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. 1997 Jun;17(6):3315–3322. doi: 10.1128/mcb.17.6.3315

Differential requirements for DNA replication in the activation of mitotic checkpoints in Saccharomyces cerevisiae.

P A Tavormina 1, Y Wang 1, D J Burke 1
PMCID: PMC232184  PMID: 9154830

Abstract

Checkpoints prevent inaccurate chromosome segregation by inhibiting cell division when errors in mitotic processes are encountered. We used a temperature-sensitive mutation, dbf4, to examine the requirement for DNA replication in establishing mitotic checkpoint arrest. We used gamma-irradiation to induce DNA damage and hydroxyurea to limit deoxyribonucleotides in cells deprived of DBF4 function to investigate the requirement for DNA replication in DNA-responsive checkpoints. In the absence of DNA replication, mitosis was not inhibited by these treatments, which normally activate the DNA damage and DNA replication checkpoints. Our results support a model that indicates that the assembly of replication structures is critical for cells to respond to defects in DNA metabolism. We show that activating the spindle checkpoint with nocodazole does not require prior progression through S phase but does require a stable kinetochore.

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Selected References

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  1. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amon A., Surana U., Muroff I., Nasmyth K. Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S. cerevisiae. Nature. 1992 Jan 23;355(6358):368–371. doi: 10.1038/355368a0. [DOI] [PubMed] [Google Scholar]
  3. Araki H., Leem S. H., Phongdara A., Sugino A. Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11791–11795. doi: 10.1073/pnas.92.25.11791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen R. H., Waters J. C., Salmon E. D., Murray A. W. Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores. Science. 1996 Oct 11;274(5285):242–246. doi: 10.1126/science.274.5285.242. [DOI] [PubMed] [Google Scholar]
  5. Cocker J. H., Piatti S., Santocanale C., Nasmyth K., Diffley J. F. An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast. Nature. 1996 Jan 11;379(6561):180–182. doi: 10.1038/379180a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. D'Urso G., Grallert B., Nurse P. DNA polymerase alpha, a component of the replication initiation complex, is essential for the checkpoint coupling S phase to mitosis in fission yeast. J Cell Sci. 1995 Sep;108(Pt 9):3109–3118. doi: 10.1242/jcs.108.9.3109. [DOI] [PubMed] [Google Scholar]
  8. Deshaies R. J. Phosphorylation and proteolysis: partners in the regulation of cell division in budding yeast. Curr Opin Genet Dev. 1997 Feb;7(1):7–16. doi: 10.1016/s0959-437x(97)80103-7. [DOI] [PubMed] [Google Scholar]
  9. Diffley J. F. Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells. Genes Dev. 1996 Nov 15;10(22):2819–2830. doi: 10.1101/gad.10.22.2819. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Dowell S. J., Romanowski P., Diffley J. F. Interaction of Dbf4, the Cdc7 protein kinase regulatory subunit, with yeast replication origins in vivo. Science. 1994 Aug 26;265(5176):1243–1246. doi: 10.1126/science.8066465. [DOI] [PubMed] [Google Scholar]
  12. Elledge S. J. Cell cycle checkpoints: preventing an identity crisis. Science. 1996 Dec 6;274(5293):1664–1672. doi: 10.1126/science.274.5293.1664. [DOI] [PubMed] [Google Scholar]
  13. Enoch T., Nurse P. Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication. Cell. 1990 Feb 23;60(4):665–673. doi: 10.1016/0092-8674(90)90669-6. [DOI] [PubMed] [Google Scholar]
  14. Gorbsky G. J., Ricketts W. A. Differential expression of a phosphoepitope at the kinetochores of moving chromosomes. J Cell Biol. 1993 Sep;122(6):1311–1321. doi: 10.1083/jcb.122.6.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guacci V., Hogan E., Koshland D. Chromosome condensation and sister chromatid pairing in budding yeast. J Cell Biol. 1994 May;125(3):517–530. doi: 10.1083/jcb.125.3.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hardwick K. G., Weiss E., Luca F. C., Winey M., Murray A. W. Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption. Science. 1996 Aug 16;273(5277):953–956. doi: 10.1126/science.273.5277.953. [DOI] [PubMed] [Google Scholar]
  17. Hartwell L. H. Macromolecule synthesis in temperature-sensitive mutants of yeast. J Bacteriol. 1967 May;93(5):1662–1670. doi: 10.1128/jb.93.5.1662-1670.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hartwell L. H., Weinert T. A. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989 Nov 3;246(4930):629–634. doi: 10.1126/science.2683079. [DOI] [PubMed] [Google Scholar]
  19. Hofmann J. F., Beach D. cdt1 is an essential target of the Cdc10/Sct1 transcription factor: requirement for DNA replication and inhibition of mitosis. EMBO J. 1994 Jan 15;13(2):425–434. doi: 10.1002/j.1460-2075.1994.tb06277.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Holloway S. L., Glotzer M., King R. W., Murray A. W. Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor. Cell. 1993 Jul 2;73(7):1393–1402. doi: 10.1016/0092-8674(93)90364-v. [DOI] [PubMed] [Google Scholar]
  21. Hoyt M. A., Totis L., Roberts B. T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell. 1991 Aug 9;66(3):507–517. doi: 10.1016/0092-8674(81)90014-3. [DOI] [PubMed] [Google Scholar]
  22. Hyman A. A., Sorger P. K. Structure and function of kinetochores in budding yeast. Annu Rev Cell Dev Biol. 1995;11:471–495. doi: 10.1146/annurev.cb.11.110195.002351. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Jacobs C. W., Adams A. E., Szaniszlo P. J., Pringle J. R. Functions of microtubules in the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1988 Oct;107(4):1409–1426. doi: 10.1083/jcb.107.4.1409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jang J. K., Messina L., Erdman M. B., Arbel T., Hawley R. S. Induction of metaphase arrest in Drosophila oocytes by chiasma-based kinetochore tension. Science. 1995 Jun 30;268(5219):1917–1919. doi: 10.1126/science.7604267. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. Leatherwood J., Lopez-Girona A., Russell P. Interaction of Cdc2 and Cdc18 with a fission yeast ORC2-like protein. Nature. 1996 Jan 25;379(6563):360–363. doi: 10.1038/379360a0. [DOI] [PubMed] [Google Scholar]
  30. Lew D. J., Kornbluth S. Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control. Curr Opin Cell Biol. 1996 Dec;8(6):795–804. doi: 10.1016/s0955-0674(96)80080-9. [DOI] [PubMed] [Google Scholar]
  31. Li J. J., Deshaies R. J. Exercising self-restraint: discouraging illicit acts of S and M in eukaryotes. Cell. 1993 Jul 30;74(2):223–226. doi: 10.1016/0092-8674(93)90413-k. [DOI] [PubMed] [Google Scholar]
  32. Li R., Murray A. W. Feedback control of mitosis in budding yeast. Cell. 1991 Aug 9;66(3):519–531. doi: 10.1016/0092-8674(81)90015-5. [DOI] [PubMed] [Google Scholar]
  33. Li X., Nicklas R. B. Mitotic forces control a cell-cycle checkpoint. Nature. 1995 Feb 16;373(6515):630–632. doi: 10.1038/373630a0. [DOI] [PubMed] [Google Scholar]
  34. Li Y., Benezra R. Identification of a human mitotic checkpoint gene: hsMAD2. Science. 1996 Oct 11;274(5285):246–248. doi: 10.1126/science.274.5285.246. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Lydall D., Weinert T. From DNA damage to cell cycle arrest and suicide: a budding yeast perspective. Curr Opin Genet Dev. 1996 Feb;6(1):4–11. doi: 10.1016/s0959-437x(96)90003-9. [DOI] [PubMed] [Google Scholar]
  37. Lydall D., Weinert T. Yeast checkpoint genes in DNA damage processing: implications for repair and arrest. Science. 1995 Dec 1;270(5241):1488–1491. doi: 10.1126/science.270.5241.1488. [DOI] [PubMed] [Google Scholar]
  38. Navas T. A., Zhou Z., Elledge S. J. DNA polymerase epsilon links the DNA replication machinery to the S phase checkpoint. Cell. 1995 Jan 13;80(1):29–39. doi: 10.1016/0092-8674(95)90448-4. [DOI] [PubMed] [Google Scholar]
  39. Nicklas R. B., Ward S. C., Gorbsky G. J. Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint. J Cell Biol. 1995 Aug;130(4):929–939. doi: 10.1083/jcb.130.4.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pangilinan F., Spencer F. Abnormal kinetochore structure activates the spindle assembly checkpoint in budding yeast. Mol Biol Cell. 1996 Aug;7(8):1195–1208. doi: 10.1091/mbc.7.8.1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Paulovich A. G., Hartwell L. H. A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage. Cell. 1995 Sep 8;82(5):841–847. doi: 10.1016/0092-8674(95)90481-6. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Rhind N., Furnari B., Russell P. Cdc2 tyrosine phosphorylation is required for the DNA damage checkpoint in fission yeast. Genes Dev. 1997 Feb 15;11(4):504–511. doi: 10.1101/gad.11.4.504. [DOI] [PubMed] [Google Scholar]
  45. Rieder C. L., Cole R. W., Khodjakov A., Sluder G. The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. J Cell Biol. 1995 Aug;130(4):941–948. doi: 10.1083/jcb.130.4.941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rieder C. L., Schultz A., Cole R., Sluder G. Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle. J Cell Biol. 1994 Dec;127(5):1301–1310. doi: 10.1083/jcb.127.5.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rudner A. D., Murray A. W. The spindle assembly checkpoint. Curr Opin Cell Biol. 1996 Dec;8(6):773–780. doi: 10.1016/s0955-0674(96)80077-9. [DOI] [PubMed] [Google Scholar]
  48. Saka Y., Fantes P., Sutani T., McInerny C., Creanor J., Yanagida M. Fission yeast cut5 links nuclear chromatin and M phase regulator in the replication checkpoint control. EMBO J. 1994 Nov 15;13(22):5319–5329. doi: 10.1002/j.1460-2075.1994.tb06866.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Saka Y., Yanagida M. Fission yeast cut5+, required for S phase onset and M phase restraint, is identical to the radiation-damage repair gene rad4+. Cell. 1993 Jul 30;74(2):383–393. doi: 10.1016/0092-8674(93)90428-s. [DOI] [PubMed] [Google Scholar]
  50. Sorger P. K., Doheny K. F., Hieter P., Kopski K. M., Huffaker T. C., Hyman A. A. Two genes required for the binding of an essential Saccharomyces cerevisiae kinetochore complex to DNA. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12026–12030. doi: 10.1073/pnas.92.26.12026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sorger P. K., Murray A. W. S-phase feedback control in budding yeast independent of tyrosine phosphorylation of p34cdc28. Nature. 1992 Jan 23;355(6358):365–368. doi: 10.1038/355365a0. [DOI] [PubMed] [Google Scholar]
  52. Straight A. F., Belmont A. S., Robinett C. C., Murray A. W. GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr Biol. 1996 Dec 1;6(12):1599–1608. doi: 10.1016/s0960-9822(02)70783-5. [DOI] [PubMed] [Google Scholar]
  53. Sudakin V., Ganoth D., Dahan A., Heller H., Hershko J., Luca F. C., Ruderman J. V., Hershko A. The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. Mol Biol Cell. 1995 Feb;6(2):185–197. doi: 10.1091/mbc.6.2.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sugimoto K., Shimomura T., Hashimoto K., Araki H., Sugino A., Matsumoto K. Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7048–7052. doi: 10.1073/pnas.93.14.7048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. 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]
  57. Tomkiel J., Cooke C. A., Saitoh H., Bernat R. L., Earnshaw W. C. CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase. J Cell Biol. 1994 May;125(3):531–545. doi: 10.1083/jcb.125.3.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Toyn J. H., Johnson A. L., Johnston L. H. Segregation of unreplicated chromosomes in Saccharomyces cerevisiae reveals a novel G1/M-phase checkpoint. Mol Cell Biol. 1995 Oct;15(10):5312–5321. doi: 10.1128/mcb.15.10.5312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tugendreich S., Tomkiel J., Earnshaw W., Hieter P. CDC27Hs colocalizes with CDC16Hs to the centrosome and mitotic spindle and is essential for the metaphase to anaphase transition. Cell. 1995 Apr 21;81(2):261–268. doi: 10.1016/0092-8674(95)90336-4. [DOI] [PubMed] [Google Scholar]
  60. Wang Y., Burke D. J. Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint in Saccharomyces cerevisiae. Mol Cell Biol. 1997 Feb;17(2):620–626. doi: 10.1128/mcb.17.2.620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wang Y., Burke D. J. Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1995 Dec;15(12):6838–6844. doi: 10.1128/mcb.15.12.6838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Weinert T. A., Hartwell L. H. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science. 1988 Jul 15;241(4863):317–322. doi: 10.1126/science.3291120. [DOI] [PubMed] [Google Scholar]
  63. Weiss E., Winey M. The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint. J Cell Biol. 1996 Jan;132(1-2):111–123. doi: 10.1083/jcb.132.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wells W. A. The spindle-assembly checkpoint: aiming for a perfect mitosis, every time. Trends Cell Biol. 1996 Jun;6(6):228–234. doi: 10.1016/0962-8924(96)10018-0. [DOI] [PubMed] [Google Scholar]
  65. Yamamoto A., Guacci V., Koshland D. Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s). J Cell Biol. 1996 Apr;133(1):99–110. doi: 10.1083/jcb.133.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. 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]
  67. 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]

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