Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 Oct 1;16(19):5988–5997. doi: 10.1093/emboj/16.19.5988

Is the yeast anaphase promoting complex needed to prevent re-replication during G2 and M phases?

S Pichler 1, S Piatti 1, K Nasmyth 1
PMCID: PMC1170229  PMID: 9312056

Abstract

The Anaphase Promoting Complex (APC) is required for anaphase progression and B-type cyclin proteolysis. The recent finding that inactivation of the APC allows 'over-replication' of DNA has led to the proposal that the APC might also be required for preventing reduplication of chromosomes during G2 and M phases. In this report we re-investigate the phenotype of apc mutant cells and find that they do not re-replicate their DNA during the period taken for wild-type cells to traverse G2 and M phases. apc mutants do, however, gradually increase their DNA content after long periods of cell cycle arrest. Such DNA synthesis occurs almost exclusively in the cytoplasm and neither occurs in cells lacking mitochondrial DNA nor depends on Cdc6, a protein which is essential for the initiation of chromosomal but not mitochondrial DNA replication. ARS1, a chromosomal replication origin, is not re-fired in cells deprived of APC function, confirming that the 'over-replicated' DNA in apc mutant cells is of mitochondrial origin. Furthermore, we find that APC function is required to promote but not to prevent re-replication in ndc10 mutant cells. We therefore propose that the APC is not involved in preventing re-duplication of chromosomes during G2 and M phases.

Full Text

The Full Text of this article is available as a PDF (690.0 KB).

Selected References

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

  1. Amon A., Irniger S., Nasmyth K. Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell. 1994 Jul 1;77(7):1037–1050. doi: 10.1016/0092-8674(94)90443-x. [DOI] [PubMed] [Google Scholar]
  2. Anachkova B., Hamlin J. L. Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element. Mol Cell Biol. 1989 Feb;9(2):532–540. doi: 10.1128/mcb.9.2.532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Basi G., Draetta G. p13suc1 of Schizosaccharomyces pombe regulates two distinct forms of the mitotic cdc2 kinase. Mol Cell Biol. 1995 Apr;15(4):2028–2036. doi: 10.1128/mcb.15.4.2028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baum P., Yip C., Goetsch L., Byers B. A yeast gene essential for regulation of spindle pole duplication. Mol Cell Biol. 1988 Dec;8(12):5386–5397. doi: 10.1128/mcb.8.12.5386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Booher R. N., Alfa C. E., Hyams J. S., Beach D. H. The fission yeast cdc2/cdc13/suc1 protein kinase: regulation of catalytic activity and nuclear localization. Cell. 1989 Aug 11;58(3):485–497. doi: 10.1016/0092-8674(89)90429-7. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Cohen-Fix O., Peters J. M., Kirschner M. W., Koshland D. Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev. 1996 Dec 15;10(24):3081–3093. doi: 10.1101/gad.10.24.3081. [DOI] [PubMed] [Google Scholar]
  9. Dahmann C., Diffley J. F., Nasmyth K. A. S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state. Curr Biol. 1995 Nov 1;5(11):1257–1269. doi: 10.1016/s0960-9822(95)00252-1. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Doheny K. F., Sorger P. K., Hyman A. A., Tugendreich S., Spencer F., Hieter P. Identification of essential components of the S. cerevisiae kinetochore. Cell. 1993 May 21;73(4):761–774. doi: 10.1016/0092-8674(93)90255-O. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Engle D. B., Osmani S. A., Osmani A. H., Rosborough S., Xin X. N., Morris N. R. A negative regulator of mitosis in Aspergillus is a putative membrane-spanning protein. J Biol Chem. 1990 Sep 25;265(27):16132–16137. [PubMed] [Google Scholar]
  13. 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]
  14. Fisher D. L., Nurse P. A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins. EMBO J. 1996 Feb 15;15(4):850–860. [PMC free article] [PubMed] [Google Scholar]
  15. Fitch I., Dahmann C., Surana U., Amon A., Nasmyth K., Goetsch L., Byers B., Futcher B. Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae. Mol Biol Cell. 1992 Jul;3(7):805–818. doi: 10.1091/mbc.3.7.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Funabiki H., Yamano H., Kumada K., Nagao K., Hunt T., Yanagida M. Cut2 proteolysis required for sister-chromatid seperation in fission yeast. Nature. 1996 May 30;381(6581):438–441. doi: 10.1038/381438a0. [DOI] [PubMed] [Google Scholar]
  17. Glotzer M., Murray A. W., Kirschner M. W. Cyclin is degraded by the ubiquitin pathway. Nature. 1991 Jan 10;349(6305):132–138. doi: 10.1038/349132a0. [DOI] [PubMed] [Google Scholar]
  18. Goh P. Y., Kilmartin J. V. NDC10: a gene involved in chromosome segregation in Saccharomyces cerevisiae. J Cell Biol. 1993 May;121(3):503–512. doi: 10.1083/jcb.121.3.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Heichman K. A. Cdc6 and DNA replication: limited to humble origins. Bioessays. 1996 Nov;18(11):859–862. doi: 10.1002/bies.950181103. [DOI] [PubMed] [Google Scholar]
  21. Heichman K. A., Roberts J. M. The yeast CDC16 and CDC27 genes restrict DNA replication to once per cell cycle. Cell. 1996 Apr 5;85(1):39–48. doi: 10.1016/s0092-8674(00)81080-6. [DOI] [PubMed] [Google Scholar]
  22. Hirano T., Hiraoka Y., Yanagida M. A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase. J Cell Biol. 1988 Apr;106(4):1171–1183. doi: 10.1083/jcb.106.4.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hirano T., Kinoshita N., Morikawa K., Yanagida M. Snap helix with knob and hole: essential repeats in S. pombe nuclear protein nuc2+. Cell. 1990 Jan 26;60(2):319–328. doi: 10.1016/0092-8674(90)90746-2. [DOI] [PubMed] [Google Scholar]
  24. Irniger S., Piatti S., Michaelis C., Nasmyth K. Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast. Cell. 1995 Apr 21;81(2):269–278. doi: 10.1016/0092-8674(95)90337-2. [DOI] [PubMed] [Google Scholar]
  25. Jackson P. K., Chevalier S., Philippe M., Kirschner M. W. Early events in DNA replication require cyclin E and are blocked by p21CIP1. J Cell Biol. 1995 Aug;130(4):755–769. doi: 10.1083/jcb.130.4.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jiang W., Lechner J., Carbon J. Isolation and characterization of a gene (CBF2) specifying a protein component of the budding yeast kinetochore. J Cell Biol. 1993 May;121(3):513–519. doi: 10.1083/jcb.121.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Juang Y. L., Huang J., Peters J. M., McLaughlin M. E., Tai C. Y., Pellman D. APC-mediated proteolysis of Ase1 and the morphogenesis of the mitotic spindle. Science. 1997 Feb 28;275(5304):1311–1314. doi: 10.1126/science.275.5304.1311. [DOI] [PubMed] [Google Scholar]
  28. King R. W., Deshaies R. J., Peters J. M., Kirschner M. W. How proteolysis drives the cell cycle. Science. 1996 Dec 6;274(5293):1652–1659. doi: 10.1126/science.274.5293.1652. [DOI] [PubMed] [Google Scholar]
  29. King R. W., Peters J. M., Tugendreich S., Rolfe M., Hieter P., Kirschner M. W. A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B. Cell. 1995 Apr 21;81(2):279–288. doi: 10.1016/0092-8674(95)90338-0. [DOI] [PubMed] [Google Scholar]
  30. Neff M. W., Burke D. J. Random segregation of chromatids at mitosis in Saccharomyces cerevisiae. Genetics. 1991 Mar;127(3):463–473. doi: 10.1093/genetics/127.3.463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nurse P. Ordering S phase and M phase in the cell cycle. Cell. 1994 Nov 18;79(4):547–550. doi: 10.1016/0092-8674(94)90539-8. [DOI] [PubMed] [Google Scholar]
  32. O'Donnell K. L., Osmani A. H., Osmani S. A., Morris N. R. bimA encodes a member of the tetratricopeptide repeat family of proteins and is required for the completion of mitosis in Aspergillus nidulans. J Cell Sci. 1991 Aug;99(Pt 4):711–719. doi: 10.1242/jcs.99.4.711. [DOI] [PubMed] [Google Scholar]
  33. Ohtsubo M., Theodoras A. M., Schumacher J., Roberts J. M., Pagano M. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol Cell Biol. 1995 May;15(5):2612–2624. doi: 10.1128/mcb.15.5.2612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Peters J. M., King R. W., Hög C., Kirschner M. W. Identification of BIME as a subunit of the anaphase-promoting complex. Science. 1996 Nov 15;274(5290):1199–1201. doi: 10.1126/science.274.5290.1199. [DOI] [PubMed] [Google Scholar]
  35. Piatti S., Böhm T., Cocker J. H., Diffley J. F., Nasmyth K. Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast. Genes Dev. 1996 Jun 15;10(12):1516–1531. doi: 10.1101/gad.10.12.1516. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Samejima I., Yanagida M. Bypassing anaphase by fission yeast cut9 mutation: requirement of cut9+ to initiate anaphase. J Cell Biol. 1994 Dec;127(6 Pt 1):1655–1670. doi: 10.1083/jcb.127.6.1655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schwob E., Böhm T., Mendenhall M. D., Nasmyth K. The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae. Cell. 1994 Oct 21;79(2):233–244. doi: 10.1016/0092-8674(94)90193-7. [DOI] [PubMed] [Google Scholar]
  39. Schwob E., Nasmyth K. CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae. Genes Dev. 1993 Jul;7(7A):1160–1175. doi: 10.1101/gad.7.7a.1160. [DOI] [PubMed] [Google Scholar]
  40. Starborg M., Brundell E., Gell K., Hög C. A novel murine gene encoding a 216-kDa protein is related to a mitotic checkpoint regulator previously identified in Aspergillus nidulans. J Biol Chem. 1994 Sep 30;269(39):24133–24137. [PubMed] [Google Scholar]
  41. Strausfeld U. P., Howell M., Descombes P., Chevalier S., Rempel R. E., Adamczewski J., Maller J. L., Hunt T., Blow J. J. Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts. J Cell Sci. 1996 Jun;109(Pt 6):1555–1563. doi: 10.1242/jcs.109.6.1555. [DOI] [PubMed] [Google Scholar]
  42. Surana U., Amon A., Dowzer C., McGrew J., Byers B., Nasmyth K. Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. EMBO J. 1993 May;12(5):1969–1978. doi: 10.1002/j.1460-2075.1993.tb05846.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Surana U., Robitsch H., Price C., Schuster T., Fitch I., Futcher A. B., Nasmyth K. The role of CDC28 and cyclins during mitosis in the budding yeast S. cerevisiae. Cell. 1991 Apr 5;65(1):145–161. doi: 10.1016/0092-8674(91)90416-v. [DOI] [PubMed] [Google Scholar]
  44. Vassilev L., Johnson E. M. Mapping initiation sites of DNA replication in vivo using polymerase chain reaction amplification of nascent strand segments. Nucleic Acids Res. 1989 Oct 11;17(19):7693–7705. doi: 10.1093/nar/17.19.7693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wuarin J., Nurse P. Regulating S phase: CDKs, licensing and proteolysis. Cell. 1996 Jun 14;85(6):785–787. doi: 10.1016/s0092-8674(00)81261-1. [DOI] [PubMed] [Google Scholar]
  46. Zachariae W., Nasmyth K. TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast. Mol Biol Cell. 1996 May;7(5):791–801. doi: 10.1091/mbc.7.5.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zachariae W., Shin T. H., Galova M., Obermaier B., Nasmyth K. Identification of subunits of the anaphase-promoting complex of Saccharomyces cerevisiae. Science. 1996 Nov 15;274(5290):1201–1204. doi: 10.1126/science.274.5290.1201. [DOI] [PubMed] [Google Scholar]

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

RESOURCES