Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1989 Jun;8(6):1849–1854. doi: 10.1002/j.1460-2075.1989.tb03580.x

Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III.

A Boulet 1, M Simon 1, G Faye 1, G A Bauer 1, P M Burgers 1
PMCID: PMC401032  PMID: 2670563

Abstract

Saccharomyces cerevisiae cdc2 mutants arrest in the S-phase of the cell cycle when grown at the non-permissive temperature, implicating this gene product as essential for DNA synthesis. The CDC2 gene has been cloned from a yeast genomic library in vector YEp13 by complementation of a cdc2 mutation. An open reading frame coding for a 1093 amino acid long protein with a calculated mol. wt of 124,518 was determined from the sequence. This putative protein shows significant homology with a class of eukaryotic DNA polymerases exemplified by human DNA polymerase alpha and herpes simplex virus DNA polymerase. Fractionation of extracts from cdc2 strains showed that these mutants lacked both the polymerase and proofreading 3'-5' exonuclease activity of DNA polymerase III, the yeast analog of mammalian DNA polymerase delta. These studies indicate that DNA polymerase III is an essential component of the DNA replication machinery.

Full text

PDF
1849

Images in this article

1852Image
on p.1852

Selected References

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

  1. Arndt K. T., Styles C., Fink G. R. Multiple global regulators control HIS4 transcription in yeast. Science. 1987 Aug 21;237(4817):874–880. doi: 10.1126/science.3303332. [DOI] [PubMed] [Google Scholar]
  2. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  3. Baldari C., Cesareni G. Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences. Gene. 1985;35(1-2):27–32. doi: 10.1016/0378-1119(85)90154-4. [DOI] [PubMed] [Google Scholar]
  4. Barker D. G., White J. H., Johnston L. H. The nucleotide sequence of the DNA ligase gene (CDC9) from Saccharomyces cerevisiae: a gene which is cell-cycle regulated and induced in response to DNA damage. Nucleic Acids Res. 1985 Dec 9;13(23):8323–8337. doi: 10.1093/nar/13.23.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bauer G. A., Burgers P. M. Protein-protein interactions of yeast DNA polymerase III with mammalian and yeast proliferating cell nuclear antigen (PCNA)/cyclin. Biochim Biophys Acta. 1988 Dec 20;951(2-3):274–279. doi: 10.1016/0167-4781(88)90097-8. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Bauer G. A., Heller H. M., Burgers P. M. DNA polymerase III from Saccharomyces cerevisiae. I. Purification and characterization. J Biol Chem. 1988 Jan 15;263(2):917–924. [PubMed] [Google Scholar]
  8. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  9. Bennetzen J. L., Hall B. D. Codon selection in yeast. J Biol Chem. 1982 Mar 25;257(6):3026–3031. [PubMed] [Google Scholar]
  10. Birkenmeyer L. G., Hill J. C., Dumas L. B. Saccharomyces cerevisiae CDC8 gene and its product. Mol Cell Biol. 1984 Apr;4(4):583–590. doi: 10.1128/mcb.4.4.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  12. Breeden L. Cell cycle-regulated promoters in budding yeast. Trends Genet. 1988 Sep;4(9):249–253. doi: 10.1016/0168-9525(88)90031-5. [DOI] [PubMed] [Google Scholar]
  13. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  14. Burgers P. M., Bauer G. A. DNA polymerase III from Saccharomyces cerevisiae. II. Inhibitor studies and comparison with DNA polymerases I and II. J Biol Chem. 1988 Jan 15;263(2):925–930. [PubMed] [Google Scholar]
  15. Burgers P. M. Mammalian cyclin/PCNA (DNA polymerase delta auxiliary protein) stimulates processive DNA synthesis by yeast DNA polymerase III. Nucleic Acids Res. 1988 Jul 25;16(14A):6297–6307. doi: 10.1093/nar/16.14.6297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Byrnes J. J., Downey K. M., Black V. L., So A. G. A new mammalian DNA polymerase with 3' to 5' exonuclease activity: DNA polymerase delta. Biochemistry. 1976 Jun 29;15(13):2817–2823. doi: 10.1021/bi00658a018. [DOI] [PubMed] [Google Scholar]
  17. Campbell J. L. Eukaryotic DNA replication. Annu Rev Biochem. 1986;55:733–771. doi: 10.1146/annurev.bi.55.070186.003505. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Celis J. E., Madsen P., Celis A., Nielsen H. V., Gesser B. Cyclin (PCNA, auxiliary protein of DNA polymerase delta) is a central component of the pathway(s) leading to DNA replication and cell division. FEBS Lett. 1987 Aug 10;220(1):1–7. doi: 10.1016/0014-5793(87)80865-7. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Culotti J., Hartwell L. H. Genetic control of the cell division cycle in yeast. 3. Seven genes controlling nuclear division. Exp Cell Res. 1971 Aug;67(2):389–401. doi: 10.1016/0014-4827(71)90424-1. [DOI] [PubMed] [Google Scholar]
  22. Focher F., Spadari S., Ginelli B., Hottiger M., Gassmann M., Hübscher U. Calf thymus DNA polymerase delta: purification, biochemical and functional properties of the enzyme after its separation from DNA polymerase alpha, a DNA dependent ATPase and proliferating cell nuclear antigen. Nucleic Acids Res. 1988 Jul 25;16(14A):6279–6295. doi: 10.1093/nar/16.14.6279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hartwell L. H. Sequential function of gene products relative to DNA synthesis in the yeast cell cycle. J Mol Biol. 1976 Jul 15;104(4):803–817. doi: 10.1016/0022-2836(76)90183-2. [DOI] [PubMed] [Google Scholar]
  24. Johnston L. H., Williamson D. H. An alkaline sucrose gradient analysis of the mechanism of nuclear DNA synthesis in the yeast Saccharomyces cerevisiae. Mol Gen Genet. 1978 Aug 17;164(2):217–225. doi: 10.1007/BF00267387. [DOI] [PubMed] [Google Scholar]
  25. Johnston M. A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiol Rev. 1987 Dec;51(4):458–476. doi: 10.1128/mr.51.4.458-476.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kammerer B., Guyonvarch A., Hubert J. C. Yeast regulatory gene PPR1. I. Nucleotide sequence, restriction map and codon usage. J Mol Biol. 1984 Dec 5;180(2):239–250. doi: 10.1016/s0022-2836(84)80002-9. [DOI] [PubMed] [Google Scholar]
  27. Kouzarides T., Bankier A. T., Satchwell S. C., Weston K., Tomlinson P., Barrell B. G. Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene. J Virol. 1987 Jan;61(1):125–133. doi: 10.1128/jvi.61.1.125-133.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kuo C., Nuang H., Campbell J. L. Isolation of yeast DNA replication mutants in permeabilized cells. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6465–6469. doi: 10.1073/pnas.80.21.6465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Lee M. Y., Toomey N. L. Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting. Biochemistry. 1987 Feb 24;26(4):1076–1085. doi: 10.1021/bi00378a014. [DOI] [PubMed] [Google Scholar]
  31. Lin H. C., Lei S. P., Wilcox G. An improved DNA sequencing strategy. Anal Biochem. 1985 May 15;147(1):114–119. doi: 10.1016/0003-2697(85)90016-8. [DOI] [PubMed] [Google Scholar]
  32. Maki H., Kornberg A. Proofreading by DNA polymerase III of Escherichia coli depends on cooperative interaction of the polymerase and exonuclease subunits. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4389–4392. doi: 10.1073/pnas.84.13.4389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nasmyth K. A., Tatchell K. The structure of transposable yeast mating type loci. Cell. 1980 Mar;19(3):753–764. doi: 10.1016/s0092-8674(80)80051-1. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Patterson M., Sclafani R. A., Fangman W. L., Rosamond J. Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae. Mol Cell Biol. 1986 May;6(5):1590–1598. doi: 10.1128/mcb.6.5.1590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Prelich G., Kostura M., Marshak D. R., Mathews M. B., Stillman B. The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro. Nature. 1987 Apr 2;326(6112):471–475. doi: 10.1038/326471a0. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Sabatino R. D., Myers T. W., Bambara R. A., Kwon-Shin O., Marraccino R. L., Frickey P. H. Calf thymus DNA polymerases alpha and delta are capable of highly processive DNA synthesis. Biochemistry. 1988 Apr 19;27(8):2998–3004. doi: 10.1021/bi00408a050. [DOI] [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Scheuermann R. H., Echols H. A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7747–7751. doi: 10.1073/pnas.81.24.7747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schild D., Byers B. Meiotic effects of DNA-defective cell division cycle mutations of Saccharomyces cerevisiae. Chromosoma. 1978 Dec 21;70(1):109–130. doi: 10.1007/BF00292220. [DOI] [PubMed] [Google Scholar]
  43. Sclafani R. A., Fangman W. L. Yeast gene CDC8 encodes thymidylate kinase and is complemented by herpes thymidine kinase gene TK. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5821–5825. doi: 10.1073/pnas.81.18.5821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sengstag C., Hinnen A. The sequence of the Saccharomyces cerevisiae gene PHO2 codes for a regulatory protein with unusual aminoacid composition. Nucleic Acids Res. 1987 Jan 12;15(1):233–246. doi: 10.1093/nar/15.1.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Simon M., Seraphin B., Faye G. KIN28, a yeast split gene coding for a putative protein kinase homologous to CDC28. EMBO J. 1986 Oct;5(10):2697–2701. doi: 10.1002/j.1460-2075.1986.tb04553.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. So A. G., Downey K. M. Mammalian DNA polymerases alpha and delta: current status in DNA replication. Biochemistry. 1988 Jun 28;27(13):4591–4595. doi: 10.1021/bi00413a001. [DOI] [PubMed] [Google Scholar]
  47. Syvaoja J., Linn S. Characterization of a large form of DNA polymerase delta from HeLa cells that is insensitive to proliferating cell nuclear antigen. J Biol Chem. 1989 Feb 15;264(5):2489–2497. [PubMed] [Google Scholar]
  48. Taylor G. R., Lagosky P. A., Storms R. K., Haynes R. H. Molecular characterization of the cell cycle-regulated thymidylate synthase gene of Saccharomyces cerevisiae. J Biol Chem. 1987 Apr 15;262(11):5298–5307. [PubMed] [Google Scholar]
  49. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wong S. W., Wahl A. F., Yuan P. M., Arai N., Pearson B. E., Arai K., Korn D., Hunkapiller M. W., Wang T. S. Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J. 1988 Jan;7(1):37–47. doi: 10.1002/j.1460-2075.1988.tb02781.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  52. Yochem J., Byers B. Structural comparison of the yeast cell division cycle gene CDC4 and a related pseudogene. J Mol Biol. 1987 May 20;195(2):233–245. doi: 10.1016/0022-2836(87)90646-2. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES