Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Feb;13(2):1051–1058. doi: 10.1128/mcb.13.2.1051

DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae.

Z Wang 1, X Wu 1, E C Friedberg 1
PMCID: PMC358990  PMID: 8423775

Abstract

Base excision repair is an important mechanism for correcting DNA damage produced by many physical and chemical agents. We have examined the effects of the REV3 gene and the DNA polymerase genes POL1, POL2, and POL3 of Saccharomyces cerevisiae on DNA repair synthesis is nuclear extracts. Deletional inactivation of REV3 did not affect repair synthesis in the base excision repair pathway. Repair synthesis in nuclear extracts of pol1, pol2, and pol3 temperature-sensitive mutants was normal at permissive temperatures. However, repair synthesis in pol2 nuclear extracts was defective at the restrictive temperature of 37 degrees C and could be complemented by the addition of purified yeast DNA polymerase epsilon. Repair synthesis in pol1 nuclear extracts was proficient at the restrictive temperature unless DNA polymerase alpha was inactivated prior to the initiation of DNA repair. Thermal inactivation of DNA polymerase delta in pol3 nuclear extracts enhanced DNA repair synthesis approximately 2-fold, an effect which could be specifically reversed by the addition of purified yeast DNA polymerase delta to the extract. These results demonstrate that DNA repair synthesis in the yeast base excision repair pathway is catalyzed by DNA polymerase epsilon but is apparently modulated by the presence of DNA polymerases alpha and delta.

Full text

PDF
1051

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Araki H., Ropp P. A., Johnson A. L., Johnston L. H., Morrison A., Sugino A. DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae. EMBO J. 1992 Feb;11(2):733–740. doi: 10.1002/j.1460-2075.1992.tb05106.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauer G. A., Burgers P. M. The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen interacts with mammalian DNA polymerase delta. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7506–7510. doi: 10.1073/pnas.85.20.7506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Brooke R. G., Singhal R., Hinkle D. C., Dumas L. B. Purification and characterization of the 180- and 86-kilodalton subunits of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex. The 180-kilodalton subunit has both DNA polymerase and 3'----5'-exonuclease activities. J Biol Chem. 1991 Feb 15;266(5):3005–3015. [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. Dianov G., Price A., Lindahl T. Generation of single-nucleotide repair patches following excision of uracil residues from DNA. Mol Cell Biol. 1992 Apr;12(4):1605–1612. doi: 10.1128/mcb.12.4.1605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fien K., Stillman B. Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex. Mol Cell Biol. 1992 Jan;12(1):155–163. doi: 10.1128/mcb.12.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Hansson J., Munn M., Rupp W. D., Kahn R., Wood R. D. Localization of DNA repair synthesis by human cell extracts to a short region at the site of a lesion. J Biol Chem. 1989 Dec 25;264(36):21788–21792. [PubMed] [Google Scholar]
  12. Hardt N., Pedrali-Noy G., Focher F., Spadari S. Aphidicolin does not inhibit DNA repair synthesis in ultraviolet-irradiated HeLa cells. A radioautographic study. Biochem J. 1981 Nov 1;199(2):453–455. doi: 10.1042/bj1990453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huang J. C., Svoboda D. L., Reardon J. T., Sancar A. Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5' and the 6th phosphodiester bond 3' to the photodimer. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3664–3668. doi: 10.1073/pnas.89.8.3664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hübscher U., Thömmes P. DNA polymerase epsilon: in search of a function. Trends Biochem Sci. 1992 Feb;17(2):55–58. doi: 10.1016/0968-0004(92)90499-y. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Lemontt J. F. Mutants of yeast defective in mutation induced by ultraviolet light. Genetics. 1971 May;68(1):21–33. doi: 10.1093/genetics/68.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Morrison A., Christensen R. B., Alley J., Beck A. K., Bernstine E. G., Lemontt J. F., Lawrence C. W. REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase. J Bacteriol. 1989 Oct;171(10):5659–5667. doi: 10.1128/jb.171.10.5659-5667.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Perrino F. W., Loeb L. A. Animal cell DNA polymerases in DNA repair. Mutat Res. 1990 Sep-Nov;236(2-3):289–300. doi: 10.1016/0921-8777(90)90012-t. [DOI] [PubMed] [Google Scholar]
  21. Popanda O., Thielmann H. W. The function of DNA polymerases in DNA repair synthesis of ultraviolet-irradiated human fibroblasts. Biochim Biophys Acta. 1992 Jan 6;1129(2):155–160. doi: 10.1016/0167-4781(92)90480-n. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Smith C. A., Okumoto D. S. Nature of DNA repair synthesis resistant to inhibitors of polymerase alpha in human cells. Biochemistry. 1984 Mar 27;23(7):1383–1391. doi: 10.1021/bi00302a008. [DOI] [PubMed] [Google Scholar]
  24. Syväoja J. E. DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases. Bioessays. 1990 Nov;12(11):533–536. doi: 10.1002/bies.950121106. [DOI] [PubMed] [Google Scholar]
  25. Wang Z., Wu X., Friedberg E. C. Excision repair of DNA in nuclear extracts from the yeast Saccharomyces cerevisiae. Biochemistry. 1992 Apr 14;31(14):3694–3702. doi: 10.1021/bi00129a019. [DOI] [PubMed] [Google Scholar]
  26. Yoder B. L., Burgers P. M. Saccharomyces cerevisiae replication factor C. I. Purification and characterization of its ATPase activity. J Biol Chem. 1991 Nov 25;266(33):22689–22697. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES