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. 1992 Feb;11(2):733–740. doi: 10.1002/j.1460-2075.1992.tb05106.x

DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae.

H Araki 1, P A Ropp 1, A L Johnson 1, L H Johnston 1, A Morrison 1, A Sugino 1
PMCID: PMC556506  PMID: 1537345

Abstract

Two temperature-sensitive DNA polymerase II mutants (pol2-9 and pol2-18) of the yeast Saccharomyces cerevisiae were isolated by the plasmid shuffling method. DNA polymerase II activity partially purified from both mutants was thermolabile, while DNA polymerase I and III activities remained thermotolerant. At the restrictive temperature, the pol2 mutants were defective in chromosomal DNA replication and exhibited the dumbbell terminal morphology typical of DNA replication mutants. The POL2 transcript accumulated periodically during the cell cycle, peaking at the G1/S boundary in the same manner as the transcripts of more than 10 other DNA replication genes. These results indicate that DNA polymerase II participates in nuclear DNA replication. The similarities in structure and activities between the DNA polymerases of yeast and mammals make it likely that mammalian DNA polymerase epsilon too is required for chromosomal DNA replication.

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Selected References

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  1. Araki H., Hamatake R. K., Johnston L. H., Sugino A. DPB2, the gene encoding DNA polymerase II subunit B, is required for chromosome replication in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4601–4605. doi: 10.1073/pnas.88.11.4601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Araki H., Hamatake R. K., Morrison A., Johnson A. L., Johnston L. H., Sugino A. Cloning DPB3, the gene encoding the third subunit of DNA polymerase II of Saccharomyces cerevisiae. Nucleic Acids Res. 1991 Sep 25;19(18):4867–4872. doi: 10.1093/nar/19.18.4867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bambara R. A., Jessee C. B. Properties of DNA polymerases delta and epsilon, and their roles in eukaryotic DNA replication. Biochim Biophys Acta. 1991 Jan 17;1088(1):11–24. doi: 10.1016/0167-4781(91)90147-e. [DOI] [PubMed] [Google Scholar]
  4. Bauer G. A., Burgers P. M. Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene. Nucleic Acids Res. 1990 Jan 25;18(2):261–265. doi: 10.1093/nar/18.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blanco L., Bernad A., Blasco M. A., Salas M. A general structure for DNA-dependent DNA polymerases. Gene. 1991 Apr;100:27–38. doi: 10.1016/0378-1119(91)90346-d. [DOI] [PubMed] [Google Scholar]
  6. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  7. Boulet A., Simon M., Faye G., Bauer G. A., Burgers P. M. Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III. EMBO J. 1989 Jun;8(6):1849–1854. doi: 10.1002/j.1460-2075.1989.tb03580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bravo R., Frank R., Blundell P. A., Macdonald-Bravo H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature. 1987 Apr 2;326(6112):515–517. doi: 10.1038/326515a0. [DOI] [PubMed] [Google Scholar]
  9. Brill S. J., Stillman B. Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication. Nature. 1989 Nov 2;342(6245):92–95. doi: 10.1038/342092a0. [DOI] [PubMed] [Google Scholar]
  10. Budd M. E., Wittrup K. D., Bailey J. E., Campbell J. L. DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):365–376. doi: 10.1128/mcb.9.2.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Budd M., Campbell J. L. Temperature-sensitive mutations in the yeast DNA polymerase I gene. Proc Natl Acad Sci U S A. 1987 May;84(9):2838–2842. doi: 10.1073/pnas.84.9.2838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bullock P. A., Seo Y. S., Hurwitz J. Initiation of simian virus 40 DNA synthesis in vitro. Mol Cell Biol. 1991 May;11(5):2350–2361. doi: 10.1128/mcb.11.5.2350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Burgers P. M., Bambara R. A., Campbell J. L., Chang L. M., Downey K. M., Hübscher U., Lee M. Y., Linn S. M., So A. G., Spadari S. Revised nomenclature for eukaryotic DNA polymerases. Eur J Biochem. 1990 Aug 17;191(3):617–618. doi: 10.1111/j.1432-1033.1990.tb19165.x. [DOI] [PubMed] [Google Scholar]
  14. Burgers P. M. Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon. J Biol Chem. 1991 Nov 25;266(33):22698–22706. [PubMed] [Google Scholar]
  15. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  16. Chapman J. W., Johnston L. H. The yeast gene, DBF4, essential for entry into S phase is cell cycle regulated. Exp Cell Res. 1989 Feb;180(2):419–428. doi: 10.1016/0014-4827(89)90068-2. [DOI] [PubMed] [Google Scholar]
  17. Conrad M. N., Newlon C. S. Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome. Mol Cell Biol. 1983 Jun;3(6):1000–1012. doi: 10.1128/mcb.3.6.1000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Eberly S. L., Sakai A., Sugino A. Mapping and characterizing a new DNA replication mutant in Saccharomyces cerevisiae. Yeast. 1989 Mar-Apr;5(2):117–129. doi: 10.1002/yea.320050207. [DOI] [PubMed] [Google Scholar]
  19. Eki T., Murakami Y., Enomoto T., Hanaoka F., Yamada M. Characterization of DNA replication at a restrictive temperature in a mouse DNA temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha activity. J Biol Chem. 1986 Jul 5;261(19):8888–8893. [PubMed] [Google Scholar]
  20. Gibbs J. S., Chiou H. C., Bastow K. F., Cheng Y. C., Coen D. M. Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6672–6676. doi: 10.1073/pnas.85.18.6672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  22. Hall J. D., Wang Y. S., Pierpont J., Berlin M. S., Rundlett S. E., Woodward S. Aphidicolin resistance in herpes simplex virus type I reveals features of the DNA polymerase dNTP binding site. Nucleic Acids Res. 1989 Nov 25;17(22):9231–9244. doi: 10.1093/nar/17.22.9231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hamatake R. K., Hasegawa H., Clark A. B., Bebenek K., Kunkel T. A., Sugino A. Purification and characterization of DNA polymerase II from the yeast Saccharomyces cerevisiae. Identification of the catalytic core and a possible holoenzyme form of the enzyme. J Biol Chem. 1990 Mar 5;265(7):4072–4083. [PubMed] [Google Scholar]
  24. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  25. Ishimi Y., Claude A., Bullock P., Hurwitz J. Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J Biol Chem. 1988 Dec 25;263(36):19723–19733. [PubMed] [Google Scholar]
  26. Johnson L. M., Snyder M., Chang L. M., Davis R. W., Campbell J. L. Isolation of the gene encoding yeast DNA polymerase I. Cell. 1985 Nov;43(1):369–377. doi: 10.1016/0092-8674(85)90042-x. [DOI] [PubMed] [Google Scholar]
  27. Johnston L. H., White J. H., Johnson A. L., Lucchini G., Plevani P. The yeast DNA polymerase I transcript is regulated in both the mitotic cell cycle and in meiosis and is also induced after DNA damage. Nucleic Acids Res. 1987 Jul 10;15(13):5017–5030. doi: 10.1093/nar/15.13.5017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jung G. H., Leavitt M. C., Schultz M., Ito J. Site-specific mutagenesis of PRD1 DNA polymerase: mutations in highly conserved regions of the family B DNA polymerase. Biochem Biophys Res Commun. 1990 Aug 16;170(3):1294–1300. doi: 10.1016/0006-291x(90)90534-t. [DOI] [PubMed] [Google Scholar]
  29. Kaczmarek L., Miller M. R., Hammond R. A., Mercer W. E. A microinjected monoclonal antibody against human DNA polymerase-alpha inhibits DNA replication in human, hamster, and mouse cell lines. J Biol Chem. 1986 Aug 15;261(23):10802–10807. [PubMed] [Google Scholar]
  30. Kornberg A. DNA replication. J Biol Chem. 1988 Jan 5;263(1):1–4. [PubMed] [Google Scholar]
  31. Larder B. A., Kemp S. D., Darby G. Related functional domains in virus DNA polymerases. EMBO J. 1987 Jan;6(1):169–175. doi: 10.1002/j.1460-2075.1987.tb04735.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lee S. H., Eki T., Hurwitz J. Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7361–7365. doi: 10.1073/pnas.86.19.7361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Linn S. How many pols does it take to replicate nuclear DNA? Cell. 1991 Jul 26;66(2):185–187. doi: 10.1016/0092-8674(91)90608-2. [DOI] [PubMed] [Google Scholar]
  34. Lowndes N. F., Johnson A. L., Johnston L. H. Coordination of expression of DNA synthesis genes in budding yeast by a cell-cycle regulated trans factor. Nature. 1991 Mar 21;350(6315):247–250. doi: 10.1038/350247a0. [DOI] [PubMed] [Google Scholar]
  35. Matsumoto T., Eki T., Hurwitz J. Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9712–9716. doi: 10.1073/pnas.87.24.9712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Morrison A., Araki H., Clark A. B., Hamatake R. K., Sugino A. A third essential DNA polymerase in S. cerevisiae. Cell. 1990 Sep 21;62(6):1143–1151. doi: 10.1016/0092-8674(90)90391-q. [DOI] [PubMed] [Google Scholar]
  37. Morrison A., Bell J. B., Kunkel T. A., Sugino A. Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9473–9477. doi: 10.1073/pnas.88.21.9473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Murakami Y., Wobbe C. R., Weissbach L., Dean F. B., Hurwitz J. Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1986 May;83(9):2869–2873. doi: 10.1073/pnas.83.9.2869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nethanel T., Kaufmann G. Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40. J Virol. 1990 Dec;64(12):5912–5918. doi: 10.1128/jvi.64.12.5912-5918.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nishida C., Reinhard P., Linn S. DNA repair synthesis in human fibroblasts requires DNA polymerase delta. J Biol Chem. 1988 Jan 5;263(1):501–510. [PubMed] [Google Scholar]
  41. Pizzagalli A., Valsasnini P., Plevani P., Lucchini G. DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3772–3776. doi: 10.1073/pnas.85.11.3772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Prelich G., Stillman B. Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA. Cell. 1988 Apr 8;53(1):117–126. doi: 10.1016/0092-8674(88)90493-x. [DOI] [PubMed] [Google Scholar]
  43. Prelich G., Tan C. K., Kostura M., Mathews M. B., So A. G., Downey K. M., Stillman B. Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein. Nature. 1987 Apr 2;326(6112):517–520. doi: 10.1038/326517a0. [DOI] [PubMed] [Google Scholar]
  44. Sitney K. C., Budd M. E., Campbell J. L. DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene. Cell. 1989 Feb 24;56(4):599–605. doi: 10.1016/0092-8674(89)90582-5. [DOI] [PubMed] [Google Scholar]
  45. Syväoja J., Suomensaari S., Nishida C., Goldsmith J. S., Chui G. S., Jain S., Linn S. DNA polymerases alpha, delta, and epsilon: three distinct enzymes from HeLa cells. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6664–6668. doi: 10.1073/pnas.87.17.6664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tsurimoto T., Melendy T., Stillman B. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature. 1990 Aug 9;346(6284):534–539. doi: 10.1038/346534a0. [DOI] [PubMed] [Google Scholar]
  47. Tsurimoto T., Stillman B. Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta. EMBO J. 1989 Dec 1;8(12):3883–3889. doi: 10.1002/j.1460-2075.1989.tb08567.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wang T. S., Wong S. W., Korn D. Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases. FASEB J. 1989 Jan;3(1):14–21. doi: 10.1096/fasebj.3.1.2642867. [DOI] [PubMed] [Google Scholar]
  49. Weinberg D. H., Collins K. L., Simancek P., Russo A., Wold M. S., Virshup D. M., Kelly T. J. Reconstitution of simian virus 40 DNA replication with purified proteins. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8692–8696. doi: 10.1073/pnas.87.22.8692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weinberg D. H., Kelly T. J. Requirement for two DNA polymerases in the replication of simian virus 40 DNA in vitro. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9742–9746. doi: 10.1073/pnas.86.24.9742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. White J. H., Barker D. G., Nurse P., Johnston L. H. Periodic transcription as a means of regulating gene expression during the cell cycle: contrasting modes of expression of DNA ligase genes in budding and fission yeast. EMBO J. 1986 Jul;5(7):1705–1709. doi: 10.1002/j.1460-2075.1986.tb04414.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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