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. 1996 Feb 20;93(4):1566–1570. doi: 10.1073/pnas.93.4.1566

cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe.

M Muzi Falconi 1, G W Brown 1, T J Kelly 1
PMCID: PMC39981  PMID: 8643672

Abstract

In the fission yeast Schizosaccharomyces pombe the cdc18'+gene is required both for initiation of DNA replication and for coupling mitosis to the completion of S phase. Cells lacking Cdc18 fail to enter S phase but still undergo nuclear division. Expression of cdc18+ is sufficient to drive a G1-arrested cdc10ts mutant into the S phase of the cell cycle, indicating that cdc18+ represents a critical link between passage through START and the initiation of DNA replication. Here we show that Cdcl8 is a highly unstable protein that is expressed only once per cell cycle at the boundary between GI and S phase. De novo synthesis of Cdc18 is required before, but not after, the initiation of DNA replication, indicating that Cdc18 function is not necessary once the initiation event has occurred. Overproduction of the protein results in an accumulation of cells with DNA content of greater than 2C and delays mitosis, suggesting that Cdc18 is sufficient to cause reinitiation of DNA replication within a given cell cycle. Our data indicate that the synthesis of Cdc18 protein is a critical rate-limiting step in the initiation of DNA replication during each cell cycle. The extreme lability of the protein may contribute to the prevention of reinitiation.

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Selected References

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

  1. Basi G., Schmid E., Maundrell K. TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene. 1993 Jan 15;123(1):131–136. doi: 10.1016/0378-1119(93)90552-e. [DOI] [PubMed] [Google Scholar]
  2. Bell S. P., Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature. 1992 May 14;357(6374):128–134. doi: 10.1038/357128a0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Broek D., Bartlett R., Crawford K., Nurse P. Involvement of p34cdc2 in establishing the dependency of S phase on mitosis. Nature. 1991 Jan 31;349(6308):388–393. doi: 10.1038/349388a0. [DOI] [PubMed] [Google Scholar]
  5. Caligiuri M., Beach D. Sct1 functions in partnership with Cdc10 in a transcription complex that activates cell cycle START and inhibits differentiation. Cell. 1993 Feb 26;72(4):607–619. doi: 10.1016/0092-8674(93)90079-6. [DOI] [PubMed] [Google Scholar]
  6. Coverley D., Laskey R. A. Regulation of eukaryotic DNA replication. Annu Rev Biochem. 1994;63:745–776. doi: 10.1146/annurev.bi.63.070194.003525. [DOI] [PubMed] [Google Scholar]
  7. Diffley J. F., Cocker J. H., Dowell S. J., Rowley A. Two steps in the assembly of complexes at yeast replication origins in vivo. Cell. 1994 Jul 29;78(2):303–316. doi: 10.1016/0092-8674(94)90299-2. [DOI] [PubMed] [Google Scholar]
  8. Diffley J. F., Cocker J. H. Protein-DNA interactions at a yeast replication origin. Nature. 1992 May 14;357(6374):169–172. doi: 10.1038/357169a0. [DOI] [PubMed] [Google Scholar]
  9. Erdile L. F., Collins K. L., Russo A., Simancek P., Small D., Umbricht C., Virshup D., Cheng L., Randall S., Weinberg D. Initiation of SV40 DNA replication: mechanism and control. Cold Spring Harb Symp Quant Biol. 1991;56:303–313. doi: 10.1101/sqb.1991.056.01.037. [DOI] [PubMed] [Google Scholar]
  10. Hayles J., Fisher D., Woollard A., Nurse P. Temporal order of S phase and mitosis in fission yeast is determined by the state of the p34cdc2-mitotic B cyclin complex. Cell. 1994 Sep 9;78(5):813–822. doi: 10.1016/s0092-8674(94)90542-8. [DOI] [PubMed] [Google Scholar]
  11. Kelly T. J., Martin G. S., Forsburg S. L., Stephen R. J., Russo A., Nurse P. The fission yeast cdc18+ gene product couples S phase to START and mitosis. Cell. 1993 Jul 30;74(2):371–382. doi: 10.1016/0092-8674(93)90427-r. [DOI] [PubMed] [Google Scholar]
  12. Kelly T. J., Nurse P., Forsburg S. L. Coupling DNA replication to the cell cycle. Cold Spring Harb Symp Quant Biol. 1993;58:637–644. doi: 10.1101/sqb.1993.058.01.071. [DOI] [PubMed] [Google Scholar]
  13. Li J. J., Herskowitz I. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science. 1993 Dec 17;262(5141):1870–1874. doi: 10.1126/science.8266075. [DOI] [PubMed] [Google Scholar]
  14. Liang C., Weinreich M., Stillman B. ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome. Cell. 1995 Jun 2;81(5):667–676. doi: 10.1016/0092-8674(95)90528-6. [DOI] [PubMed] [Google Scholar]
  15. Lowndes N. F., McInerny C. J., Johnson A. L., Fantes P. A., Johnston L. H. Control of DNA synthesis genes in fission yeast by the cell-cycle gene cdc10+. Nature. 1992 Jan 30;355(6359):449–453. doi: 10.1038/355449a0. [DOI] [PubMed] [Google Scholar]
  16. Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
  17. Micklem G., Rowley A., Harwood J., Nasmyth K., Diffley J. F. Yeast origin recognition complex is involved in DNA replication and transcriptional silencing. Nature. 1993 Nov 4;366(6450):87–89. doi: 10.1038/366087a0. [DOI] [PubMed] [Google Scholar]
  18. Moreno S., Hayles J., Nurse P. Regulation of p34cdc2 protein kinase during mitosis. Cell. 1989 Jul 28;58(2):361–372. doi: 10.1016/0092-8674(89)90850-7. [DOI] [PubMed] [Google Scholar]
  19. Moreno S., Klar A., Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. [DOI] [PubMed] [Google Scholar]
  20. Moreno S., Labib K., Correa J., Nurse P. Regulation of the cell cycle timing of Start in fission yeast by the rum1+ gene. J Cell Sci Suppl. 1994;18:63–68. doi: 10.1242/jcs.1994.supplement_18.9. [DOI] [PubMed] [Google Scholar]
  21. Moreno S., Nurse P. Regulation of progression through the G1 phase of the cell cycle by the rum1+ gene. Nature. 1994 Jan 20;367(6460):236–242. doi: 10.1038/367236a0. [DOI] [PubMed] [Google Scholar]
  22. Muzi Falconi M., Piseri A., Ferrari M., Lucchini G., Plevani P., Foiani M. 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. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10519–10523. doi: 10.1073/pnas.90.22.10519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Muzi-Falconi M., Kelly T. J. Orp1, a member of the Cdc18/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12475–12479. doi: 10.1073/pnas.92.26.12475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Niman H. L., Houghten R. A., Walker L. E., Reisfeld R. A., Wilson I. A., Hogle J. M., Lerner R. A. Generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4949–4953. doi: 10.1073/pnas.80.16.4949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nishitani H., Nurse P. p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast. Cell. 1995 Nov 3;83(3):397–405. doi: 10.1016/0092-8674(95)90117-5. [DOI] [PubMed] [Google Scholar]
  26. Piatti S., Lengauer C., Nasmyth K. Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a 'reductional' anaphase in the budding yeast Saccharomyces cerevisiae. EMBO J. 1995 Aug 1;14(15):3788–3799. doi: 10.1002/j.1460-2075.1995.tb00048.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rao P. N., Johnson R. T. Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis. Nature. 1970 Jan 10;225(5228):159–164. doi: 10.1038/225159a0. [DOI] [PubMed] [Google Scholar]
  28. Reymond A., Marks J., Simanis V. The activity of S.pombe DSC-1-like factor is cell cycle regulated and dependent on the activity of p34cdc2. EMBO J. 1993 Nov;12(11):4325–4334. doi: 10.1002/j.1460-2075.1993.tb06117.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sazer S., Sherwood S. W. Mitochondrial growth and DNA synthesis occur in the absence of nuclear DNA replication in fission yeast. J Cell Sci. 1990 Nov;97(Pt 3):509–516. doi: 10.1242/jcs.97.3.509. [DOI] [PubMed] [Google Scholar]
  30. Su T. T., Follette P. J., O'Farrell P. H. Qualifying for the license to replicate. Cell. 1995 Jun 16;81(6):825–828. doi: 10.1016/0092-8674(95)90000-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tyers M., Tokiwa G., Nash R., Futcher B. The Cln3-Cdc28 kinase complex of S. cerevisiae is regulated by proteolysis and phosphorylation. EMBO J. 1992 May;11(5):1773–1784. doi: 10.1002/j.1460-2075.1992.tb05229.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang T. A., Li J. J. Eukaryotic DNA replication. Curr Opin Cell Biol. 1995 Jun;7(3):414–420. doi: 10.1016/0955-0674(95)80098-0. [DOI] [PubMed] [Google Scholar]
  33. Woods A., Sherwin T., Sasse R., MacRae T. H., Baines A. J., Gull K. Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J Cell Sci. 1989 Jul;93(Pt 3):491–500. doi: 10.1242/jcs.93.3.491. [DOI] [PubMed] [Google Scholar]

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