Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Nov 15;90(22):10519–10523. doi: 10.1073/pnas.90.22.10519

De novo synthesis of budding yeast DNA polymerase alpha and POL1 transcription at the G1/S boundary are not required for entrance into S phase.

M M Falconi 1, A Piseri 1, M Ferrari 1, G Lucchini 1, P Plevani 1, M Foiani 1
PMCID: PMC47808  PMID: 8248139

Abstract

The POL1 gene, encoding DNA polymerase alpha (pol alpha) in Saccharomyces cerevisiae, is transiently transcribed during the cell cycle at the G1/S phase boundary. Here we show that yeast pol alpha is present at every stage of the cell cycle, and its level only slightly increases following the peak of POL1 transcription. POL1 mRNA synthesis driven by a GAL1 promoter can be completely abolished without affecting the growth rate of logarithmically growing yeast cultures for several cell divisions, although the amount of the pol alpha polypeptide drops below the physiological level. Moreover, alpha-factor-arrested cells can enter S phase and divide synchronously even if POL1 transcription is abolished. These results indicate that the level of yeast pol alpha is not rate limiting and de novo synthesis of the enzyme is not required for entrance into S phase.

Full text

PDF
10519

Images in this article

Selected References

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

  1. Blow J. J., Laskey R. A. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature. 1988 Apr 7;332(6164):546–548. doi: 10.1038/332546a0. [DOI] [PubMed] [Google Scholar]
  2. Bouvier D., Pignede G., Damagnez V., Tillit J., de Recondo A. M., Baldacci G. DNA polymerase alpha in the fission yeast Schizosaccharomyces pombe: identification and tracing of the catalytic subunit during the cell cycle. Exp Cell Res. 1992 Feb;198(2):183–190. doi: 10.1016/0014-4827(92)90370-n. [DOI] [PubMed] [Google Scholar]
  3. Brill S. J., Stillman B. Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase. Genes Dev. 1991 Sep;5(9):1589–1600. doi: 10.1101/gad.5.9.1589. [DOI] [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. Bueno A., Russell P. Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division. EMBO J. 1992 Jun;11(6):2167–2176. doi: 10.1002/j.1460-2075.1992.tb05276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Davis J. L., Kunisawa R., Thorner J. A presumptive helicase (MOT1 gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1992 Apr;12(4):1879–1892. doi: 10.1128/mcb.12.4.1879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Din S., Brill S. J., Fairman M. P., Stillman B. Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev. 1990 Jun;4(6):968–977. doi: 10.1101/gad.4.6.968. [DOI] [PubMed] [Google Scholar]
  9. Dirick L., Moll T., Auer H., Nasmyth K. A central role for SWI6 in modulating cell cycle Start-specific transcription in yeast. Nature. 1992 Jun 11;357(6378):508–513. doi: 10.1038/357508a0. [DOI] [PubMed] [Google Scholar]
  10. Epstein C. B., Cross F. R. CLB5: a novel B cyclin from budding yeast with a role in S phase. Genes Dev. 1992 Sep;6(9):1695–1706. doi: 10.1101/gad.6.9.1695. [DOI] [PubMed] [Google Scholar]
  11. Foiani M., Cigan A. M., Paddon C. J., Harashima S., Hinnebusch A. G. GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):3203–3216. doi: 10.1128/mcb.11.6.3203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Foiani M., Santocanale C., Plevani P., Lucchini G. A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Jul;9(7):3081–3087. doi: 10.1128/mcb.9.7.3081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gordon C. B., Campbell J. L. A cell cycle-responsive transcriptional control element and a negative control element in the gene encoding DNA polymerase alpha in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6058–6062. doi: 10.1073/pnas.88.14.6058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hennessy K. M., Clark C. D., Botstein D. Subcellular localization of yeast CDC46 varies with the cell cycle. Genes Dev. 1990 Dec;4(12B):2252–2263. doi: 10.1101/gad.4.12b.2252. [DOI] [PubMed] [Google Scholar]
  15. Johnson A. L., Barker D. G., Johnston L. H. Induction of yeast DNA ligase genes in exponential and stationary phase cultures in response to DNA damaging agents. Curr Genet. 1986;11(2):107–112. doi: 10.1007/BF00378201. [DOI] [PubMed] [Google Scholar]
  16. Johnston L. H. Cell cycle control of gene expression in yeast. Trends Cell Biol. 1992 Dec;2(12):353–357. doi: 10.1016/0962-8924(92)90041-k. [DOI] [PubMed] [Google Scholar]
  17. Johnston L. H., Lowndes N. F. Cell cycle control of DNA synthesis in budding yeast. Nucleic Acids Res. 1992 May 25;20(10):2403–2410. doi: 10.1093/nar/20.10.2403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lasko D. D., Tomkinson A. E., Lindahl T. Mammalian DNA ligases. Biosynthesis and intracellular localization of DNA ligase I. J Biol Chem. 1990 Jul 25;265(21):12618–12622. [PubMed] [Google Scholar]
  21. Lowndes N. F., Johnson A. L., Breeden L., Johnston L. H. SWI6 protein is required for transcription of the periodically expressed DNA synthesis genes in budding yeast. Nature. 1992 Jun 11;357(6378):505–508. doi: 10.1038/357505a0. [DOI] [PubMed] [Google Scholar]
  22. Morris G. F., Mathews M. B. Regulation of proliferating cell nuclear antigen during the cell cycle. J Biol Chem. 1989 Aug 15;264(23):13856–13864. [PubMed] [Google Scholar]
  23. Nasheuer H. P., Moore A., Wahl A. F., Wang T. S. Cell cycle-dependent phosphorylation of human DNA polymerase alpha. J Biol Chem. 1991 Apr 25;266(12):7893–7903. [PubMed] [Google Scholar]
  24. Nasmyth K., Dirick L. The role of SWI4 and SWI6 in the activity of G1 cyclins in yeast. Cell. 1991 Sep 6;66(5):995–1013. doi: 10.1016/0092-8674(91)90444-4. [DOI] [PubMed] [Google Scholar]
  25. Norbury C., Nurse P. Animal cell cycles and their control. Annu Rev Biochem. 1992;61:441–470. doi: 10.1146/annurev.bi.61.070192.002301. [DOI] [PubMed] [Google Scholar]
  26. Pizzagalli A., Piatti S., Derossi D., Gander I., Plevani P., Lucchini G. Positive cis-acting regulatory sequences mediate proper control of POL1 transcription in Saccharomyces cerevisiae. Curr Genet. 1992 Mar;21(3):183–189. doi: 10.1007/BF00336839. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Plevani P., Foiani M., Valsasnini P., Badaracco G., Cheriathundam E., Chang L. M. Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex. J Biol Chem. 1985 Jun 10;260(11):7102–7107. [PubMed] [Google Scholar]
  29. Pringle J. R., Preston R. A., Adams A. E., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. doi: 10.1016/s0091-679x(08)61620-9. [DOI] [PubMed] [Google Scholar]
  30. Richardson H., Lew D. J., Henze M., Sugimoto K., Reed S. I. Cyclin-B homologs in Saccharomyces cerevisiae function in S phase and in G2. Genes Dev. 1992 Nov;6(11):2021–2034. doi: 10.1101/gad.6.11.2021. [DOI] [PubMed] [Google Scholar]
  31. Roberts J. M. Turning DNA replication on and off. Curr Opin Cell Biol. 1993 Apr;5(2):201–206. doi: 10.1016/0955-0674(93)90103-w. [DOI] [PubMed] [Google Scholar]
  32. Santocanale C., Foiani M., Lucchini G., Plevani P. The isolated 48,000-dalton subunit of yeast DNA primase is sufficient for RNA primer synthesis. J Biol Chem. 1993 Jan 15;268(2):1343–1348. [PubMed] [Google Scholar]
  33. 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]
  34. Verma R., Smiley J., Andrews B., Campbell J. L. Regulation of the yeast DNA replication genes through the Mlu I cell cycle box is dependent on SWI6. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9479–9483. doi: 10.1073/pnas.89.20.9479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Virshup D. M., Russo A. A., Kelly T. J. Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A. Mol Cell Biol. 1992 Nov;12(11):4883–4895. doi: 10.1128/mcb.12.11.4883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wahl A. F., Geis A. M., Spain B. H., Wong S. W., Korn D., Wang T. S. Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle. Mol Cell Biol. 1988 Nov;8(11):5016–5025. doi: 10.1128/mcb.8.11.5016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wang T. S. Eukaryotic DNA polymerases. Annu Rev Biochem. 1991;60:513–552. doi: 10.1146/annurev.bi.60.070191.002501. [DOI] [PubMed] [Google Scholar]
  38. Waseem N. H., Labib K., Nurse P., Lane D. P. Isolation and analysis of the fission yeast gene encoding polymerase delta accessory protein PCNA. EMBO J. 1992 Dec;11(13):5111–5120. doi: 10.1002/j.1460-2075.1992.tb05618.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES