Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1994-02-14
2001-07-03
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S194000, C435S091100, C435S091300, C435S091500, C435S325000, C435S320100, C435S252300, C536S023200
Reexamination Certificate
active
06255062
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel DNA polymerases of the &bgr;-family, called &bgr;-type DNA polymerases. Nucleic acids encoding &bgr;-type polymerases are also provided, as well as expression vectors and host cells containing the nucleic acid encoding &bgr;-type DNA polymerase. The invention further relates to methods for synthesizing nucleic acid using &bgr;-type DNA polymerases.
BACKGROUND OF THE INVENTION
Three nuclear DNA polymerases, designated &agr;, &dgr;, and &egr;, have been identified in
Saccharomyces cerevisiae
(Campbell et al.
Genome Dynamics, Protein Synthesis and Energetic
, J. R. Broach, J. R. Pringle, and E. R. Jones, eds. (1991)). The corresponding genes, POL1 (a.k.a. CDC17), POL2,and POL3 (a.k.a. CDC2), (Johnson et al.,
Cell
43, 369-377 (1985); Pizzagalli et al.,
Proc. Natl. Acad. Sci. USA
85, 3772-3776 (1988); Boulet et al.,
EMBO J
. 8, 1849-1854 (1989); Morrison et al.,
Cell
62, 1143-1151 (1990)) have been cloned and sequenced and the effect of mutations in these genes has been studied. The genes contain considerable sequence similarity, including a signature set of six highly conserved stretches in the central part of the proteins, and are often referred to as members of the &agr;-class of DNA polymerases. Failure to synthesize chromosomal sized DNA at the restrictive temperature in temperature sensitive mutants affecting DNA polymerases &agr;, &dgr;and &egr; provides direct evidence that these three distinct polymerases are required during DNA replication (Budd et al.,
Moll. Cell. Biol
. 9. 365-367 (1989); Budd et al.,
Mol. Cell. Biol
. 13, 496-505 (1993)). It has been proposed, based largely on studies in the simian virus 40 system, that during DNA replication DNA polymerase &agr; initiates synthesis of leading and lagging strand primers with either pol&dgr; or &egr; extending the primers. Another yeast gene, REV3, has significant homology to &agr;-class DNA polymerases (Morrison et al.,
J. Bacteriol
. 171, 5659-5667 (1989)). Strains with deletions of the REV3 gene are viable, grow at a normal rate, show decreased induced mutation rates, and are slightly sensitive to UV, suggesting that the REV3 protein functions in mutagenic repair but not replication. However, no DNA polymerase activity corresponding to the REV3 gene has been identified to date.
Yet another nuclear eukaryotic DNA polymerase, DNA polymerase &bgr;, has been identified in mammalian cells but not in lower eukaryotes such as yeast (Rein et al., In
The Eukaryotic Nucleus
, P. R. Strauss, and S. H. Wilson, eds. pp. 95-123 (1990); Wilson, In
The Eukaryotic Nucleus
, P. R. Strauss, and S. H. Wilson, eds. pp. 199-233 (1990)). Genes encoding DNA polymerase &bgr; have been cloned from several sources, and the sequence of the predicted proteins do not share significant similarity with the &agr;-class. Mammalian DNA polymerase &bgr; is a small protein of only 40 kDa, and has received much attention from investigators interested in structure/function relationships in DNA polymerases, because it comprises the minimal functional DNA polymerase domain of any known naturally occurring polymerase. Little is known about its in vivo function, however. Since DNA polymerase &bgr; is expressed in non-mitotic, fully differentiated tissues such as the brain (Zmudzka et al.,
Nucleic Acids Research
16, 9587-9596 (1988); Hirose et al.
Experimental Cell Research
181, 169-180 (1989); Nowak et al.,
Biochem. Biophys. Acta
1008, 203-207 (1989)), it is believed to play a role in DNA repair rather than DNA replication. In further support of this idea, the replicative polymerase, DNA polymerase &agr;, increases dramatically in liver or human lymphocytes stimulated to undergo DNA synthesis, whereas DNA polymerase &bgr; does not (Chang et al.,
J. Mol. Biol
. 74, 1-8 (1973); Bertazzoni et al.,
Proc. Natl. Acad. Sci
. 73, 783-789 (1976)). A role for DNA polymerase &bgr; in DNA repair is also suggested by its biochemical properties. DNA polymerase &bgr; preferentially catalyzes repair DNA synthesis at small gaps in duplex DNA substrates (Mosbaugh et al.,
J. Biol. Chem
. 258, 108-118 (1982); Randahl et al.,
J. Biol. Chem
. 263, 12228-12234 (1988); Singhad et al.
J. Biol. Chem
. 268, 15906-15911 (1993)). In HeLa cell extracts, DNA polymerase &bgr; repairs the single nucleotide gap created by glycosylase repair of G/T mispairs (Wiebauer et al.,
Proc. Natl. Acad. Sci. USA
87, 5842-5845 (1990)). Similarly, an aphidicolin resistant polymerase in Hela cell extracts repairs a single nucleotide gap created by excision of uracil residues from duplex DNA, implying that mammalian DNA polymerase &bgr;, the only known nuclear aphidicolin resistant DNA polymerase, is involved (Dianov et al.,
Mol. Cell. Biol
. 12, 1605-1612 (1992)). A replicative function has not been entirely excluded, however, as mammalian DNA polymerase &bgr; appears to be required to replicate single stranded DNA injected into Xenopus oocytes (Jenkins et al.,
Science
258, 475-477 (1992)). Also, when expressed in
E. coli
, DNA polymerase &bgr; can complement the gap filling deficiency of a DNA polymerase I mutant, perhaps suggesting a role in processing Okazaki fragments (Sweasy et al.,
J. Biol. Chem
. 267 1407-1410 (1992)).
The mammalian DNA polymerase &bgr;s share several properties, including low molecular weights (32,000 to 40,000), alkaline pI and pH optima, stimulation by 50-100 mM salt, inhibition by phosphate, resistance to aphidicolin, sensitivity to dideoxythymidine triphosphate, and general resistance to N-ethylmaleimide (NEM). In addition, the &bgr;-polymerases are considered to be substantially non-processive, synthesizing only short stretches of DNA in a single binding event to the primed template. &bgr;-polymerases also copy RNA as well as DNA templates, although the rat &bgr;-polymerase only exhibits reverse transcriptase activity in the presence of Mn++ (Wang et al., Biochemistry 16, 4927-4934 (1977), Ono et al.,
Nucleic Acids Research
7, 715-726 (1979), Yoshida et al.,
J. Biochem
. 85, 1387-1395 (1979)).
In addition, mammalian &bgr;-polymerases show the highest error rate measured for cellular DNA polymerases. This may be due to the reduced ability of &bgr;-polymerases to discriminate among substrates compared to &agr; polymerases, since they may exhibit up to 20 to 50% of normal activity in the presence of a single dNTP as in the presence of all four dNTPs (Rein et al., supra).
An &agr;-DNA polymerase and a &bgr;-DNA polymerase were reported in
Tetrahymena pyriformis
, a protozoa (Sakai et al., J. Biochem. 91, 845-853 (1982); Sakai et al., J. Biochem. 91, 855-863 (1982)). The two polymerases were purified from exponentially growing cells and purified. The &bgr;-DNA polymerase had a molecular weight of about 70,000,was inhibited by NEM, resistant to aphidicolin, and could not synthesize DNA in the presence of Mn++.
The complete DNA sequence of the
Saccharomyces cerevisiae
chromosome III has been elucidated as a result of the European yeast genome project (Oliver et al.,
Nature
357:38-46 (1992). Low-stringency homology searching identified an open reading frame (ORF) of 582 amino acids which has 26% homology to rat DNA polymerase-&bgr; over the 393 amino acids of the C-terminal portion of the ORF. (Bork et al.,
Nature
, 358:287 (1992)).
SUMMARY OF THE INVENTION
It is an object of the invention to provide a new form of DNA polymerase, &bgr;-type DNA polymerase, as well as variants of &bgr;-type DNA polymerases, and to produce useful quantities of these &bgr;-type DNA polymerases using recombinant DNA techniques.
It is a further object of the invention to provide recombinant or isolated nucleic acids encoding &bgr;-type DNA polymerases, and expression vectors and host cells containing the nucleic acid encoding the &bgr;-type DNA polymerase.
An additional object of the invention is to provide methods for synthesizing nucleic acids and for producing nucleic acids which have synthetic, modified or labelled nucleotides incorporated into the synthesized strand.
In accordance with t
Budd Martin E.
Campbell Judith L.
California Institute of Technology
Flehr Hohbach Test Albritton & Herbert LLP
Prouty Rebecca E.
Trecartin Richard F.
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