinant JDF-3 polymerase,
the intein was removed by inverse PCR. PCR primers were designed to
prime immediately upstream and downstream to the sequence coding
for the intein termini, and were oriented such that the 3' ends of
the primers were pointed away from the intein. The primers were
also modified with 5'-phosphate groups to facilitate ligation. The
plasmid/insert sequence was PCR amplified and circularized by
standard methods.
[0199] C. Construction of a JDF-3 DNA Polymerase Mutant with
Diminished 3'-5' Exonuclease Activity.
[0200] DNA polymerases lacking 3'-5' exonuclease (proofreading)
activity are preferred for applications requiring nucleotide analog
incorporation (e.g., DNA sequencing) to prevent removal of
nucleotide analogs after incorporation. The 3'-5' exonuclease
activity associated with proofreading DNA polymerases can be
reduced or abolished by mutagenesis. Sequence comparisons have
identified three conserved motifs (exo I, II, III) in the 3'-5'
exonuclease domain of DNA polymerases (reviewed V. Derbyshire, J.
K. Pinsonneault, and C. M. Joyce, Methods Enzymol. 262, 363
(1995)). Replacement of any of the conserved aspartic or glutamic
acid residues with alanine has been shown to abolish the
exonuclease activity of numerous DNA polymerases, including
archaeal DNA polymerases such as Vent (H. Kong, R. B. Kucera, and
W. E. Jack, J. Biol. Chem. 268, 1965 (1993)) and Pfu (Stratagene,
unpublished). Conservative substitutions lead to reduced
exonuclease activity, as shown for mutants of the archaeal 9 N-7
DNA polymerase (M. W. Southworth, H. Kong, R. B. Kucera, J. Ware,
H. Jannasch, and F. B. Perler, Proc. Natl. Acad. Sci. 93, 5281
(1996)).
[0201] JDF-3 DNA polymerase mutants exhibiting substantially
reduced 3'-5' exonuclease activity were prepared by introducing
amino acid substitutions at the conserved 141D or 143E residues in
the exo I domain. Using the CHAMELEON.RTM. Double-Stranded,
Site-Directed Mutagenesis Kit (Stratagene), the following JDF-3
mutants were constructed: D141A, D141N, D141S, D141T, D141E and
E143A.
[0202] To analyze JDF-3 mutant proteins, the DNA sequence encoding
JDF-3 DNA polymerase was PCR amplified using primers GGG AAA CAT
ATG ATC CTT GAC GTT GAT TAC (where NdeI site in bold and start
codon underlined) and GGG AAA GGA TCC TCA CTT CTT CTT CCC CTT C
(where BamHI site shown in bold type). The PCR products were
digested, purified, and ligated into a high expression level vector
using standard methods. Plasmid clones were transformed into
BL21(DE3). Recombinant bacterial clones were grown using standard
procedures and JDF-3 polymerase mutants were expressed in the
absence of induction. The exonuclease and polymerase activities of
recombinant clones were assayed using bacterial lysates. Typically,
crude extracts were heated at 70C for 15-30 minutes and then
centrifuged to obtain a cleared lysate.
[0203] There are several methods of measuring 3' to 5' exonuclease
activity known in the art, including that of Kong et al. (Kong et
al., 1993, J. Biol. Chem. 268: 1965) and that of Southworth et al.
(Southworth et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93: 5281),
the full contents of both of which are hereby incorporated by
reference. The exonuclease activity of wild type and active mutant
polymerases as measured by the Kong et al. method were as
follows:
[0204] Exo activity (U/mg):
[0205] Wt 915
[0206] D141A7
[0207] D141N953
[0208] D141S 954
[0209] D141T0.5
[0210] D141E940
[0211] E143A 0.3
[0212] The combination exonuclease mutant D141A+E143A was made as
described in section L.
[0213] The E143A JDF-3 mutant (clone #550) exhibited significantly
reduced 3'-5' exo activity and was chosen for further mutagenesis
to improve incorporation of ddNTP and other nucleotide analogs.
Other JDF-3 mutants with substantially reduced exonuclease activity
could have been used for this purpose, such as the JDF-3 D141T
mutant for experiment or applications requiring the absolute
elimination of 3' to 5' exonuclease activity, the double mutant
D141A +E143A was preferred.
[0214] D. Error-Prone PCR Amplification of the JDF-3 DNA Polymerase
Gene.
[0215] Random mutations were introduced into exo.sup.- JDF-3 by
amplifying the entire gene (clone #550) under conditions which did
not support high fidelity replication. To broaden the spectrum of
potential mutations, three different PCR enzymes were used under
error-prone conditions.
[0216] In the preferred mode, ten reactions of 100 .mu.l each were
amplified with each PCR enzyme.
[0217] i. Amplifiction with Taq DNA Polymerase:
[0218] Reaction Mixture
5 Reaction Mixture 1.times. magnesium free Taq Buffer (Stratagene
catalog #200530) 1 mM each TTP and dCTP 0.2 mM each dGTP and dATP 2
ng/.mu.l Primer 923 (also called 490) 2 ng/.mu.l Primer 721 0.05
u/.mu.l Taq2000 (Stratagene catalog #600195) 1.5 mM MgCl.sub.2 0.5
mM MnCl.sub.2 0.1 pM plasmid DNA (clone #550)
[0219] Cycling Parameters
[0220] PCRs were carried out using Stratagene's ROBOCYCLER.TM.40
Temperature Cycler with a Hot Top assembly. The following cycling
conditions were used:
[0221] 1) 95.degree. C. for 1 minute
[0222] 2) 95.degree. C. for 1 minute
[0223] 3) 54.degree. C. for 1 minute
[0224] 4) 72.degree. C. for 2.5 minutes
[0225] 5) Repeat steps 2 to 4 thirty times.
[0226] ii. Amplification with exo.sup.- JDF-3 DNA Polymerase
6 Reaction Mixture 1.times. magnesium free Taq Buffer (Stratagene
catalog #200530) 450 .mu.M each deoxynucleotide (dGTP, dATP, TTP
and dCTP) 2 ng/.mu.l Primer 923 (also called 490) 2 ng/.mu.l Primer
721 0.1 u/.mu.l exo.sup.- JDF-3 DNA polymerase 0.5 mM MnCl.sub.2
0.1 pM plasmid DNA (clone #550)
[0227] Cycling Parameters
[0228] PCRs were carried out using Stratagene's ROBOCYCLER.TM.40
Temperature Cycler with a Hot Top assembly. The following cycling
conditions were used:
[0229] 1) 95.degree. C. for 1 minute
[0230] 2) 95.degree. C. for 1 minute
[0231] 3) 54.degree. C. for 1 minute
[0232] 4) 72.degree. C. for 2.5 minutes
[0233] 5) Repeat steps 2 through 4 thirty times.
[0234] iii. Amplifiction with exo.sup.- Pfu DNA Polymerase
7 Reaction Mixture 1.times. TAQPLUS .RTM. Precision Buffer
(Stratagene catalog #600210) 200 .mu.M each deoxynucleotide (dGTP,
dATP, TTP, dCTP) 2 ng/.mu.l Primer 923 (also called 490) 2 ng/.mu.l
Primer 721 0.05 u/.mu.l exo.sup.- Pfu DNA polymerase (Stratagene
catalog number 600163) 0.1 pM plasmid DNA (clone #550)
[0235] Cycling Parameters
[0236] PCRs were carried out using Perkin-Elmer's 9600 Temperature
Cycler. The following cycling conditions were used:
[0237] 1) 95.degree. C. for 1 minute
[0238] 2) 95.degree. C. for 1 minute
[0239] 3) 53.degree. C. for 1 minute
[0240] 4) 72.degree. C. for 5 minutes
[0241] 5) Repeat steps 2 through 4 thirty times.
[0242] Forward Primers
[0243] Earlier versions of the mutant libraries were made with the
forward primer 461, which contains an EcoR I site. When products
amplified with primers 461 and 923 were restriction digested and
cloned into the lambda vector as described in the following
section, JDF-3 DNA polymerase was synthesized as a fusion protein
with the first 39 amino acids of the vector-encoded -galactosidase
(lacZ) protein.
8 Primer 461 5' TCAGATGAATTCGATGATCCTTGACGTTGATTAC 3' EcoR I JDF-3
specific sequence
[0244] The clones isolated using primer 461 were designed as
p#.
[0245] The preferred mode of amplification and cloning utilizes the
forward primer 721, which also contains an EcoR I site followed by
three consecutive in-frame stop codons and a ribosome binding site.
This arrangement allows the JDF-3 DNA polymerase to be translated
without any vector-derived residues at the amino terminus. The
clones isolated from libraries constructed with the forward primer
721 were designated as 1-# to differentiate them from the p# series
of clones.
9 Primer 721 5' GAGAGAATTCATAATGATAAGGAGGAAAAAATTATGATCCTTG-
ACGTTGATTAC3' EcoR I 3x STOP JDF-3 specific sequence Reverse
Primers Primer 923 (490) 5' TCAGATCTCGAGTCACTTCTTCTTCCCCTTC 3' Xho
I JDF-3 specific sequence
[0246] E. Preparing PCR Products for Cloning.
[0247] PCR products were purified and concentrated with the
STRATAPREP.TM. PCR Purification kit (Stratagene catalog number
400771). The PCR products were then digested with 50 units of Xho I
and 50 units of EcoR I in 1.5.times. Universal buffer (10.times.
Universal Buffer: 1M KOAc, 250 mM Tris-Acetate (pH 7.6), 10 mM
MgOAc, 5 mM -mercaptoethanol and 100 .mu.g/ml BSA) for one hour at
37.degree. C. The digested samples were run on a 1% agarose,
1.times.TBE gel and visualized with ethidium bromide staining. The
2.3 kb amplification product was gel isolated and purified with the
STRATAPREP.TM. DNA Gel Extraction Kit (Stratagene catalog number
400766).
[0248] F. Cloning PCR Inserts into the Uni-Zap.RTM.XR Lambda
Vector.
[0249] 200 ng of purified amplification product was ligated with 11
g of UNI-ZAP.RTM.XR Lambda Vector (Stratagene catalog #239213),
which had been predigested with EcoR I and Xho I and then
dephosphorylated with alkaline phosphatase (Stratagene catalog
number 237211). The DNAs were ligated using 2 units of T4 DNA
ligase (Stratagene catalog number 600011) and 0.5 mM ATP in
1.times. ligase buffer (50 mM Tris-HCL (pH 7.5), 7 mM MgCl.sub.2, 1
mM DTT) in reaction volumes of 10 to 15 .mu.l. Ligations were
carried out at 16.degree. C. for a minimum of 16 hours.
[0250] G. Lambda Packaging and Bacterial Infection.
[0251] Two microliters of each ligation reaction were packaged with
GIGAPACK.RTM. III Gold Packaging extract (Stratagene catalog
#200201) for 90 minutes at room temperature before being stopped
with 500 l SM buffer (50 mM Tris pH 7.5, 100 mM NaCl, 8 mM
MgSO.sub.4 and 0.01% gelatin) and 20 l of chloroform. The packaged
lambda vectors were plated on E. coli XL1-Blue MRF' host cells.
[0252] H. Dideoxynucleotide Screening.
[0253] Mutant polymerase libraries were screened using a variation
of the technique used by Sagner et al (Sagner, G., Ruger, R., and
Kessler, C. (1991) Gene 97: 119-123). Lambda phage clones were
plated at a density of 10-20 plaques per square centimeter.
Proteins present in the plaques were transferred to filters and
moistened with polymerase screening buffer (50 mM Tris (pH 8.0), 7
mM MgCl.sub.2, 3 mM -ME). The filters were kept between layers of
plastic wrap and glass while the host cell proteins were
heat-inactivated by incubation at 65.degree. C. for 30 minutes. The
heat-treated filters were transferred to fresh plastic wrap and
approximately 35 .mu.l of the polymerase assay cocktail was added
for every square centimeter of filter. Polymerase assay cocktail
consisted of 1.times.cloned Pfu magnesium-free buffer (Stratagene
catalog #200534), 125 ng/ml activated calf thymus or salmon sperm
DNA, 1.29 .mu.Ci/ml .alpha.-.sup.33P ddNTP (Amersham), and 0.5 mM
MnCl.sub.2. Initial screening was done in the presence of
MnCl.sub.2, but the preferred method was to screen in 1.times.Taq
Polymerase buffer (1.5 mM MgCl.sub.2). The filters were sandwiched
between plastic wrap and glass again and incubated at 65.degree. C.
for one hour, and then at 70.degree. C. for one hour and 15
minutes. The filters were washed three times in 2.times.SSC for
five minutes each time before being rinsed twice in 100% ethanol
and dried on a vacuum dryer. The filters were exposed to X-ray film
for approximately 16 hours. Plaques corresponding to strong signals
were cored and placed in SM buffer. The positive primary plaques
were replated at more dilute concentrations and assayed under
essentially similar conditions to allow the purification of
isolated plaques.
[0254] Dye-Dideoxynucleotide Screening
[0255] To detect mutant polymerases with improved capacity for
dye-deoxynucleotide and dye-dideoxynucleotide utilization, the
JDF-3 mutant DNA polymerase library was screened as described
previously with the following exceptions:
[0256] Polymerase Assay Cocktail for Flu-12-dUTP Screening:
[0257] 0.9.times.Taq Buffer (Stratagene Catalog #200435), 65 .mu.M
dATP, 65 .mu.M dCTP, 65 .mu.M dGTP, 65 .mu.MdTTP, 0.3 .mu.M
Fluoresceince-12-dUTP (Stratagene in-house production), 0.75
.mu.g/.mu.l activated calf thymus DNA.
[0258] Polymerase Assay Cocktail for ROX ddNTP
[0259] 1.times.Taq Buffer, 0.9 .mu.M dATP, 0.9 .mu.M dCTP, 0.9
.mu.M dGTP, 0.9 .mu.l TTP, 0.6 .mu.M ROX ddATP (New England Nuclear
(NEN) NEN478), 0.06 .mu.M ROX ddGTP (NEN NEL479), 0.06 .mu.M ROX
ddCTP (NEN NEL477), 0.06 .mu.M ROX ddUTP (NEN NEL476), 0.84
.mu.g/.mu.l activated calf thymus DNA. (Note: A screening system
without ROX ddGTP is the preferred method since DNA polymerases do
not discriminate against ddGTP).
[0260] Polymerase Assay Cocktail for Fluroesceine ddUTP
[0261] 1.times.Taq Buffer, 70 .mu.M dATP, 70 .mu.M dTTP, 70 .mu.M
dCTP, 15 .mu.M dTTP, 1 .mu.M Fluroesceine-12-ddUTP (NEN NEL401),
0.84 .mu.g/.mu.l activated calf thymus DNA.
[0262] Antibody Binding to Fluroesceine
[0263] The filters were blocked overnight with 1% non-fat dry milk
dissolved in TBST (50 mM Tris pH 8.0, 150 mM NaCl, 0.05% Tween-20)
at 4.degree. C. The filters were washed briefly in TBST before
alkaline phosphatase conjugated anti-fluoresceine antibody from the
Illuminator kit (Stratagene catalog #300360) was added at a
{fraction (1/10,000)} dilution in 50 ml TBST. The antibody was
detected with NBT/BCIP at concentrations of 0.3 mg/ml and 0.15
mg/ml respectively in a buffer composed of 100 mM Tris pH 9.5, 100
mM NaCl, and 5 mM MgCl.sub.2.
[0264] Antibody Binding to Rhodamine
[0265] Anti-ROX antibody (Zymed cat. no. 71-3600 rabbit Rhodamine
(5-ROX polyclonal, 1 mg/ml)) was diluted to 1:1000 in TBST. The
bfocked filters were blotted briefly to remove excess moisture then
laid on plastic wrap and covered with 2.5 ml of the diluted
antibody solution. An additional sheet of plastic wrap was laid
over the filters before incubation at room temperature for 1 hour.
The filters were washed briefly three times with TBST, then washed
three times with gentle agitation for 15 minutes each time. The
washed filters were incubated with alkaline phosphatase conjugated
goat anti-rabbit antibodies diluted 1:5000 in TBST. The filters
were incubated with the antibody for one hour then detected with
NBT/BCIP as described previously.
[0266] I. Dideoxynucleotide Qualification
[0267] Lambda phage clones which incorporated .sup.33P-labeled
ddNTPs in the primary library screen were re-screened to verify
polymerase activity and to assess the contribution of the divalent
metal ion to .sup.33P-ddNTP incorporation. The clones selected
during this round of screening were designated as p#. These clones
all contained an amino-terminal tag, as discussed in the section
entitled "Forward Primers". FIG. 5 shows that clones p1, p2, p3,
p6, p7, p8, p9, p10, p11, p12, p14, p15, and p16 exhibited wild
type levels of DNA polymerase activity, based upon similarity in
signal strength to the parental #550 clone (FIG. 5, panel 3).
Although initial screening was carried out in the presence of 0.5
mM MnCl.sub.2, all of the clones except p9 and p10 were able to
incorporate .sup.33P-labeled ddNTPs to at least some extent in the
presence of 1.5 mM MgCl.sub.2 (panel 2), with clones p2, p4, p8,
p11, p12, p13, p14, p15, p17, and p18 producing the highest
signals.
[0268] Eighteen mutants were chosen for evaluation. One microliter
of phage isolated from each purified plaque was placed on each of
three E. coli XL1-Blue MRF' lawns. Phage containing a parental copy
of exo.sup.- JDF3 DNA (#550 clone) were also spotted on the grid.
The plaques formed by the phage were transferred to filters and
treated as described in the preceding screening section with the
exception of the final buffer composition. The buffers used for
each filter (filters 1-3) are as follows:
[0269] Filter 1: Dideoxynucleotide Screen with Manganese
Chloride
[0270] 1.times.Taq DNA polymerase magnesium-free buffer
[0271] 1.28 .mu.Ci/ml .sup.33P ddNTPs
[0272] 0.5 .mu.g/.mu.l Activated Calf Thymus DNA (Sigma)
[0273] 0.5 mM MnCl.sub.2
[0274] Filter 2: Dideoxynucleotide Screen with Magnesium
Chloride
[0275] 1.times.Taq DNA polymerase buffer (containing 1.5 mM
MgCl.sub.2, catalog #200435)
[0276] 1.28 .mu.Ci/ml .sup.33P ddNTP
[0277] 0.5 .mu.g/.mu.l Activated Calf Thymus DNA (Sigma)
[0278] Filter 3: Deoxynucleotide Screen with Magnesium Chloride
[0279] 1.times.Taq DNA polymerase buffer
[0280] 0.072 mM dGTP, dCTP and TTP
[0281] 40 .mu.M dATP
[0282] 0.5 .mu.g/ml Activated Calf Thymus DNA (Sigma)
[0283] 0.01 .mu.Ci .alpha.-.sup.33P dATP.
[0284] Results are shown in FIG. 5.
[0285] Dye-Dideoxynucleotide Qualification
[0286] As described in the previous segments, primary lambda clones
were spotted on an E. coli lawn and re-screened with the
appropriate antibody or antibodies.
[0287] J. Excision of Lambda Clones.
[0288] When incubated with helper phage under suitable conditions,
Lambda Zap.TM. vectors are designed to produce phagemid copies of
the part of the vector containing pBluescript (SK-) and the insert.
This process yields a plasmid (pBluescript SK-) vector carrying the
same insert that was contained in the lambda clone. Excision of
clones with the desired phenotype was carried out according to the
instructions in the EXASSIST.TM. system (Stratagene catalog
#200253).
[0289] K. Sequence Analysis of Mutants.
[0290] The mutants were sequenced by Sequetech Corporation
(Mountain View, Calif.) using the following primers:
10 Primer 3 (or primer G) 5' CCAGCTTTCCAGACTAGTCGGCCAAGG- CC 3'
Primer 5 (or JDF3-1128) 5' AACTCTCGACCCGCTG 3'
[0291] L. Dideoxynucleotide Mutagenesis.
[0292] To conclusively identify the amino acids contributing to
reduced ddNTP discrimination, individual point mutations were
introduced into the exo.sup.- JDF-3 #550 clone using the
QUIKCHANGE.TM. Site-Directed Mutagenesis Kit (Stratagene catalog
#200518). The following mutants were prepared: L408H, L408F, P410L,
A485T, S345P, D373Y, A619V, and L631V. In addition, a double mutant
(P410L/A485T) was constructed by introducing the A485T mutation
into the exo.sup.- JDF-3 P410L mutant clone. To completely
eliminate all 3' to 5' exonuclease activity, the mutation D141A was
added to all clones. A pre-existing 5' to 3' exonuclease mutation
(E143A) was present in the parental template JDF-3 550.
[0293] Dye-Dideoxynucleotide Mutagenesis
[0294] To conclusively identify amino acids responsible for
contributing to reduced discrimination of dye nucleotides, the
mutation S345P was generated alone and in combination with the
P410L and P410L+A485T.
[0295] M. Preparation of Heat-Treated Bacterial Extracts.
[0296] E. coli SOLR cells containing the excised plasmid were grown
overnight at 37.degree. C. The cells contained in 500 .mu.l of
culture were collected by microcentrifugation. The cell pellets
were resuspended in 501 .mu.l of 50 mM Tris (pH 8.0). Lysozyme was
added to a final concentration of 11 .mu.g/.mu.l, and the cells
were lysed during a 10 minute incubation at 37.degree. C., followed
by 10 minutes at 65.degree. C. The heat-inactivated cell material
was collected by microcentrifugation and the supernatants were
assayed for dNTP and ddNTP incorporation as described below. N.
Purification of JDF-3 and JDF-3 polymerase mutants.
[0297] One method for purifying exo.sup.- JDF-3 DNA polymerase
involves ammonium sulfate fractionation, followed by Q Sepharose
and DNA cellulose columns. A second method has been developed to
allow rapid purification of JDF-3 polymerase mutants, and entails
adsorption of contaminants on a HiTrap Q column, followed by
gradient elution from a HiTrap heparin column (section iii).
[0298] i. Preparation of Bacterial Lysate.
[0299] Frozen cell paste (3-14 grams) was resuspended with 3.times.
volume of lysis buffer, consisting of 50 mM Tris-HCl (pH 8.0), 1 mM
EDTA, and 10 mM -mercaptoethanol. Lysozyme was added to 0.2 mg/ml
and PMSF was added to 1 mM final concentration. The cells were
lysed on ice over a period of 1 hour. The lysate was then sonicated
for 2 minutes (90% duty, level of 2.times.2.5, 1.times.3.0).
Following sonication, the lysate was heated at 65C for 15 minutes
to denature bacterial proteins. The heated lysate was then
centrifuged for 30 minutes at 14.5K rpm in a Sorvall RC-2B
centrifuge using a Sorvall SS-34 rotor, and the supernatant was
recovered.
[0300] ii. Ammonium Sulfate Fractionation and Q Sepharose/DNA
Cellulose Chromatography (Method 1)
[0301] Ammonium sulfate was added to the bacterial lysate to a
final concentration of 45%. The ammonium sulfate was added over a
period of 15 minutes, and the mixture was stirred for an additional
30 minutes. The mixture was centrifuged as described above, and the
supernatant was recovered. Additional ammonium sulfate was then
added to bring the final concentration to 65%. The mixture was
centrifuged as described above, and the supernatant removed. The
pellet was resuspended in 10 ml of buffer A consisting of 50 mM
Tris-HCl (pH 7.5), 1 mM EDTA, 10 mM -mercaptoethanol, 0.1% (v/v)
Tween 20, and 10% (v/v) glycerol. The supernatant was dialyzed
overnight against 2 changes of buffer A (3 liters each).
[0302] The dialysate was loaded onto a 2.6.times.9.4 cm Q-Sepharose
Fast Flow column (50 mls), pre-equilibrated in buffer A. The column
was washed with buffer A until the absorbence (OD.sub.280)
approached baseline. The column was then eluted with a gradient
from 0 to 1M NaCl/buffer A. Fractions were collected, and analyzed
by SDS-PAGE and DNA polymerase activity assays (see below). Active
protein typically eluted between 130 and 240 mM NaCl. Active
fractions were pooled and dialyzed overnight against 2 changes of
buffer B (3 liters each), consisting of 50 mM Tris-HCl (pH 7.5), 1
mM EDTA, 10 mM -mercaptoethanol, 0.1% (v/v) Tween 20,
10%(v/v)glycerol, and 50 mM NaCl.
[0303] The Q-Sepharose eluate was then loaded onto a 1.6.times.4.9
cm (10 mls) DNA cellulose column, equilibrated in buffer B. The
column was washed with buffer B until the absorbence (OD.sub.280)
approached baseline. The column was then eluted with a gradient
from 50 to 1000 mM NaCl/buffer A. Fractions were collected, and
analyzed by SDS-PAGE and DNA polymerase activity assays. Active
protein typically eluted between 280 and 360 mM NaCl. Active
fractions were pooled and dialyzed overnight against JDF-3 final
dialysis buffer, consisting of 25 mM Tris-HCl (pH 7.5), 100 mM KCl,
0.1 mM EDTA, 1 mM DTT, 0.1% (v/v) Tween 20, 0.1% (v/v) Igepal 630,
10 g/ml BSA, and 50% (v/v) glycerol.
[0304] iii. HiTrap Q/HiTrap Heparin Chromatography (Method 2)
[0305] The preferable method for rapid purification of multiple
mutants is as follows. Bacterial cell lysates were prepared as
described for method 1, except that Tween 20 and Igepal CA 630 were
added to a final concentration of 0.01% (v/v) just prior to the
heat denaturation step, and a heat denaturation temperature of 72C
was used.
[0306] The lysate was loaded onto a 1.6.times.2.5 cm (5 mls) HiTrap
Q column (pre-packed column from Pharmacia), pre-equilibrated in
buffer C consisting of 50 mM Tris-HCl (pH 8.2), 1 mM EDTA, 10 mM
-mercaptoethanol, 0.1% (v/v) Tween 20, and 0.1% (v/v) Igepal CA
630. The column was washed with buffer C until the absorbence
(OD.sub.280) approached baseline. The flow through fractions
(OD.sub.280 absorbence above background) were collected and then
loaded onto a 1.6.times.2.5 cm (5 mls) HiTrap heparin column
(pre-packed column from Pharmacia), pre-equilibrated in buffer D
consisting of 50 mM Tris-HCl (pH 8.2), 1 mM EDTA, 1 mM DTT, 0.1%
(v/v) Tween 20, 0.1% (v/v) Igepal CA 630, and 10% glycerol (v/v).
The column was washed with buffer D until the absorbence
(OD.sub.280) approached baseline. The column was then eluted with a
gradient from 0 to 1M KCl/buffer D. Fractions were collected, and
analyzed by SDS-PAGE and DNA polymerase activity assays. Active
protein typically eluted between 390 and 560 mM NaCl. Active
fractions were pooled and dialyzed overnight against JDF-3 final
dialysis buffer (see above). Purified polymerases were stored at
-20 C.
[0307] iv. Analysis of Purified Proteins
[0308] The concentrations of JDF-3 and mutant DNA polymerases were
determined relative to a BSA standard (Pierce), using Pierce's
Coumassie Blue Protein assay reagent. In addition, the purity and
relative protein concentrations of different polymerase
preparations were verified by SDS-PAGE. Polymerase samples were
electrophoresed on 4-20% Tris-glycine gels (Novex), and the gels
were silver-stained using standard procedures.
[0309] O. Nucleotide Incorporation Assay.
[0310] DNA polymerase activity was measured using purified JDF-3
polymerase mutants or heat-treated bacterial extracts prepared from
various mutant clones. DNA polymerase activity was measured by
monitoring the incorporation of .sup.3H-TTP into activated calf
thymus DNA. A typical DNA polymerase reaction cocktail
contained:
[0311] 10 mM Tris-HCl, pH 8.8
[0312] 1.5 mM MgCl.sub.2
[0313] 0.001% gelatin
[0314] 200 .mu.M each dATP, dCTP, dGTP
[0315] 195 .mu.M TTP
[0316] 5 M [.sup.3H]TTP (NEN #NET-221H, 20.5Ci/mmole; partially
evaporated to remove EtOH)
[0317] 250 g/ml of activated calf thymus DNA (e.g., Pharmacia
#27-4575-01)
[0318] Incorporation was measured by adding 1 .mu.l of polymerase
samples to 110 .mu.l aliquots of polymerase cocktail. DNA
polymerase samples were diluted in a suitable storage buffer (e.g.,
25 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1%
(v/v) Tween 20, 0.1% (v/v) Igepal 630, 10 .mu.g/ml BSA, and 50%
(v/v) glycerol). Polymerization reactions were conducted for 30
minutes at 72C. Extension reactions were quenched on ice, and then
5 .mu.l aliquots were spotted immediately onto DE81 ion-exchange
filters (2.3 cm; Whatman #3658323). Unincorporated [.sup.3H]TTP was
removed by 6 washes with 2.times.SCC (0.3M NaCl, 30 mM sodium
citrate, pH 7.0), followed by a brief wash with 100% ethanol.
Incorporated radioactivity was measured by scintillation counting.
Reactions that lacked enzyme were also set up along with sample
incubations to determine "total cpms" (omit filter wash steps) and
"minimum cpms" (wash filters as above).
[0319] Cpms bound is proportional to amount of polymerase activity
present per volume of bacterial extract. The volume of bacterial
extract (0.25-1 .mu.l) which brought about incorporation of
approximately 10,000 cpms was determined for use in subsequent
nucleotide analog incorporation testing.
[0320] P. Quantitating ddNTP Incorporation Efficiency.
[0321] JDF-3 polymerase mutants were evaluated to assess relative
ddNTP incorporation efficiency. Nucleotide incorporation was
measured in the presence of varying concentrations of each ddNTP
terminator (ddATP, ddCTP, ddGTP, and ddTTP). Since ddNTP
incorporation produces non-extendable termini, polymerization is
strongly inhibited for polymerases that incorporate ddNTPs
efficiently. Comparisons of ddNTP concentrations that bring about
50% inhibition of nucleotide incorporation (I.sub.50%) can be used
to compare ddNTP incorporation efficiency of different polymerases
or polymerase mutants. Comparisons of I.sub.50% values for ddATP,
ddCTP, ddGTP, and ddTTP can be used to identify mutants with
reduced selectivity for particular bases. Such mutants would be
expected to produce more uniform DNA sequencing ladders.
[0322] To measure incorporation of individual ddNTPs, cocktails
were prepared which consisted of varying concentrations of the
ddNTP of interest, and a total of 200 .mu.M of each nucleotide
triphosphate. For example, the incorporation of ddATP by wild type
JDF-3 polymerase was measured at 0, 40, 80, 120 and 160 .mu.M
ddATP. In these reactions, dATP concentrations were adjusted to
200, 160, 120, 80, and 40 .mu.M, respectively, so that the total
amount of adenine nucleotide triphosphate was 200 .mu.M. In
comparison, mutants were assayed using ddATP concentrations of 0,
5, 10, and 20 .mu.M ddATP, and adjusted dATP concentrations of 200,
195, 190, and 180 .mu.M, respectively (dATP+ddATP=200 .mu.M).
Additional cocktails were prepared to measure ddCTP, ddGTP, and
ddTTP incorporation. To assess ddNTP incorporation by JDF-3 mutants
at 3 different ddNTP concentrations, 12 reaction cocktails were
prepared consisting of:
[0323] 10 mM Tris-HCl, pH 8.8
[0324] 1.5 mM MgCl.sub.2
[0325] 50 mM KCl
[0326] 0.001% gelatin
[0327] 5 .mu.M [.sup.3H]TTP (NEN #NET-221H, 20.5Ci/mmole; partially
evaporated to remove EtOH)
[0328] 250 .mu.g/ml of activated calf thymus DNA (e.g., Pharmacia
#27-4575-01)
[0329] To each of 12 reaction cocktails was added the appropriate
amounts of dNTPs and ddNTPs as summarized below:
11 Cocktail DGTP dDATP dCTP TTP ddGTP ddATP ddCTP ddTTP G-0 200
.mu.M 200 .mu.M 200 .mu.M 195 .mu.M 0 0 0 0 G-5 195 .mu.M 200 .mu.M
200 .mu.M 195 .mu.M 5 0 0 0 G-10 190 .mu.M 200 .mu.M 200 .mu.M 195
.mu.M 10 0 0 0 G-20 180 .mu.M 200 .mu.M 200 .mu.M 195 .mu.M 20 0 0
0 A-0 200 .mu.M 200 .mu.M 200 .mu.M 195 .mu.M 0 0 0 0 A-5 200 .mu.M
195 .mu.M 200 .mu.M 195 .mu.M 0 5 0 0 A-10 200 .mu.M 190 .mu.M 200
.mu.M 195 .mu.M 0 10 0 0 A-20 200 .mu.M 180 .mu.M 200 .mu.M 195
.mu.M 0 20 0 0 C-0 200 .mu.M 200 .mu.M 200 .mu.M 195 .mu.M 0 0 0 0
C-5 200 .mu.M 200 .mu.M 195 .mu.M 195 .mu.M 0 0 5 0 C-10 200 .mu.M
200 .mu.M 190 .mu.M 195 .mu.M 0 0 10 0 C-20 200 .mu.M 200 .mu.M 180
.mu.M 195 .mu.M 0 0 20 0 T-0 200 .mu.M 200 .mu.M 200 .mu.M 195
.mu.M 0 0 0 0 T-5 200 .mu.M 200 .mu.M 200 .mu.M 190 .mu.M 0 0 0 5
T-10 200 .mu.M 200 .mu.M 200 .mu.M 185 .mu.M 0 0 0 10 T-20 200
.mu.M 200 .mu.M 200 .mu.M 175 .mu.M 0 0 0 20
[0330] Incorporation was measured by adding 1 .mu.l of
appropriately diluted bacterial extract (10,000 cpms) to 10 .mu.l
of each polymerase cocktail. Polymerization reactions were
conducted for 30 minutes at 72C. The extension reactions were
counted as described above.
[0331] Reactions that lacked enzyme were also set up along with
sample incubations to determine "minimum cpms" (wash filters as
above). To determine % activity as a function of ddNTP
concentration, background ("minimum cpms" value) was first
subtracted from each of the sample cpms. "Total cpms", which are
equivalent to 100% activity (0 ddNTPs), are determined by averaging
the corrected cpms for the 4 reactions lacking ddNTPs (A-0, G-0,
C-0, and T-0). Percent remaining activity was then calculated by
dividing corrected sample cpms (with ddNTPs) by the corrected total
cpms (average 0 ddNTPs).
[0332] Percent activity was plotted as a function of ddNTP
concentration. I.sub.50% values for each ddNTP (ddNTP concentration
which inhibits nucleotide incorporation by 50%) were determined for
each mutant. Comparisons allowed the identification of mutants with
improved ddNTP incorporation relative to wild type JDF-3.
[0333] Initial studies used purified enzymes, and I.sub.50% values
were determined from inhibition plots employing 40-160 .mu.ddNTPs.
The results in Table V show that mutants p8 (P41OL), p11 (P410L),
and p12(A485T) are inhibited by lower concentrations of ddNTPs than
the parental exo.sup.- JDF-3 polymerase. Greater sensitivity
indicates that the mutants incorporate all four ddNTPs more
efficiently than the original JDF-3 polymerase.
[0334] For enzymes which preferentially incorporate TTP over ddTTP
(exo.sup.- JDF-3, exo.sup.- Pfu), the use of increasingly higher
concentrations of ddTTP (80-160 .mu.M) and correspondingly lower
concentrations of TTP (115-35 .mu.M), in combination with a
constant amount of [H]TTP (5 .mu.M), leads to an increase in cpms
incorporated with increasing ddNTP concentration. Therefore, in
these initial experiments (where ddTTP>120 .mu.M), 150% values
for TTP are artificially high. While they can be used to compare
ddTTP incorporation among different polymerase mutants, they can
not be used to assess reduced/enhanced preference for ddTTP
relative to ddCTP, ddGTP, or ddATP.
12TABLE V I.sub.50% Values for Purified JDF-3 and JDF-3 Mutants.
Primary I.sub.50% Values (.mu.M) Purified Polymerase Mutation ddATP
ddGTP ddCTP ddTTP Exo.sup.-JDF-3 -- 160 110 >160 >>160
Exo.sup.-Pfu -- >160 >160 >160 >>160 JDF-3 mutant p8
P410L 30 25 40 40 JDF-3 mutant p11 P410L 30 30 60 >160 JDF-3
mutant p12 A485T 40 25 25 150
[0335] To allow a larger number of mutant clones to be screened,
subsequent experiments employed bacterial extracts containing JDF-3
polymerase mutants. In addition, sensitivity was improved by using
lower concentrations of each ddNTP inhibitor (5-20 .mu.M). The
results in Table VI demonstrate that all of the mutants selected
from the primary filter screen exhibited improved incorporation of
ddNTPs. Improvements in ddNTP incorporation were as high as
>20-fold. All of the mutants containing a mutation at amino acid
408 (L408H/F), 410 (P410L), or 485 (A485T) (referred to as the
"primary mutation") exhibited reduced discrimination against all
four ddNTPs. Most, but not all, mutants with the L408H/F primary
mutation produced very similar I.sub.50% values (<2-fold
difference) for all four ddNTPs, indicating that base selectivity
is diminished or absent.
13TABLE VI I.sub.50% Values for JDF-3 Mutants (Bacterial Extracts).
Primary I.sub.50% Values (.mu.M) JDF-3 mutant clones mutation ddATP
ddGTP ddCTP ddTTP Exo.sup.-JDF-3 -- >80 >80 >80 >80
1-1, 1-4, 1-18 L408H 8 to >20 4 to 5 6 to 13 5.5. to 10 1-25,
1-28, 1-29, 1-17 L408F 4.5 to >20 3.5 to 10 4 to 6.5 4 to 8 p8
P410L 18.5 12 9.5 >20 1-5, 1-6, 1-17 P410L 10 to >20 3.5 to 9
16.5 to >20 11 to >20 1-41, 1-38, 1-37, 1-3, Not 7 to >20
3.5 to 12 4 to >20 5 to >20 1-19, 1-30, 1-27, 1,20 determined
1-26, 1-32, 1-16, 1-12
[0336] Q. Sequencing with Purified JDF-3 Polymerase Mutants.
[0337] i. Sequencing with Radioactively Labeled
Dideoxynucleotides
[0338] 1 to 2 .mu.l of purified enzyme was substituted into the
Thermo Sequenase radiolabeled terminator cycle sequencing kit
(Amersham-Pharmacia #US79750). The samples were processed according
to the manufacturer's instructions using the control primer and
template provided with the kit. Three microliters of each
sequencing reaction were loaded onto a 6% acylamide-7M urea,
1.times.TBE CASTAWAY.TM. Precast gel (Stratagene catalog #s 401090
and 401094). When the bromophenol blue indicator dye reached the
end of the gel, the gel was fixed, dried and exposed to film for
24-72 hours (FIG. 6).
[0339] The results in FIG. 6 show that clones p11 (panel D) and p8
(panel E) exhibit a dramatic improvement in the incorporation of
all four ddNTPs compared to the parental #550 clone (panel B).
Mutants p11 and p8 both contain the primary P410L mutation and an
amino tag, but differ with respect to the number and types of
ancillary mutations. Mutant p12 (panel C) produced a faint
sequencing ladder, presumably due to the use of an insufficient
amount of enzyme or the presence of ancillary mutations which
reduce thermal stability. There is evidence of termination products
in all lanes, suggesting an improvement in the incorporation of all
four ddNTPs relative to the parental clone. Mutant p12 contains the
primary mutation A485T in addition to ancillary mutations. In
contrast to JDF-3 mutants identified here, the parental clone shows
a strong preference to incorporate ddGTP, as evidenced both in
primer extension (FIG. 6) and ddNTP inhibition assays (Tables V and
VI).
[0340] ii. Sequencing with a Radioactively Labeled Primer and
Fluorescent Dideoxynucleotides
[0341] Different DNA polymerases and polymerase mutants will
exhibit varying degrees of discrimination against the dye moieties
on the dideoxynucleotide analogs. An assessment of usage of
dye-labeled dideoxynucleotide analogs by the JDF-3 polymerase
mutants was carried out. The procedure used was as follows:
[0342] a. Primer Labeling
[0343] The sequencing primer SK was radioactively labeled with the
KINACE-IT.TM. Kinasing Kit (Stratagene catalog #200390). The
incubation reaction (40 .mu.l) contained the following
components:
[0344] 1.times. kinase buffer #1
[0345] 0.75 .mu.Ci/.mu.l -.sup.33P ATP
[0346] 0.375 u/.mu.l T4 polynucleotide kinase
[0347] 2.5 pmol/.mu.l SK primer
[0348] The reaction was incubated at 37C for 45 minutes. The primer
was purified away from free nucleotides with a size exclusion
matrix (NUC TRAPS Stratagene catalog number 400701).
[0349] b. Dye Labeled-Dideoxynucleotide: dNTP Ratios
[0350] Fluorescent dideoxynucleotides were purchased from New
England Nuclear (NEN):
14 R6G-ddATP NEN catalog number NEL-490 R110-ddTP NEN catalog
number NEL-495 TAMRA-ddUTP NEN catalog number NEL-472 ROX-ddCTP NEN
catalog number NEL-477
[0351] Incorporation was measured using 3 different concentrations
of dye labeled dideoxynucleotides (ddNTPs) and a constant amount of
deoxynucleotides (dNTPs; 2.14 .mu.M):
15 Condition 3) 1:1 (2.14 .mu.M each dNTP:2.14 .mu.M dye-labeled
ddNTP) Condition 2) 1:0.1 (2.14 .mu.M each dNTP:0.214 .mu.M
dye-labeled ddNTP) Condition 1) 1:0.01 (2.14 .mu.M each dNTP:0.0214
.mu.M dye-labeled ddNTP)
[0352] c. Preparation of the DNA Sequencing Reaction Mixtures
[0353] Four polymerases were tested for utilization of dye-labeled
ddNTPs, exo.sup.- JDF-3 (#550 clone), Thermo Sequenase (4u/.mu.l),
JDF-3 P410L (clone p8 with ancillary mutations and an
amino-terminal tag) and JDF-3 L408H (clone 1-1). A mixture
containing the following reagents was assembled:
[0354] 13.7 .mu.l H.sub.2O
[0355] 1 .mu.l labeled SK primer (2 pmol/.mu.l)
[0356] 1 .mu.l pBluescript KS (0.2 .mu.g/.mu.l)
[0357] 1 .mu.l polymerase (.about.1.5 u/.mu.l)
[0358] 2 .mu.l 10.times. buffer (reaction buffer 1 for all but
L408H which uses 1.5 mM MgCl.sub.2, buffer (see below)
[0359] 10.times. Reaction Buffer 1
[0360] 260 mM Tris pH 9.5
[0361] 65 mM MgCl.sub.2
[0362] 10.times. 1.5 mM MgCl.sub.2 Buffer
[0363] 24 mM MgCl.sub.2
[0364] 260 mM Tris pH 9.5
[0365] 2.5 .mu.l of each dye-labeled ddNTP terminator (ddGTP,
ddATP, ddTTP and
[0366] ddGTP was aliquotted separately into one of four tubes. 4.5
.mu.l of each polymerase reaction was added to each of the four
tubes, to give a final reaction volume of 7 .mu.l.
[0367] d. Cycle Sequencing Reactions
[0368] The samples were cycled in a RoboCycler.RTM.96 Temperature
Cycler with a Hot Top Assembly (Stratagene Catalog #400870 and
#400894) using the following conditions:
[0369] 1) 1 minute at 95C
[0370] 2) 1 minute at 95C
[0371] 3) 1 minute at 50C
[0372] 4) 2 minutes at 72C
[0373] 5) Repeat steps 2-4 thirty times.
[0374] 4 .mu.l of stop solution (95% formamide, 20 mM EDTA, 0.05%
bromophenol blue, 0.05% xylene cyanol FF) was added to each of the
amplified reactions before heating them to 99.degree. C. for five
minutes. The samples were electrophoresed on a 6% CASTAWAY.TM. gel
as described above. The gels were dried and then exposed to film
for 72 hours (FIG. 7).
[0375] The results of studies designed to assess utilization of
dye-labeled ddNTPs by the different polymerase clones are shown in
FIG. 7. Clones p8 (panel C) and 1-1 (panel D) exhibited
significantly improved incorporation of R6G-ddATP and R110-ddGTP,
compared to the parental clone (panel A). Improvement was evidenced
by the synthesis of sequencing ladders at 0.01.times. (1) and
0.1.times. (2) dye-ddNTP/dNTP ratios. Optimization of reaction
conditions and/or dye moieties may be performed to realize
improvements in the incorporation of ddTTP and ddCTP.
[0376] iii. Sequencing with Double-Mutant exo.sup.- JDF-3 DNA
Polymerase.
[0377] To verify that changes at residues 408, 410, and 485 were
sufficient to improve ddNTP incorporation, individual mutations
were introduced into the parental 550 (JDF-3 exo.sup.- DNA
polymerase) clone by site-directed mutagenesis. In addition, point
mutations were combined to examine whether they resulted in further
improvements in dideoxynucleotide incorporation over polymerases
bearing single mutations.
[0378] DNA sequencing reactions consisting of 1.times. reaction
buffer, 0.15 pmol/.mu.l long -20 primer, and 10 ng/.mu.g
pBluescript KS were prepared as follows:
16 81 .mu.l H.sub.2O 9 .mu.l -20 long primer (2 pmol/.mu.l) 6 .mu.l
pBluescript KS (0.2 .mu.g/.mu.l) ** .mu.l polymerase 12 .mu.l
10.times. buffer (260 mM Tris pH 9.5, 65 mM MgCl.sub.2)
[0379] 18 .mu.l of the cocktail listed above was aliquotted into
the appropriate number of tubes (one per polymerase). Each
polymerase (2 .mu.l) was added to an aliquot of cocktail and the
tubes were mixed well. Each resulting polymerase mixture (4.5
.mu.l) was then added to each of four tubes, already containing
0.06 mM of one of the four -.sup.33P-dideoxynucleotides (ddATP,
ddTTP, ddGTP or ddTTP; 1500Ci/mmol; 450 .mu.Ci/ml) and 6 mM each
deoxynucleotide in a volume of 2.5 .mu.l.
[0380] The sequencing reactions were cycled in a ROBOCYCLER.RTM.96
temperature cycler with a Hot Top Assembly using the following
conditions:
[0381] 1) 1 minute at 95C
[0382] 2) 45 seconds at 95C
[0383] 3) 45 seconds at 60C
[0384] 4) 1.5 minutes at 72C
[0385] 5) Repeat steps 2-4 thirty times.
[0386] Stop solution (.mu.l; 95% formamide, 20 mM EDTA, 0.05%
bromophenol blue, 0.05% xylene cyanol FF) was added to each
reaction before heating to 99C for five minutes. Each sample (4
.mu.l) was loaded onto a 6% acrylamide denaturing CastAway gel. The
gel was run and treated as described previously.
[0387] FIG. 8 shows that the P410L/A485T double mutant exhibits
exceptionally even signals. Band uniformity was improved compared
to mutant p8 (P410L mutation plus ancillary mutations that do not
include A485T) and mutant A485T (data not shown). Mutant p8
exhibited a tendency to preferentially incorporate ddGTP and ddCTP
in a sequence-dependent fashion. The optimal amount of enzyme may
be higher than the quantity tested in this experiment. Sequence
produced by the commercially available Family A DNA polymerase
mutant, Thermo Sequenase, is shown in panel E.
[0388] iv. Ribonucleotide Incorporation by JDF-3 Polymerase
Mutants.
[0389] A primer annealed to single stranded DNA template was
extended in a mixture containing all ribonucleotides or all
deoxynucleotides with the mutant and progenitor polymerases.
[0390] M13 mp18+single stranded DNA was annealed to 95.times. molar
excess of the 38mer primer by heating the mixture to 95.degree. C.
and cooling slowly at room temperature.
17 38mer primer: 5' GGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCA- GT 3'
[0391] Preliminary assays were carried out to determine what
dilutions of enzyme would be necessary to examine the incorporation
activity at non-maximal levels. The final assay solutions were
composed as described below:
[0392] Ribonucleotide Mixture
18 20 ng/.mu.l annealed primer/template 1.times. Cloned Pfu buffer
(Stratagene catalog #200532) 200 .mu.M each GTP, UTP, ATP 50 .mu.M
CTP 1 .mu.M 5-.sup.3H CTP 20.2 Ci/mmole 0.05-0.3 units JDF-3
polymerase*
[0393] Deoxyribonucleotide Mixture
19 20 ng/.mu.l annealed primer template 1.times. Cloned Pfu buffer
200 .mu.M each dGTP, dATP, dCTP 50 .mu.M TTP (deoxyribonucleotide)
1 .mu.M Thymidine 5'-triphosphate, [methyl-.sup.3H] 20.5 Ci/mmole
0.05-0.3 units JDF-3 polymerase* *Added separately
[0394] Nine microliters of the polymerase-free mixtures were placed
in 0.2 ml tubes before the polymerases were added. The samples were
incubated at 72.degree. C. in a ROBOCYCLER.RTM.996 temperature
cycler with Hot Top Assembly (Stratagene Catalog Nos. 400870 and
400894). The deoxyribonucleotide mixture was removed at 2 minutes
and placed at approximately 2.degree. C. The ribonucleotide mixture
was incubated for 30 minutes. Seven microliters of the assay
mixture were spotted onto DE81 filter circles (Whatmann) and dried
prior to being washed three times in 2.times.SSC (0.3M NaCl, 0.03M
sodium citrate) for five minutes each wash. The filters were rinsed
twice in ethanol and allowed to dry before being quantified with a
scintillation counter.
[0395] Background counts per minute (CPM) for the
deoxyribonucleotide and the ribonucleotide reactions were
subtracted from the respective averaged CPM value of duplicate
samples for each enzyme. The background-corrected ribonucleotide
CPM value was divided by the background-corrected
deoxyribonucleotide CPM value (FIG. 9).
20 Polymerase Ratio NTP/dNTP Relative to JDF-3 550 JDF-3 550
0.000165162 1 JDF-3 L408H 0.041087258 249 JDF-3 L408F 0.051703924
313 JDF-3 A485T 0.007628583 46
[0396] v. Ribonucleotide Sequencing with JDF-3 Polymerase
Mutants.
[0397] Ribonucleotides incorporated into a deoxyribonucleotide
polymer are susceptible to alkali hydrolysis which can produce a
sub-population of polymer lengths. When labeled primer is extended
in the presence of a particular ribonucleotide base (for example
ATP) and the four deoxyribonucleotide bases, the fragments
resulting from alkali hydrolysis create a population of different
lengths, which correspond to all the possible positions where ATP
was incorporated. When those fragments are size separated, their
migration pattern, with respect to other ribonucleotide base (CTP,
UTP and GTP) hydrolysis products allows the template sequence to be
read. As described previously, most DNA polymerases discriminate
against non-conventional deoxynucleotides. A subset of the JDF-3
DNA polymerase mutants which allow improved uptake of the
unconventional dideoxynucleotides also show improved tolerance for
ribonucleotide incorporation.
[0398] 100 ng of the 38mer primer was kinased with -.sup.33P
according to the instructions in the KINACE-IT.TM. Kinasing Kit
(Stratagene catalog #300390).
21 38mer primer: 5' GGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCA- GT 3'
[0399] The labeled oligonucleotide was purified from contaminating
free nucleotides with a NUC TRAP.RTM. Probe Purification Column
(Statagene catalog #400701) in 10T.1E (10 mM Tris pH 8.0, 0.1 mM
EDTA). Labeled oligonucleotide (.about.7 picomoles) was annealed to
0.09 pmoles M13 mp18+ by heating to 95.degree. C. then cooling to
room temperature in the presence of 0.32 mM MgCl.sub.2.
[0400] Extension Components
22 0.054 pM annealed primer/template 200 .mu.M each dNTP 1.times.
cPfu DNA polymerase buffer (Stratagene catalog #200532) 4-200 ATP*
0.1-5 Units JDF-3 polymerase* *Added separately
[0401] Eight microliters of a cocktail containing the first three
components listed above were aliquoted into a 0.2 ml tube. 1 .mu.l
of polymerase and 1 .mu.l of 2 mM, 0.2 mM or 0.4 mM ATP were added
and the reaction was incubated at 72.degree. C. for 15 minutes. The
reaction volume was brought to 10011 with 1.times.cPfu polymerase
buffer and transferred to a 1.5 ml tube. After heating the
reactions in the presence of 70 mM NaOH for 15 minutes at
100.degree. C., the reaction was neutralized with 70 mM HCl and
precipitated through the addition of 10 .mu.l 3M sodium acetate and
327.5 .mu.l of ethanol. The samples were microcentrifuged for 30
minutes at 14krpm before the supernatant was removed and the pellet
washed in 80% ethanol. After vacuum drying, the samples were
resuspended in 5 .mu.l of sequencing stop solution (95% formamide,
20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF) and 2.5
.mu.l was loaded on a 6% acylamide-7M urea, 1.times. TBE
CASTAWAY.TM. Precast gel (Stratagene catalog numbers 401090 and
401094). The gels were run at 50 watts until the bromophenol blue
dye migrated past the bottom of the gel after which the gel was
fixed, dried and exposed to film for 72 hours.
[0402] Sequencing ladders for JDF-3 550 (wild-type nucleotide
incorporation) and all the mutants tested were visible at the 200
.mu.M and 20 .mu.M ATP level. At the 4 .mu.M level, only the L408H
and L408F mutants produced ladders (data not shown).
[0403] vi. Sequencing with Dye-dideoxynucleotide Terminators
[0404] Primer was extended in the presence of FAM ddCTP
(NENNEL481). The sequence reactions were purified and run on an ABI
370.
[0405] Reaction conditions for cycle-sequencing were as described
below:
[0406] 1.times.cPFU buffer, 200 ng pBluescript II KS plasmid, 3
pmole T7 primer, 0.23 mM dCTP, 0.23 mM dATP, 0.23 mM dTTP, 0.23 mM
dGTP with 0.046 mM FAM ddCTP. The samples were cycled in a
Perkin-Elmer cycler in 10 .mu.l volumes for 25 cycles of the
temperatures and times described below:
23 95.degree. C. 30 s 55.degree. C. 30 s 72.degree. C. 2 min
[0407] The samples were purified using CentriSep columns according
to the manufacturer's instructions. After drying, the samples were
resuspended in 3 .mu.l of a loading dye comprised of 66.7%
deionized formamide, 16.7 mg/ml Blue Dextran, and 8.3 mM EDTA.
Samples were heated at 95.degree. C. for three minutes and loaded
on a 5% LongRangen gel in an ABI PRISM 377 DNA sequencer.
[0408] Data was processed in Gene Scan 2.1.
Example 2
[0409] Labeling of DNA.
[0410] The modified DNA polymerases of the invention are applicable
to labeling of DNA. It is known to those skilled in the art that
there are several means by which to label DNA, including the
incorporation of radiolabeled nucleotides. One such common means is
by random priming, which enables one of skill in the art to
generate labeled DNA fragments, typically about 50 to about 1000
bases long. The procedure described herein are adapted from F.
Ausubel et al., Short Protocols in Molecular Biology, Third
Edition, John Wiley and Sons, Inc., 1995.
[0411] As a first step toward random priming DNA, a reaction mix
containing 2.5 microliters 0.5 mM 3dNTP (dCTP, dGTP, TTP, each at
0.5 mM), 50 .mu.Ci [-.sup.32P]dATP, 1 microliter of 3 to 8
units/microliter DNA polymerase in 50 mM Tris-HCl, pH 7.5, 10 mM
MgCl.sub.2, 1 mM dithiothreitol, 0.05 mg/ml bovine serum albumin is
prepared in a total volume of 11 microliters and incubated on ice.
Next, about 30 to about 100 ng of DNA is mixed with about with 1 to
5 .mu.g of random hexanucleotides in 14 microliters and boiled for
2 to 3 minutes and then placed on ice. The 11 microliter reaction
mix is then added to the DNA/random hexamer mix, and the random
priming reaction is incubated over 10 minutes to as much as 4 hours
at room temperature. To stop the reaction, 1 microliter 0.5 M EDTA,
3 microliters 10 mg/ml tRNA, and 100 microliters 10 mM Tris-HCl, pH
7.4 is added and the mixture is extracted with phenol. The labeled
DNA is then separated from unincorporated radioactive precursors by
chromatography.
[0412] R. Gel Assay for Dye-dideoxynucleotide Incorporation.
[0413] A labeled oligonucleotide duplex was extended with a mixture
of dideoxynucleotides and dye-dideoxynucleotides. When the duplex
was separated on a denaturing 20% Acrylamide/7 M urea gel, labeled
oligonucleotides terminated with a dideoxynucleotide could be
resolved from oligonucleotides terminated with
dye-deoxynucleotides.
[0414] Oligonucleotides:
24 259C .sup.32P-TAACGTTGGGGGGGGGCA 258C TGCAACCCCCCCCCGTAT
[0415] The 5' end of 259C was labeled and purified as described in
Section Q.ii.a except that .sup.32P-ATP was used. The labeled
oligonucleotide 259C was at a concentration of approximately 0.7
ng/.mu.l. The complimentary oligonucleotide (258C) was added as an
equal concentration, heated to 95.degree. C. for three minutes,
50.degree. C. for 5 minutes and room temperature for 20 minutes.
Heat killed lysates of the relevant mutants were prepared as
described in Example section C. The reactions were incubated in a 5
.mu.l volume composed of 30 mM Tris pH 8.0 and 3 mM MgCl.sub.2 with
a nucleotide mixture totaling 0.1 mM. The ratio of ddTTP to FLU
ddUTP or ROXddUTP was 10:1. The dimer was present at a
concentration of 1.2 picomoles and 0.5 .mu.l of enzyme or crude
lysate or purified enzyme was added to the reaction before
incubation at 50.degree. C. in the RobeCycler.RTM. Gradient 96
Temperature Cycler with Hot Top. The samples were incubated for 20s
before 3 .mu.l of a formamide based loading dye was added and the
samples were heat-denatured at 95.degree. C. for 3 minutes then
loaded onto a 20% acrylamide/7 M urea gel and subjected to
electrophoresis at a constant 60 watts. The gel was exposed to
X-ray film and the film was analyzed in the EagleEye.RTM. Eagle
Sight software package.
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Other Embodiments
[0486] Other embodiments will be evident to those of skill in the
art. It should be understood that the foregoing detailed
description is provided for clarity only and is merely exemplary.
The spirit and scope of the present invention are not limited to
the above examples, but are encompassed by the following claims.
Sequence CWU 1
1
48 1 2331 DNA Thermococcus sp. JDF-3 1 atgatccttg acgttgatta
catcaccgag aatggaaagc ccgtcatcag ggtcttcaag 60 aaggagaacg
gcgagttcag gattgaatac gaccgcgagt tcgagcccta cttctacgcg 120
ctcctcaggg acgactctgc catcgaagaa atcaaaaaga taaccgcgga gaggcacggc
180 agggtcgtta aggttaagcg cgcggagaag gtgaagaaaa agttcctcgg
caggtctgtg 240 gaggtctggg tcctctactt cacgcacccg caggacgttc
cggcaatccg cgacaaaata 300 aggaagcacc ccgcggtcat cgacatctac
gagtacgaca tacccttcgc caagcgctac 360 ctcatagaca agggcctaat
cccgatggaa ggtgaggaag agcttaaact catgtccttc 420 gacatcgaga
cgctctacca cgagggagaa gagtttggaa ccgggccgat tctgatgata 480
agctacgccg atgaaagcga ggcgcgcgtg ataacctgga agaagatcga ccttccttac
540 gttgaggttg tctccaccga gaaggagatg attaagcgct tcttgagggt
cgttaaggag 600 aaggacccgg acgtgctgat aacatacaac ggcgacaact
tcgacttcgc ctacctgaaa 660 aagcgctgtg agaagcttgg cgtgagcttt
accctcggga gggacgggag cgagccgaag 720 atacagcgca tgggggacag
gtttgcggtc gaggtgaagg gcagggtaca cttcgacctt 780 tatccagtca
taaggcgcac cataaacctc ccgacctaca cccttgaggc tgtatacgag 840
gcggttttcg gcaagcccaa ggagaaggtc tacgccgagg agatagccac cgcctgggag
900 accggcgagg ggcttgagag ggtcgcgcgc tactcgatgg aggacgcgag
ggttacctac 960 gagcttggca gggagttctt cccgatggag gcccagcttt
ccaggctcat cggccaaggc 1020 ctctgggacg tttcccgctc cagcaccggc
aacctcgtcg agtggttcct cctaaggaag 1080 gcctacgaga ggaacgaact
cgctcccaac aagcccgacg agagggagct ggcgaggaga 1140 agggggggct
acgccggtgg ctacgtcaag gagccggagc ggggactgtg ggacaatatc 1200
gtgtatctag actttcgtag tctctaccct tcaatcataa tcacccacaa cgtctcgcca
1260 gatacgctca accgcgaggg gtgtaggagc tacgacgttg cccccgaggt
cggtcacaag 1320 ttctgcaagg acttccccgg cttcattccg agcctgctcg
gaaacctgct ggaggaaagg 1380 cagaagataa agaggaagat gaaggcaact
ctcgacccgc tggagaagaa tctcctcgat 1440 tacaggcaac gcgccatcaa
gattctcgcc aacagctact acggctacta cggctatgcc 1500 agggcaagat
ggtactgcag ggagtgcgcc gagagcgtta cggcatgggg aagggagtac 1560
atcgaaatgg tcatcagaga gcttgaggaa aagttcggtt ttaaagtcct ctatgcagac
1620 acagacggtc tccatgccac cattcctgga gcggacgctg aaacagtcaa
gaaaaaggca 1680 atggagttct taaactatat caatcccaaa ctgcccggcc
ttctcgaact cgaatacgag 1740 ggcttctacg tcaggggctt cttcgtcacg
aagaaaaagt acgcggtcat cgacgaggag 1800 ggcaagataa ccacgcgcgg
gcttgagata gtcaggcgcg actggagcga gatagcgaag 1860 gagacgcagg
cgagggtttt ggaggcgata ctcaggcacg gtgacgttga agaggccgtc 1920
agaattgtca gggaagtcac cgaaaagctg agcaagtacg aggttccgcc ggagaagctg
1980 gttatccacg agcagataac gcgcgagctc aaggactaca aggccaccgg
cccgcacgta 2040 gccatagcga agcgtttggc cgccagaggt gttaaaatcc
ggcccggaac tgtgataagc 2100 tacatcgttc tgaagggctc cggaaggata
ggcgacaggg cgattccctt cgacgagttc 2160 gacccgacga agcacaagta
cgatgcggac tactacatcg agaaccaggt tctgccggca 2220 gttgagagaa
tcctcagggc cttcggctac cgcaaggaag acctgcgcta ccagaagacg 2280
aggcaggtcg ggcttggcgc gtggctgaag ccgaagggga agaagaagtg a 2331 2 776
PRT Thermococcus sp. JDF-3 2 Met Ile Leu Asp Val Asp Tyr Ile Thr
Glu Asn Gly Lys Pro Val Ile 1 5 10 15 Arg Val Phe Lys Lys Glu Asn
Gly Glu Phe Arg Ile Glu Tyr Asp Arg 20 25 30 Glu Phe Glu Pro Tyr
Phe Tyr Ala Leu Leu Arg Asp Asp Ser Ala Ile 35 40 45 Glu Glu Ile
Lys Lys Ile Thr Ala Glu Arg His Gly Arg Val Val Lys 50 55 60 Val
Lys Arg Ala Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Ser Val 65 70
75 80 Glu Val Trp Val Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala
Ile 85 90 95 Arg Asp Lys Ile Arg Lys His Pro Ala Val Ile Asp Ile
Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp
Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Glu Glu Glu Leu Lys Leu
Met Ser Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu
Phe Gly Thr Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp
Glu Ser Glu Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu
Pro Tyr Val Glu Val Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190
Arg Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195
200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys
Glu 210 215 220 Lys Leu Gly Val Ser Phe Thr Leu Gly Arg Asp Gly Ser
Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val
Glu Val Lys Gly Arg Val 245 250 255 His Phe Asp Leu Tyr Pro Val Ile
Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val
Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Ala
Glu Glu Ile Ala Thr Ala Trp Glu Thr Gly Glu Gly 290 295 300 Leu Glu
Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Arg Val Thr Tyr 305 310 315
320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu
325 330 335 Ile Gly Gln Gly Leu Trp Asp Val Ser Arg Ser Ser Thr Gly
Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg
Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala
Arg Arg Arg Gly Gly Tyr 370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro
Glu Arg Gly Leu Trp Asp Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe
Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 Asn Val Ser
Pro Asp Thr Leu Asn Arg Glu Gly Cys Arg Ser Tyr Asp 420 425 430 Val
Ala Pro Glu Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe 435 440
445 Ile Pro Ser Leu Leu Gly Asn Leu Leu Glu Glu Arg Gln Lys Ile Lys
450 455 460 Arg Lys Met Lys Ala Thr Leu Asp Pro Leu Glu Lys Asn Leu
Leu Asp 465 470 475 480 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn
Ser Tyr Tyr Gly Tyr 485 490 495 Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr
Cys Arg Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu
Tyr Ile Glu Met Val Ile Arg Glu Leu 515 520 525 Glu Glu Lys Phe Gly
Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu 530 535 540 His Ala Thr
Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560
Met Glu Phe Leu Asn Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 565
570 575 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys
Lys 580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr
Arg Gly Leu 595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala
Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Ile Leu Arg His
Gly Asp Val Glu Glu Ala Val 625 630 635 640 Arg Ile Val Arg Glu Val
Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu
Val Ile His Glu Gln Ile Thr Arg Glu Leu Lys Asp 660 665 670 Tyr Lys
Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Ala 675 680 685
Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690
695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu
Phe 705 710 715 720 Asp Pro Thr Lys His Lys Tyr Asp Ala Asp Tyr Tyr
Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Arg
Ala Phe Gly Tyr Arg Lys 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr
Arg Gln Val Gly Leu Gly Ala Trp 755 760 765 Leu Lys Pro Lys Gly Lys
Lys Lys 770 775 3 1300 PRT Thermococcus sp. JDF-3 MISC_FEATURE
(1015)..(1015) Xaa is unknown amino acid. 3 Met Ile Leu Asp Val Asp
Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile 1 5 10 15 Arg Val Phe Lys
Lys Glu Asn Gly Glu Phe Arg Ile Glu Tyr Asp Arg 20 25 30 Glu Phe
Glu Pro Tyr Phe Tyr Ala Leu Leu Arg Asp Asp Ser Ala Ile 35 40 45
Glu Glu Ile Lys Lys Ile Thr Ala Glu Arg His Gly Arg Val Val Lys 50
55 60 Val Lys Arg Ala Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Ser
Val 65 70 75 80 Glu Val Trp Val Leu Tyr Phe Thr His Pro Gln Asp Val
Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Lys His Pro Ala Val Ile
Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu
Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Glu Glu Glu Leu
Lys Leu Met Ser Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly
Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr
Ala Asp Glu Ser Glu Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ser Thr Glu Lys Glu Met Ile Lys 180
185 190 Arg Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile
Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys
Arg Cys Glu 210 215 220 Lys Leu Gly Val Ser Phe Thr Leu Gly Arg Asp
Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe
Ala Val Glu Val Lys Gly Arg Val 245 250 255 His Phe Asp Leu Tyr Pro
Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu
Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val
Tyr Ala Glu Glu Ile Ala Thr Ala Trp Glu Thr Gly Glu Gly 290 295 300
Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Arg Val Thr Tyr 305
310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser
Arg Leu 325 330 335 Ile Gly Gln Gly Leu Trp Asp Val Ser Arg Ser Ser
Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr
Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu
Leu Ala Arg Arg Arg Gly Gly Tyr 370 375 380 Ala Gly Gly Tyr Val Lys
Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile 385 390 395 400 Val Tyr Leu
Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 Asn
Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Arg Ser Tyr Asp 420 425
430 Val Ala Pro Glu Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe
435 440 445 Ile Pro Ser Leu Leu Gly Asn Leu Leu Glu Glu Arg Gln Lys
Ile Lys 450 455 460 Arg Lys Met Lys Ala Thr Leu Asp Pro Leu Glu Lys
Asn Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Ala Ile Lys Ile Leu
Ala Asn Ser Leu Leu Pro Gly 485 490 495 Glu Trp Val Ala Val Ile Glu
Gly Gly Lys Leu Arg Pro Val Arg Ile 500 505 510 Gly Glu Leu Val Asp
Gly Leu Met Glu Ala Ser Gly Glu Arg Val Lys 515 520 525 Arg Asp Gly
Asp Thr Glu Val Leu Glu Val Glu Gly Leu Tyr Ala Ser 530 535 540 Pro
Ser Thr Gly Ser Pro Arg Lys Pro Ala Gln Cys Arg Lys Pro Gly 545 550
555 560 Thr Ala Met Pro Gly Lys Phe Thr Glu Leu Ser Thr Pro Glu Gly
Gly 565 570 575 Leu Ser Val Thr Arg Gly His Ser Leu Phe Ala Tyr Arg
Asp Ala Ser 580 585 590 Leu Trp Arg Arg Gly Arg Arg Arg Phe Lys Pro
Gly Asp Leu Leu Ala 595 600 605 Val Pro Ser Gly Pro Ser Arg Arg Gly
Gly Arg Gly Ser Thr Ser Leu 610 615 620 Asn Cys Ser Ser Asn Cys Pro
Arg Arg Lys Arg Pro Thr Cys His Arg 625 630 635 640 His Ser Gly Lys
Gly Arg Lys Asn Phe Phe Arg Gly Met Leu Arg Thr 645 650 655 Leu Arg
Trp Ile Phe Gly Glu Glu Lys Thr Gly Gly Arg Pro Gly Ala 660 665 670
Thr Trp Ser Thr Leu Arg Gly Leu Gly Tyr Val Lys Leu Arg Lys Ile 675
680 685 Gly Tyr Gly Val Val Asp Arg Glu Gly Leu Gly Lys Val Pro Arg
Phe 690 695 700 Tyr Glu Arg Leu Val Glu Val Ile Arg Tyr Asn Gly Asn
Arg Gly Glu 705 710 715 720 Phe Ile Ala Asp Phe Asn Ala Leu Arg Pro
Val Leu Arg Leu Met Met 725 730 735 Pro Glu Lys Glu Leu Glu Glu Trp
Leu Val Gly Thr Arg Asn Gly Phe 740 745 750 Arg Ile Arg Pro Phe Ile
Glu Val Asp Trp Lys Phe Ala Lys Leu Leu 755 760 765 Gly Tyr Tyr Val
Ser Glu Gly Ser Ala Gly Lys Trp Lys Asn Arg Thr 770 775 780 Gly Gly
Trp Ser Tyr Ser Val Arg Leu Tyr Asn Glu Asp Gly Ser Val 785 790 795
800 Leu Asp Asp Met Glu Arg Leu Ala Arg Ser Ser Leu Gly Ala Ala Arg
805 810 815 Gly Glu Leu Arg Arg Asp Phe Lys Glu Asp Gly Leu His Asn
Leu Arg 820 825 830 Gly Ala Leu Arg Phe Thr Gly Arg Glu Gln Glu Gly
Ser Val Ala Tyr 835 840 845 Leu His Val Pro Gly Gly Pro Leu Gly Leu
Pro Gly Val Leu His Arg 850 855 860 Arg Arg Arg Arg Ser Pro Glu Gln
Asp Gly Ser Ala Leu His Gln Glu 865 870 875 880 Arg Ala Ser Gly Arg
Pro Arg Pro Ala Pro Glu Leu Ala Gly Arg Leu 885 890 895 Ser Asp Lys
Arg Pro Pro Arg Gln Arg Gly Leu Gln Gly Leu Arg Glu 900 905 910 Arg
Gly Thr Ala Leu Tyr Arg Val Pro Glu Ala Glu Glu Arg Leu Thr 915 920
925 Tyr Ser His Val Ile Pro Arg Glu Val Leu Glu Glu Thr Ser Ala Gly
930 935 940 Pro Ser Arg Arg Thr Val Thr Gly Asn Ser Gly Ser Trp Trp
Lys Ala 945 950 955 960 Gly Ser Ser Thr Arg Lys Gly Pro Val Gly Ala
Gly Ser Ser Thr Gly 965 970 975 Ile Ser Ser Thr Gly Ser Arg Lys Ser
Gly Arg Lys Ala Thr Arg Gly 980 985 990 Thr Ser Thr Thr Ala Leu Arg
Arg Thr Arg Thr Ser Gly Gly Leu Trp 995 1000 1005 Val Pro Leu Arg
Ala Gln Xaa Ser Tyr Tyr Gly Tyr Tyr Gly Tyr 1010 1015 1020 Ala Arg
Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser Val Thr 1025 1030 1035
Ala Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu Leu Glu 1040
1045 1050 Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly
Leu 1055 1060 1065 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val
Lys Lys Lys 1070 1075 1080 Ala Met Glu Phe Leu Asn Tyr Ile Asn Pro
Lys Leu Pro Gly Leu 1085 1090 1095 Leu Glu Leu Glu Tyr Glu Gly Phe
Tyr Val Arg Gly Phe Phe Val 1100 1105 1110 Thr Lys Lys Lys Tyr Ala
Val Ile Asp Glu Glu Gly Lys Ile Thr 1115 1120 1125 Thr Arg Gly Leu
Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala 1130 1135 1140 Lys Glu
Thr Gln Ala Arg Val Leu Glu Ala Ile Leu Arg His Gly 1145 1150 1155
Asp Val Glu Glu Ala Val Arg Ile Val Arg Glu Val Thr Glu Lys 1160
1165 1170 Leu Ser Lys Tyr Glu Val Pro Pro Glu Lys Leu Val Ile His
Glu 1175 1180 1185 Gln Ile Thr Arg Glu Leu Lys Asp Tyr Lys Ala Thr
Gly Pro His 1190 1195 1200 Val Ala Ile Ala Lys Arg Leu Ala Ala Arg
Gly Val Lys Ile Arg 1205 1210 1215 Pro Gly Thr Val Ile Ser Tyr Ile
Val Leu Lys Gly Ser Gly Arg 1220 1225 1230 Ile Gly Asp Arg Ala Ile
Pro Phe Asp Glu Phe Asp Pro Thr Lys 1235 1240 1245 His Lys Tyr Asp
Ala Asp Tyr Tyr Ile Glu Asn Gln Val Leu Pro 1250 1255 1260 Ala Val
Glu Arg
Ile Leu Arg Ala Phe Gly Tyr Arg Lys Glu Asp 1265 1270 1275 Leu Arg
Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp Leu 1280 1285 1290
Lys Pro Lys Gly Lys Lys Lys 1295 1300 4 5255 DNA Thermococcus sp.
JDF-3 misc_feature (3518)..(3519) n = unknown 4 aattccactg
ccgtgtttaa cctttccacc gttgaacttg agggtgattt tctgagcctc 60
ctcaatcact taatcgagac cgcggattac cttgaactgg tacacgttca acgattcggt
120 tcttgtaatg gtcgatactg ggccgtgctg gattttctaa acgtctcaag
aacggctttc 180 atcaacggaa actgccacgt ctccgccgtc gtgagggtta
aacctgaagt tcaagacttt 240 gcaacggaat ggcgagagaa cggcgactac
cccagtggaa gagcttttga aagccaaagc 300 cgagcttcag cgaatgtgcg
gtgcccttgt tcaagagttg tgagcccttg attgttgttt 360 tctcctcttt
tctgataaca tcgatggcga agtttattag ttctcagttc gataatcagg 420
caggtgttgg tcatgatcct tgacgttgat tacatcaccg agaatggaaa gcccgtcatc
480 agggtcttca agaaggagaa cggcgagttc aggattgaat acgaccgcga
gttcgagccc 540 tacttctacg cgctcctcag ggacgactct gccatcgaag
aaatcaaaaa gataaccgcg 600 gagaggcacg gcagggtcgt taaggttaag
cgcgcggaga aggtgaagaa aaagttcctc 660 ggcaggtctg tggaggtctg
ggtcctctac ttcacgcacc cgcaggacgt tccggcaatc 720 cgcgacaaaa
taaggaagca ccccgcggtc atcgacatct acgagtacga catacccttc 780
gccaagcgct acctcataga caagggccta atcccgatgg aaggtgagga agagcttaaa
840 ctcatgtcct tcgacatcga gacgctctac cacgagggag aagagtttgg
aaccgggccg 900 attctgatga taagctacgc cgatgaaagc gaggcgcgcg
tgataacctg gaagaagatc 960 gaccttcctt acgttgaggt tgtctccacc
gagaaggaga tgattaagcg cttcttgagg 1020 gtcgttaagg agaaggaccc
ggacgtgctg ataacataca acggcgacaa cttcgacttc 1080 gcctacctga
aaaagcgctg tgagaagctt ggcgtgagct ttaccctcgg gagggacggg 1140
agcgagccga agatacagcg catgggggac aggtttgcgg tcgaggtgaa gggcagggta
1200 cacttcgacc tttatccagt cataaggcgc accataaacc tcccgaccta
cacccttgag 1260 gctgtatacg aggcggtttt cggcaagccc aaggagaagg
tctacgccga ggagatagcc 1320 accgcctggg agaccggcga ggggcttgag
agggtcgcgc gctactcgat ggaggacgcg 1380 agggttacct acgagcttgg
cagggagttc ttcccgatgg aggcccagct ttccaggctc 1440 atcggccaag
gcctctggga cgtttcccgc tccagcaccg gcaacctcgt cgagtggttc 1500
ctcctaagga aggcctacga gaggaacgaa ctcgctccca acaagcccga cgagagggag
1560 ctggcgagga gaaggggggg ctacgccggt ggctacgtca aggagccgga
gcggggactg 1620 tgggacaata tcgtgtatct agactttcgt agtctctacc
cttcaatcat aatcacccac 1680 aacgtctcgc cagatacgct caaccgcgag
gggtgtagga gctacgacgt tgcccccgag 1740 gtcggtcaca agttctgcaa
ggacttcccc ggcttcattc cgagcctgct cggaaacctg 1800 ctggaggaaa
ggcagaagat aaagaggaag atgaaggcaa ctctcgaccc gctggagaag 1860
aatctcctcg attacaggca acgcgccatc aagattctcg ccaacagcct tcttcccggg
1920 gagtgggttg cggtcattga aggggggaaa ctcaggcccg tccgcatcgg
cgagctggtt 1980 gatggactga tggaagccag cggggagagg gtgaaaagag
acggcgacac cgaggtcctt 2040 gaagtcgagg ggctttacgc ctctccttcg
acagggagtc caagaaagcc cgcacaatgc 2100 cggtgaaagc cgtgataagg
caccgctatg ccggggaagt ttacagaata gctctcaact 2160 ccggaaggag
gattaagcgt gacgcgcggc cacagcctct tcgcgtaccg ggacgcgagc 2220
ttgtggaggt gacgggggag gaggaggttc aagcccggcg acctcctggc ggtgccaagc
2280 ggataaccct cccggagagg agggagaggc tcaacatcgt tgaactgctc
ctcgaactgc 2340 ccgaggagga aacggccgac atgtcatcga cattccggca
agggtagaaa gaacttcttc 2400 aggggaatgc tcagaaccct ccgctggatt
ttcggggagg agaagaccgg agggcggcca 2460 ggcgctacct ggagcacctt
gcgtgggctc ggctacgtga agctgaggaa aatcggctac 2520 ggggtggttg
atagggaggg actgggaaag gtaccgcgct tctacgagag gctcgtggag 2580
gtaatccgct acaacggcaa caggggggag ttcatcgccg atttcaacgc gctccgcccc
2640 gtcctccgcc tgatgatgcc cgagaaggag cttgaagagt ggctcgttgg
gacgaggaac 2700 gggttcagga taaggccgtt catagaggtt gattggaagt
tcgcaaagct cctcggctac 2760 tacgtgagcg aggggagcgc cgggaagtgg
aaaaaccgga ccgggggctg gagctactcg 2820 gtgaggcttt acaacgagga
cgggagcgtt ctcgacgaca tggagagact cgcgaggagt 2880 tctttggggg
cgtgagcgcg gggggaacta cgtcgagatt tcaaagaaga tggcctacat 2940
aatcttcgag gggctctgcg gttcaccggc cgagaacaag agggttccgt ggcttatctt
3000 cacgtcccct gaggaggtcc gctgggcctt ccttgagggg tacttcatcg
gcgacggcga 3060 cgttcacccg agcaagatgg ttcggctctc caccaagagc
gagcttctgg ctaacggcct 3120 cgtcctgctc ctgaactcgc tgggcgtctc
agcgataaac gtccgccacg acagcggggt 3180 ttacagggtc tacgtgaacg
aggaactgcc ctttacagag taccggaagc ggaagaacgc 3240 ctcacttact
cccacgtcat accgagggaa gtgctggagg agacttcggc cgggccttcc 3300
agaagaacat gagtcacggg aaattcaggg agctggtgga aagcggggag ctcgacgcgg
3360 aaagggccgg taggataggc tggctcctcg acggggatat agtcctcgac
agggtctcgg 3420 aagtcaggaa ggaaagctac gaggggtacg tctacgacct
gagcgttgag gaggacgaga 3480 acttctggcg ggctttgggt tcctctacgc
gcacaacnna gctactacgg ctactacggc 3540 tatgccaggg caagatggta
ctgcagggag tgcgccgaga gcgttacggc atggggaagg 3600 gagtacatcg
aaatggtcat cagagagctt gaggaaaagt tcggttttaa agtcctctat 3660
gcagacacag acggtctcca tgccaccatt cctggagcgg acgctgaaac agtcaagaaa
3720 aaggcaatgg agttcttaaa ctatatcaat cccaaactgc ccggccttct
cgaactcgaa 3780 tacgagggct tctacgtcag gggcttcttc gtcacgaaga
aaaagtacgc ggtcatcgac 3840 gaggagggca agataaccac gcgcgggctt
gagatagtca ggcgcgactg gagcgagata 3900 gcgaaggaga cgcaggcgag
ggttttggag gcgatactca ggcacggtga cgttgaagag 3960 gccgtcagaa
ttgtcaggga agtcaccgaa aagctgagca agtacgaggt tccgccggag 4020
aagctggtta tccacgagca gataacgcgc gagctcaagg actacaaggc caccggcccg
4080 cacgtagcca tagcgaagcg tttggccgcc agaggtgtta aaatccggcc
cggaactgtg 4140 ataagctaca tcgttctgaa gggctccgga aggataggcg
acagggcgat tcccttcgac 4200 gagttcgacc cgacgaagca caagtacgat
gcggactact acatcgagaa ccaggttctg 4260 ccggcagttg agagaatcct
cagggccttc ggctaccgca aggaagacct gcgctaccag 4320 aagacgaggc
aggtcgggct tggcgcgtgg ctgaagccga aggggaagaa gaagtgagga 4380
attatctggt ttcttttccc agcattaaat gcttccgaca ttgccttatt tatgaaactc
4440 ctgttgtgcc tgagtttgtg ccagaaaaca gcctgttctg acggcgcttt
ttcttgccag 4500 gtctcttgag tttcgcaagg gtcttctcga ccagctcaat
ggtcttgtcg tcattgtttn 4560 nnnnnnnnnn nnnnnnnnnn cccggggact
tcatactggc ggtaatagac agggattcct 4620 tcctcaagga cttcccggga
ggcattggag ttttttggtg gggctttcac aggatttgct 4680 catcttgtgg
atttctcgtt cgattgaatc tgtccacttg agggtgtagg tcgagacggt 4740
ggagcgcgta ttccgggagc gggtcttgag gctccatttt tcagtcctcc tccggcgaag
4800 aagtggaact caagccgggt gttagcttat gttatgttcc caactcctcc
agcacctcca 4860 ggatcccctc aatcccggaa cctcgaagcc cctctcgtgg
atctttctaa cttcctctgc 4920 ctccgggttt atccagaccg cccacatgcc
ggctctcagc gcaccctcga aatcctccgc 4980 gtaggtgtcg ccgatgtgga
ttgcctcgtc cggctcgacc ccgaagcatc gagcggtttt 5040 ctgaacatct
cgggcatcgg cttatacgcc agaacctcgt cggcgaagaa ggttccctca 5100
atgtagtcca tcaggccgaa cctctcgagg gggggcccgg tacccaattc gccctatagt
5160 gagtcgatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa
aaccctggcg 5220 ttacccaact taagtcgctt tgcagcacat ccccc 5255 5 8 PRT
Thermococcus sp. JDF-3 MISC_FEATURE (2)..(3) Xaa can be any amino
acid 5 Lys Xaa Xaa Asn Ser Xaa Tyr Gly 1 5 6 10 PRT Thermococcus
sp. JDF-3 MISC_FEATURE (2)..(4) Xaa can be any amino acid 6 Lys Xaa
Xaa Xaa Xaa Gly Xaa Xaa Tyr Gly 1 5 10 7 10 PRT Thermococcus sp.
JDF-3 MISC_FEATURE (2)..(3) Xaa can be any amino acid 7 Asp Xaa Xaa
Ser Leu Tyr Pro Ser Ile Ile 1 5 10 8 10 PRT Thermococcus sp. JDF-3
8 Asp Phe Arg Ser Leu Tyr Leu Ser Ile Ile 1 5 10 9 10 PRT
Thermococcus sp. JDF-3 9 Asp Phe Arg Ser His Tyr Pro Ser Ile Ile 1
5 10 10 10 PRT Thermococcus sp. JDF-3 10 Asp Phe Arg Ser Phe Tyr
Pro Ser Ile Ile 1 5 10 11 30 DNA Artificial Sequence misc_feature
Synthetic oligonucleotide PCR primer 11 gggaaacata tgatccttga
cgttgattac 30 12 31 DNA Artificial Sequence misc_feature Synthetic
oligonucleotide PCR primer 12 gggaaaggat cctcacttct tcttcccctt c 31
13 34 DNA Artificial Sequence misc_feature Synthetic
oligonucleotide primer 13 tcagatgaat tcgatgatcc ttgacgttga ttac 34
14 54 DNA Artificial Sequence misc_feature Synthetic
oligonucleotide primer 14 gagagaattc ataatgataa ggaggaaaaa
attatgatcc ttgacgttga ttac 54 15 31 DNA Artificial Sequence
misc_feature Synthetic oligonucleotide primer 15 tcagatctcg
agtcacttct tcttcccctt c 31 16 29 DNA Artificial Sequence
misc_feature Synthetic oligonucleotide sequencing primer 16
ccagctttcc agactagtcg gccaaggcc 29 17 16 DNA Artificial Sequence
misc_feature Synthetic oligonucleotide sequencing primer 17
aactctcgac ccgctg 16 18 37 DNA Artificial Sequence misc_feature
Synthetic oligonucleotide primer 18 ggtttcccag tcacgacgtt
gtaaaacgac ggccagt 37 19 18 DNA Artificial Sequence misc_feature
First strand of synthetic oligonucleotide duplex 19 taacgttggg
ggggggca 18 20 18 DNA Artificial Sequence misc_feature Second
strand of synthetic oligonucleotide duplex 20 tgcaaccccc ccccgtat
18 21 139 PRT Thermococcus sp. JDF-3 MISC_FEATURE (6)..(6) Xaa is
unknown 21 Leu Val Cys Asn Ala Xaa Ser Thr Gly Asn Leu Val Glu Trp
Phe Leu 1 5 10 15 Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro
Asn Lys Pro Asp 20 25 30 Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly
Tyr Ala Gly Gly Tyr Val 35 40 45 Lys Glu Pro Glu Arg Gly Leu Trp
Asp Asn Ile Val Tyr Leu Asp Phe 50 55 60 Arg Ser Leu Tyr Pro Ser
Ile Ile Ile Thr His Asn Val Ser Pro Asp 65 70 75 80 Thr Leu Asn Arg
Glu Gly Cys Arg Ser Tyr Asp Val Ala Pro Glu Val 85 90 95 Gly His
Lys Phe Cys Lys Asp Phe Pro Gly Phe Ile Pro Ser Leu Leu 100 105 110
Gly Asn Leu Leu Glu Glu Arg Gln Lys Ile Lys Arg Lys Met Lys Ala 115
120 125 Thr Leu Asp Pro Leu Glu Lys Asn Leu Leu Asp 130 135 22 140
PRT Thermococcus sp. JDF-3 22 Val Trp Asp Val Ser Arg Ser Ser Thr
Gly Asn Leu Val Glu Arg Phe 1 5 10 15 Leu Leu Arg Lys Ala Tyr Glu
Arg Asn Glu Leu Ala Pro Asn Lys Pro 20 25 30 Asp Glu Arg Glu Leu
Ala Arg Arg Arg Gly Gly Tyr Ala Gly Gly Tyr 35 40 45 Val Lys Glu
Pro Glu Arg Gly Leu Trp Asp Asn Ile Val Tyr Leu Asp 50 55 60 Phe
Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His Ser Val Ser Pro 65 70
75 80 Asp Thr Leu Asp Arg Glu Gly Cys Arg Ser Tyr Asp Val Ala Pro
Glu 85 90 95 Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe Ile
Pro Ser Leu 100 105 110 Leu Gly Asn Leu Leu Glu Glu Arg Gln Lys Ile
Lys Arg Lys Met Lys 115 120 125 Ala Thr Leu Asp Pro Leu Glu Lys Asn
Leu Leu Asp 130 135 140 23 140 PRT Thermococcus sp. JDF-3 23 Val
Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Phe 1 5 10
15 Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro
20 25 30 Asp Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr Ala Gly
Gly Tyr 35 40 45 Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile
Val Tyr Leu Asp 50 55 60 Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile
Thr His Asn Val Ser Pro 65 70 75 80 Asp Thr Leu Asn Arg Glu Gly Cys
Arg Ser Tyr Asp Val Ala Pro Glu 85 90 95 Val Gly His Lys Phe Cys
Lys Asp Phe Pro Gly Phe Ile Pro Ser Leu 100 105 110 Leu Gly Asn Leu
Leu Glu Glu Arg Gln Lys Ile Lys Arg Lys Met Lys 115 120 125 Ala Thr
Leu Asp Pro Leu Glu Lys Asn Leu Leu Asp 130 135 140 24 140 PRT
Thermococcus sp. JDF-3 24 Val Trp Asp Val Ser Arg Ser Ser Thr Gly
Asn Leu Val Glu Trp Phe 1 5 10 15 Leu Leu Arg Lys Ala Tyr Glu Arg
Asn Glu Leu Ala Pro Asn Lys Pro 20 25 30 Asp Glu Arg Glu Leu Ala
Arg Arg Arg Gly Gly Tyr Ala Gly Gly Tyr 35 40 45 Val Lys Glu Pro
Glu Arg Gly Leu Trp Asp Asn Ile Val Tyr Leu Asp 50 55 60 Phe Arg
Ser Leu Tyr Pro Ser Ile Ile Ile Thr His Asn Val Ser Pro 65 70 75 80
Asp Thr Leu Asn Arg Glu Gly Cys Arg Ser Tyr Asp Val Ala Pro Glu 85
90 95 Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe Ile Pro Ser
Leu 100 105 110 Leu Gly Asn Leu Leu Glu Glu Arg Gln Lys Ile Lys Met
Lys Met Lys 115 120 125 Ala Thr Leu Asp Pro Leu Glu Lys Asn Leu Leu
Asp 130 135 140 25 140 PRT Thermococcus sp. JDF-3 25 Val Trp Asp
Val Ser Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Phe 1 5 10 15 Leu
Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro 20 25
30 Asp Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr Ala Gly Gly Tyr
35 40 45 Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile Val Tyr
Leu Asp 50 55 60 Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His
Asn Val Ser Pro 65 70 75 80 Asp Thr Leu Asn Arg Glu Gly Cys Arg Ser
Tyr Asp Val Ala Pro Glu 85 90 95 Val Gly His Lys Phe Cys Lys Asp
Phe Pro Gly Phe Ile Pro Ser Leu 100 105 110 Leu Gly Asn Leu Leu Glu
Glu Arg Gln Lys Ile Lys Arg Lys Met Lys 115 120 125 Ala Thr Leu Asp
Pro Leu Glu Lys Asn Leu Leu Asp 130 135 140 26 140 PRT Thermococcus
sp. JDF-3 MISC_FEATURE (5)..(5) Xaa is unknown amino acid 26 Val
Trp Asp Val Xaa Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Phe 1 5 10
15 Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro
20 25 30 Asp Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr Ala Gly
Gly Tyr 35 40 45 Val Lys Glu Pro Glu Arg Gly Gln Trp Asp Asn Ile
Ala Tyr Leu Asp 50 55 60 Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile
Thr His Asn Val Ser Pro 65 70 75 80 Asp Thr Leu Lys Arg Glu Gly Cys
Arg Ser Tyr Asp Val Ala Pro Glu 85 90 95 Val Gly His Lys Phe Cys
Lys Asp Phe Pro Gly Phe Ile Pro Ser Leu 100 105 110 Leu Gly Asn Leu
Leu Glu Glu Arg Gln Lys Ile Lys Arg Lys Met Lys 115 120 125 Ala Thr
Leu Asp Pro Leu Glu Lys Asn Leu Leu Asp 130 135 140 27 140 PRT
Thermococcus sp. JDF-3 27 Val Trp Asp Val Pro Arg Ser Ser Thr Gly
Asn Leu Val Glu Trp Phe 1 5 10 15 Leu Leu Arg Lys Ala Tyr Glu Arg
Asn Glu Leu Ala Pro Asn Lys Pro 20 25 30 Asp Glu Arg Glu Leu Ala
Arg Arg Arg Gly Gly Tyr Ala Gly Gly Tyr 35 40 45 Val Lys Glu Pro
Glu Arg Gly Leu Trp Asp Asn Ile Val Tyr Leu Asp 50 55 60 Phe Arg
Ser Leu Tyr Pro Ser Ile Ile Ile Thr His Asn Val Ser Pro 65 70 75 80
Asp Thr Leu Asn Arg Glu Gly Cys Arg Ser Tyr Asp Val Ala Pro Glu 85
90 95 Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe Ile Pro Ser
Leu 100 105 110 Leu Gly Asn Leu Leu Glu Glu Arg Gln Lys Ile Lys Arg
Lys Met Lys 115 120 125 Ala Thr Leu Asp Pro Leu Glu Lys Asn Leu Leu
Asp 130 135 140 28 140 PRT Thermococcus sp. JDF-3 MISC_FEATURE
(92)..(92) Xaa is unknown amino acid 28 Val Trp Asp Val Ser Arg Ser
Ser Thr Gly Asn Leu Val Glu Trp Phe 1 5 10 15 Leu Leu Arg Lys Ala
Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro 20 25 30 Asp Glu Arg
Glu Leu Ala Arg Arg Arg Gly Gly Tyr Ala Gly Gly Tyr 35 40 45 Val
Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile Val Tyr Leu Asp 50 55
60 Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His Asn Val Ser Pro
65 70 75 80 Asp Thr Leu Asn Arg Glu Gly Cys Arg Ser Tyr Xaa Val Ala
Pro Glu 85 90 95 Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe
Ile Pro Ser Leu 100 105 110 Leu Gly Asn Leu Leu Glu Glu Arg Gln Lys
Ile Lys Arg Lys Met Lys 115 120 125 Ala Thr Leu Asp Pro Leu Glu Lys
Asn Leu Leu Asp 130 135 140 29 140 PRT Thermococcus sp. JDF-3
MISC_FEATURE (92)..(92) Xaa is unknown amino acid 29 Val Trp Asp
Val Ser Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Phe 1 5 10 15
Leu
Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro 20 25
30 Asp Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr Ala Gly Gly Tyr
35 40 45 Val Lys Glu Pro Glu Arg Gly Pro Trp Asp Asn Ile Val Tyr
Leu Asp 50 55 60 Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His
Asn Val Ser Pro 65 70 75 80 Asp Thr Leu Asn Arg Glu Gly Cys Arg Ser
Tyr Xaa Val Ala Pro Glu 85 90 95 Val Gly His Lys Phe Cys Lys Asp
Phe Pro Gly Phe Ile Pro Ser Leu 100 105 110 Leu Gly Asn Leu Leu Glu
Val Arg Gln Lys Ile Lys Arg Lys Met Lys 115 120 125 Ala Thr Leu Asp
Pro Leu Glu Lys Asn Leu Leu Asp 130 135 140 30 140 PRT Thermococcus
sp. JDF-3 30 Val Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Val
Glu Trp Phe 1 5 10 15 Leu Leu Arg Lys Ala Tyr Glu Arg Asn Lys Leu
Ala Pro Asn Lys Pro 20 25 30 Asp Glu Arg Glu Leu Ala Arg Arg Arg
Gly Gly Tyr Ala Gly Gly Tyr 35 40 45 Val Lys Glu Pro Glu Arg Gly
Leu Trp Asp Asn Ile Val Tyr Leu Asp 50 55 60 Phe Arg Ser Leu Tyr
Pro Ser Ile Ile Ile Thr His Asn Val Ser Pro 65 70 75 80 Asp Thr Leu
Asn Arg Glu Gly Cys Arg Ser Tyr Asp Val Ala Pro Glu 85 90 95 Val
Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe Ile Pro Ser Leu 100 105
110 Leu Gly Asn Leu Leu Glu Glu Arg Gln Lys Ile Lys Arg Lys Met Lys
115 120 125 Ala Thr Leu Asp Pro Leu Glu Lys Asn Leu Leu Asp 130 135
140 31 140 PRT Thermococcus sp. JDF-3 MISC_FEATURE (4)..(4) Xaa is
unknown amino acid 31 Tyr Trp Ser Xaa Pro Xaa Leu Arg Thr Gly Asn
Leu Val Glu Trp Phe 1 5 10 15 Leu Leu Arg Lys Ala Tyr Glu Arg Asn
Glu Leu Ala Pro Asn Lys Pro 20 25 30 Asp Glu Arg Glu Leu Ala Arg
Arg Arg Gly Gly Tyr Ala Gly Gly Tyr 35 40 45 Val Lys Glu Pro Glu
Arg Gly Leu Trp Asp Asn Ile Val Tyr Leu Asp 50 55 60 Phe Arg Ser
Leu Tyr Pro Ser Ile Ile Ile Thr His Asn Val Ser Pro 65 70 75 80 Asp
Thr Leu Asn Arg Glu Gly Cys Arg Ser Tyr Asp Val Ala Pro Glu 85 90
95 Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe Ile Pro Ser Leu
100 105 110 Leu Gly Asn Pro Leu Glu Glu Arg Gln Lys Ile Lys Arg Lys
Met Lys 115 120 125 Ala Thr Leu Asp Pro Leu Glu Lys Asn Leu Leu Asp
130 135 140 32 141 PRT Thermococcus sp. JDF-3 MISC_FEATURE (5)..(5)
Xaa is unknown amino acid 32 Val Asp Gly Thr Xaa Pro Arg Ser Ser
Thr Gly Asn Leu Val Glu Trp 1 5 10 15 Phe Leu Leu Arg Lys Ala Tyr
Glu Arg Asn Glu Leu Ala Pro Asn Lys 20 25 30 Pro Asp Glu Arg Glu
Leu Ala Arg Arg Arg Gly Gly Tyr Ala Gly Gly 35 40 45 Tyr Val Lys
Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile Val Tyr Leu 50 55 60 Asp
Phe Arg Ser His Tyr Pro Ser Ile Ile Ile Thr His Asn Val Ser 65 70
75 80 Pro Asp Thr Leu Asn Arg Glu Gly Cys Arg Ser Tyr Asp Val Ala
Pro 85 90 95 Glu Asp Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe
Ile Pro Ser 100 105 110 Leu Leu Gly Asn Leu Leu Glu Glu Arg Gln Lys
Ile Lys Arg Lys Met 115 120 125 Lys Ala Thr Leu Asp Pro Leu Glu Lys
Asn His Leu Asp 130 135 140 33 143 PRT Thermococcus sp. JDF-3
MISC_FEATURE (1)..(3) Xaa is unknown amino acid 33 Xaa Xaa Xaa Phe
Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Val 1 5 10 15 Glu Trp
Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro 20 25 30
Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr Ala 35
40 45 Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile
Val 50 55 60 Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile
Thr His Asn 65 70 75 80 Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys
Arg Ser Tyr Asp Val 85 90 95 Ala Pro Glu Val Gly His Lys Phe Cys
Lys Asp Phe Pro Gly Phe Ile 100 105 110 Pro Ser Leu Leu Gly Asn Leu
Leu Glu Glu Arg Gln Lys Ile Lys Arg 115 120 125 Lys Met Lys Ala Thr
Leu Asp Pro Leu Glu Lys Asn Leu Leu Asp 130 135 140 34 180 PRT
Thermococcus sp. JDF-3 34 Thr Gly Glu Gly Leu Glu Arg Val Ala Arg
Tyr Ser Met Glu Asp Ala 1 5 10 15 Arg Val Thr Tyr Glu Leu Gly Arg
Glu Phe Phe Pro Met Glu Ala Gln 20 25 30 Leu Ser Arg Leu Ile Gly
Gln Gly Asp Trp Asp Val Ser Arg Ser Ser 35 40 45 Thr Gly Asn Leu
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg 50 55 60 Asn Glu
Leu Ala Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg 65 70 75 80
Arg Gly Gly Tyr Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu 85
90 95 Trp Asp Asn Ile Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser
Ile 100 105 110 Ile Ile Thr His Asn Val Ser Pro Asp Thr Leu Asn Arg
Glu Gly Cys 115 120 125 Arg Ser Tyr Asp Val Ala Pro Glu Val Gly His
Lys Phe Cys Lys Asp 130 135 140 Phe Pro Gly Phe Ile Pro Ser Leu Leu
Gly Asn Leu Leu Glu Glu Arg 145 150 155 160 Gln Lys Ile Lys Arg Lys
Met Lys Ala Thr Leu Asp Pro Leu Glu Lys 165 170 175 Asn Leu Leu Asp
180 35 180 PRT Thermococcus sp. JDF-3 35 Tyr Arg Gln Arg Ala Ile
Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr 1 5 10 15 Cys Gly Tyr Ala
Arg Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser 20 25 30 Val Thr
Ala Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu Leu 35 40 45
Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu 50
55 60 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys
Ala 65 70 75 80 Met Glu Phe Leu Asn Tyr Ile Asn Pro Lys Leu Pro Gly
Leu Leu Glu 85 90 95 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe
Phe Val Thr Lys Lys 100 105 110 Lys Tyr Ala Val Ile Asp Glu Glu Gly
Lys Ile Thr Thr Arg Gly Leu 115 120 125 Glu Ile Val Arg Arg Asp Trp
Ser Glu Ile Ala Lys Glu Thr Gln Ala 130 135 140 Arg Val Leu Glu Ala
Val Leu Arg His Gly Asp Val Glu Glu Ala Val 145 150 155 160 Arg Ile
Val Arg Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 165 170 175
Pro Glu Lys Leu 180 36 180 PRT Thermococcus sp. JDF-3 36 Tyr Arg
Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr 1 5 10 15
Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser 20
25 30 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu
Leu 35 40 45 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr
Asp Gly Leu 50 55 60 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr
Val Lys Lys Lys Ala 65 70 75 80 Met Glu Phe Leu Asn Tyr Ile Asn Pro
Lys Leu Pro Gly Leu Leu Glu 85 90 95 Leu Glu Tyr Glu Gly Phe Tyr
Val Arg Gly Phe Phe Val Thr Lys Lys 100 105 110 Lys Tyr Ala Val Ile
Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 115 120 125 Glu Ile Val
Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 130 135 140 Arg
Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala Val 145 150
155 160 Arg Ile Val Arg Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val
Pro 165 170 175 Pro Glu Glu Leu 180 37 180 PRT Thermococcus sp.
JDF-3 37 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr
Gly Tyr 1 5 10 15 Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Arg Glu
Cys Ala Glu Ser 20 25 30 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu
Met Val Ile Arg Glu Leu 35 40 45 Glu Glu Lys Phe Gly Phe Lys Val
Leu Tyr Ala Asp Thr Asp Gly Leu 50 55 60 His Ala Thr Ile Pro Gly
Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 65 70 75 80 Met Glu Phe Leu
Asn Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 85 90 95 Leu Glu
Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys 100 105 110
Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 115
120 125 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
Ala 130 135 140 Arg Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu
Glu Ala Val 145 150 155 160 Arg Ile Val Arg Lys Val Thr Glu Lys Leu
Ser Lys Tyr Glu Val Pro 165 170 175 Pro Glu Lys Leu 180 38 180 PRT
Thermococcus sp. JDF-3 38 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala
Asn Ser Tyr Tyr Gly Tyr 1 5 10 15 Tyr Gly Tyr Ala Arg Ala Arg Trp
Tyr Cys Arg Glu Cys Ala Glu Ser 20 25 30 Val Thr Ala Trp Gly Arg
Glu Tyr Ile Glu Met Val Ile Arg Glu Leu 35 40 45 Glu Glu Lys Phe
Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu 50 55 60 His Ala
Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 65 70 75 80
Met Glu Phe Leu Asn Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 85
90 95 Leu Lys Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys
Lys 100 105 110 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr
Arg Gly Leu 115 120 125 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala
Lys Glu Thr Gln Ala 130 135 140 Arg Val Leu Glu Ala Ile Leu Arg His
Gly Asp Val Glu Glu Ala Val 145 150 155 160 Arg Ile Val Arg Glu Val
Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 165 170 175 Pro Glu Lys Leu
180 39 180 PRT Thermococcus sp. JDF-3 39 Tyr Arg Gln Arg Ala Ile
Lys Ile Leu Ala Asn Asn Tyr Tyr Gly Tyr 1 5 10 15 Tyr Gly Tyr Ala
Arg Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser 20 25 30 Val Thr
Ala Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu Leu 35 40 45
Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu 50
55 60 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys
Ala 65 70 75 80 Met Glu Phe Leu Asn Tyr Ile Asn Pro Lys Leu Pro Gly
Leu Leu Glu 85 90 95 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe
Phe Val Thr Lys Lys 100 105 110 Lys Tyr Ala Val Ile Asp Glu Glu Gly
Lys Ile Thr Thr Arg Gly Leu 115 120 125 Glu Ile Val Arg Arg Asp Trp
Ser Glu Ile Ala Lys Glu Thr Gln Ala 130 135 140 Arg Val Leu Glu Ala
Ile Leu Arg His Asp Asp Val Glu Glu Ala Val 145 150 155 160 Arg Ile
Val Arg Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 165 170 175
Pro Glu Lys Leu 180 40 180 PRT Thermococcus sp. JDF-3 MISC_FEATURE
(114)..(114) Xaa is unknown amino acid 40 Tyr Arg Gln Arg Ala Ile
Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr 1 5 10 15 Tyr Gly Tyr Ala
Arg Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser 20 25 30 Val Thr
Ala Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu Leu 35 40 45
Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu 50
55 60 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys
Ala 65 70 75 80 Met Glu Phe Leu Asn Tyr Ile Asn Leu Lys Leu Pro Gly
Leu Leu Glu 85 90 95 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe
Phe Val Thr Lys Lys 100 105 110 Lys Xaa Ala Val Ile Asp Glu Glu Gly
Lys Ile Thr Thr Arg Gly Leu 115 120 125 Glu Ile Val Arg Arg Asp Trp
Ser Lys Ile Ala Lys Glu Thr Gln Ala 130 135 140 Arg Val Leu Glu Ala
Ile Leu Arg His Gly Asp Val Glu Glu Ala Ile 145 150 155 160 Arg Ile
Val Arg Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 165 170 175
Pro Glu Lys Leu 180 41 180 PRT Thermococcus sp. JDF-3 41 Tyr Arg
Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr 1 5 10 15
Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser 20
25 30 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu
Leu 35 40 45 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr
Asp Gly Leu 50 55 60 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr
Val Lys Lys Lys Ala 65 70 75 80 Met Glu Phe Leu Asn Tyr Ile Asn Pro
Lys Leu Pro Gly Leu Leu Glu 85 90 95 Leu Glu Tyr Glu Gly Phe Tyr
Val Arg Gly Phe Phe Val Thr Lys Lys 100 105 110 Lys Tyr Ala Val Ile
Asp Glu Glu Gly Lys Ile Ala Thr Arg Gly Leu 115 120 125 Glu Ile Val
Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 130 135 140 Arg
Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala Val 145 150
155 160 Arg Ile Val Arg Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val
Pro 165 170 175 Pro Glu Lys Leu 180 42 180 PRT Thermococcus sp.
JDF-3 42 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr
Gly Tyr 1 5 10 15 Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Arg Glu
Cys Ala Glu Ser 20 25 30 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu
Met Val Ile Arg Glu Leu 35 40 45 Glu Glu Lys Phe Gly Phe Lys Val
Leu Tyr Ala Asp Thr Asp Gly Leu 50 55 60 His Ala Thr Ile Pro Gly
Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 65 70 75 80 Met Glu Phe Leu
Asn Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 85 90 95 Leu Glu
Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys 100 105 110
Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 115
120 125 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
Ala 130 135 140 Arg Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu
Glu Ala Val 145 150 155 160 Arg Ile Val Arg Glu Val Thr Glu Lys Leu
Asn Lys Tyr Glu Val Pro 165 170 175 Pro Glu Lys Leu 180 43 180 PRT
Thermococcus sp. JDF-3 43 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala
Asn Ser Tyr Tyr Gly Tyr 1 5 10 15 Tyr Gly Tyr Ala Arg Ala Arg Trp
Tyr Cys Arg Glu Cys Ala Glu Ser 20 25 30 Val Thr Ala Trp Gly Arg
Glu Tyr Ile Glu Met Val Ile Arg Glu Leu
35 40 45 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp
Gly Leu 50 55 60 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val
Lys Lys Lys Ala 65 70 75 80 Met Glu Phe Leu Asn Tyr Ile Asn Pro Lys
Leu Pro Gly Leu Leu Glu 85 90 95 Leu Glu Tyr Glu Gly Phe Tyr Val
Arg Gly Phe Phe Val Thr Lys Lys 100 105 110 Lys Tyr Ala Val Ile Asp
Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 115 120 125 Glu Ile Val Arg
Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 130 135 140 Arg Val
Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala Val 145 150 155
160 Arg Ile Val Arg Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro
165 170 175 Pro Glu Lys Leu 180 44 180 PRT Thermococcus sp. JDF-3
44 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr
1 5 10 15 Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Arg Glu Cys Ala
Glu Ser 20 25 30 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Val
Ile Arg Glu Leu 35 40 45 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr
Ala Asp Thr Asp Gly Leu 50 55 60 His Ala Thr Ile Pro Gly Ala Asp
Ala Glu Thr Val Lys Lys Lys Ala 65 70 75 80 Met Glu Phe Leu Asn Tyr
Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 85 90 95 Pro Glu Tyr Glu
Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys 100 105 110 Lys Tyr
Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 115 120 125
Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 130
135 140 Arg Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala
Val 145 150 155 160 Arg Ile Val Arg Glu Val Thr Glu Lys Leu Ser Lys
Tyr Glu Val Pro 165 170 175 Pro Glu Lys Leu 180 45 180 PRT
Thermococcus sp. JDF-3 45 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala
Asn Ser Tyr Tyr Gly Tyr 1 5 10 15 Tyr Gly Tyr Ala Arg Ala Arg Trp
Tyr Cys Arg Glu Cys Ala Glu Ser 20 25 30 Val Thr Ala Trp Gly Arg
Glu Tyr Ile Glu Met Val Ile Arg Glu Leu 35 40 45 Glu Glu Lys Phe
Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu 50 55 60 His Ala
Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 65 70 75 80
Met Glu Phe Leu Asn Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 85
90 95 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys
Lys 100 105 110 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr
Arg Gly Leu 115 120 125 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala
Lys Glu Thr Gln Ala 130 135 140 Arg Val Leu Glu Ala Ile Leu Arg His
Gly Asp Val Glu Glu Ala Val 145 150 155 160 Arg Ile Val Arg Glu Val
Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 165 170 175 Pro Val Lys Leu
180 46 180 PRT Thermococcus sp. JDF-3 46 Tyr Arg Gln Arg Ala Ile
Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr 1 5 10 15 Tyr Gly Tyr Ala
Arg Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser 20 25 30 Val Thr
Ala Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu Leu 35 40 45
Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu 50
55 60 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys
Ala 65 70 75 80 Met Glu Phe Leu Asn Tyr Ile Asn Pro Lys Leu Pro Gly
Leu Leu Glu 85 90 95 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe
Phe Val Thr Lys Lys 100 105 110 Lys Tyr Ala Val Ile Asp Glu Glu Gly
Lys Ile Thr Thr Arg Gly Leu 115 120 125 Glu Ile Val Arg Arg Asp Trp
Ser Glu Ile Ala Lys Glu Thr Gln Ala 130 135 140 Arg Val Leu Glu Ala
Ile Leu Arg His Gly Asp Val Glu Glu Ala Val 145 150 155 160 Arg Ile
Val Arg Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 165 170 175
Pro Gly Glu Ala 180 47 180 PRT Thermococcus sp. JDF-3 47 Tyr Arg
Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Asn 1 5 10 15
Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser 20
25 30 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Val Ile Arg Glu
Leu 35 40 45 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr
Asp Gly Leu 50 55 60 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr
Val Lys Lys Lys Ala 65 70 75 80 Met Glu Phe Leu Asn Tyr Ile Asn Pro
Lys Leu Pro Gly Leu Leu Glu 85 90 95 Leu Glu Tyr Glu Gly Phe Tyr
Val Arg Gly Phe Phe Val Thr Lys Lys 100 105 110 Lys Tyr Ala Val Ile
Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 115 120 125 Glu Ile Val
Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 130 135 140 Arg
Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala Val 145 150
155 160 Arg Ile Val Arg Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val
Pro 165 170 175 Pro Glu Lys Leu 180 48 180 PRT Thermococcus sp.
JDF-3 48 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr
Gly Tyr 1 5 10 15 Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Arg Glu
Cys Ala Glu Ser 20 25 30 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu
Met Val Ile Arg Glu Leu 35 40 45 Glu Glu Lys Phe Gly Phe Lys Val
Leu Tyr Ala Asp Thr Asp Gly Leu 50 55 60 His Ala Thr Ile Pro Gly
Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 65 70 75 80 Met Glu Phe Leu
Asn Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 85 90 95 Leu Glu
Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys 100 105 110
Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 115
120 125 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
Ala 130 135 140 Arg Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu
Glu Ala Val 145 150 155 160 Arg Ile Val Arg Glu Val Thr Glu Lys Leu
Ser Lys Tyr Glu Val Pro 165 170 175 Pro Glu Lys Leu 180
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