Abstract
Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair alpha-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor.
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- Biggins S., Severin F. F., Bhalla N., Sassoon I., Hyman A. A., Murray A. W. The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast. Genes Dev. 1999 Mar 1;13(5):532–544. doi: 10.1101/gad.13.5.532. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bishop A. C., Ubersax J. A., Petsch D. T., Matheos D. P., Gray N. S., Blethrow J., Shimizu E., Tsien J. Z., Schultz P. G., Rose M. D. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature. 2000 Sep 21;407(6802):395–401. doi: 10.1038/35030148. [DOI] [PubMed] [Google Scholar]
- Brown M. T., Goetsch L., Hartwell L. H. MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae. J Cell Biol. 1993 Oct;123(2):387–403. doi: 10.1083/jcb.123.2.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burgess S., Couto J. R., Guthrie C. A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing. Cell. 1990 Mar 9;60(5):705–717. doi: 10.1016/0092-8674(90)90086-t. [DOI] [PubMed] [Google Scholar]
- Cheng S. C., Tarn W. Y., Tsao T. Y., Abelson J. PRP19: a novel spliceosomal component. Mol Cell Biol. 1993 Mar;13(3):1876–1882. doi: 10.1128/mcb.13.3.1876. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ciosk R., Shirayama M., Shevchenko A., Tanaka T., Toth A., Shevchenko A., Nasmyth K. Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol Cell. 2000 Feb;5(2):243–254. doi: 10.1016/s1097-2765(00)80420-7. [DOI] [PubMed] [Google Scholar]
- Ciosk R., Zachariae W., Michaelis C., Shevchenko A., Mann M., Nasmyth K. An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell. 1998 Jun 12;93(6):1067–1076. doi: 10.1016/s0092-8674(00)81211-8. [DOI] [PubMed] [Google Scholar]
- Francisco L., Wang W., Chan C. S. Type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation. Mol Cell Biol. 1994 Jul;14(7):4731–4740. doi: 10.1128/mcb.14.7.4731. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Freeman L., Aragon-Alcaide L., Strunnikov A. The condensin complex governs chromosome condensation and mitotic transmission of rDNA. J Cell Biol. 2000 May 15;149(4):811–824. doi: 10.1083/jcb.149.4.811. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Gardner R. D., Burke D. J. The spindle checkpoint: two transitions, two pathways. Trends Cell Biol. 2000 Apr;10(4):154–158. doi: 10.1016/s0962-8924(00)01727-x. [DOI] [PubMed] [Google Scholar]
- Guacci V., Koshland D., Strunnikov A. A direct link between sister chromatid cohesion and chromosome condensation revealed through the analysis of MCD1 in S. cerevisiae. Cell. 1997 Oct 3;91(1):47–57. doi: 10.1016/s0092-8674(01)80008-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hardwick K. G., Murray A. W. Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast. J Cell Biol. 1995 Nov;131(3):709–720. doi: 10.1083/jcb.131.3.709. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hartman T., Stead K., Koshland D., Guacci V. Pds5p is an essential chromosomal protein required for both sister chromatid cohesion and condensation in Saccharomyces cerevisiae. J Cell Biol. 2000 Oct 30;151(3):613–626. doi: 10.1083/jcb.151.3.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirano T. Chromosome cohesion, condensation, and separation. Annu Rev Biochem. 2000;69:115–144. doi: 10.1146/annurev.biochem.69.1.115. [DOI] [PubMed] [Google Scholar]
- Hirano T., Kobayashi R., Hirano M. Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell. 1997 May 16;89(4):511–521. doi: 10.1016/s0092-8674(00)80233-0. [DOI] [PubMed] [Google Scholar]
- Hirano T., Mitchison T. J. A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro. Cell. 1994 Nov 4;79(3):449–458. doi: 10.1016/0092-8674(94)90254-2. [DOI] [PubMed] [Google Scholar]
- Hollingsworth R. E., Jr, Sclafani R. A. DNA metabolism gene CDC7 from yeast encodes a serine (threonine) protein kinase. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6272–6276. doi: 10.1073/pnas.87.16.6272. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jensen S., Segal M., Clarke D. J., Reed S. I. A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1. J Cell Biol. 2001 Jan 8;152(1):27–40. doi: 10.1083/jcb.152.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson E. S., Blobel G. Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins. J Cell Biol. 1999 Nov 29;147(5):981–994. doi: 10.1083/jcb.147.5.981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lavoie B. D., Tuffo K. M., Oh S., Koshland D., Holm C. Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren. Mol Biol Cell. 2000 Apr;11(4):1293–1304. doi: 10.1091/mbc.11.4.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lechner J., Carbon J. A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell. 1991 Feb 22;64(4):717–725. doi: 10.1016/0092-8674(91)90501-o. [DOI] [PubMed] [Google Scholar]
- Loidl J., Nairz K., Klein F. Meiotic chromosome synapsis in a haploid yeast. Chromosoma. 1991 May;100(4):221–228. doi: 10.1007/BF00344155. [DOI] [PubMed] [Google Scholar]
- Mahajan R., Delphin C., Guan T., Gerace L., Melchior F. A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell. 1997 Jan 10;88(1):97–107. doi: 10.1016/s0092-8674(00)81862-0. [DOI] [PubMed] [Google Scholar]
- Matunis M. J., Coutavas E., Blobel G. A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol. 1996 Dec;135(6 Pt 1):1457–1470. doi: 10.1083/jcb.135.6.1457. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meluh P. B., Koshland D. Budding yeast centromere composition and assembly as revealed by in vivo cross-linking. Genes Dev. 1997 Dec 15;11(24):3401–3412. doi: 10.1101/gad.11.24.3401. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meluh P. B., Koshland D. Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. Mol Biol Cell. 1995 Jul;6(7):793–807. doi: 10.1091/mbc.6.7.793. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meluh P. B., Yang P., Glowczewski L., Koshland D., Smith M. M. Cse4p is a component of the core centromere of Saccharomyces cerevisiae. Cell. 1998 Sep 4;94(5):607–613. doi: 10.1016/s0092-8674(00)81602-5. [DOI] [PubMed] [Google Scholar]
- Michaelis C., Ciosk R., Nasmyth K. Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell. 1997 Oct 3;91(1):35–45. doi: 10.1016/s0092-8674(01)80007-6. [DOI] [PubMed] [Google Scholar]
- Nasmyth K., Peters J. M., Uhlmann F. Splitting the chromosome: cutting the ties that bind sister chromatids. Science. 2000 May 26;288(5470):1379–1385. doi: 10.1126/science.288.5470.1379. [DOI] [PubMed] [Google Scholar]
- Ouspenski I. I., Cabello O. A., Brinkley B. R. Chromosome condensation factor Brn1p is required for chromatid separation in mitosis. Mol Biol Cell. 2000 Apr;11(4):1305–1313. doi: 10.1091/mbc.11.4.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pidoux A. L., Allshire R. C. Centromeres: getting a grip of chromosomes. Curr Opin Cell Biol. 2000 Jun;12(3):308–319. doi: 10.1016/s0955-0674(00)00094-6. [DOI] [PubMed] [Google Scholar]
- Saitoh H., Pu R. T., Dasso M. SUMO-1: wrestling with a new ubiquitin-related modifier. Trends Biochem Sci. 1997 Oct;22(10):374–376. doi: 10.1016/s0968-0004(97)01102-x. [DOI] [PubMed] [Google Scholar]
- Schmidt C. L., Grey M., Schmidt M., Brendel M., Henriques J. A. Allelism of Saccharomyces cerevisiae genes PSO6, involved in survival after 3-CPs+UVA induced damage, and ERG3, encoding the enzyme sterol C-5 desaturase. Yeast. 1999 Oct;15(14):1503–1510. doi: 10.1002/(sici)1097-0061(199910)15:14<1503::aid-yea481>3.3.co;2-n. [DOI] [PubMed] [Google Scholar]
- Skibbens R. V., Corson L. B., Koshland D., Hieter P. Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery. Genes Dev. 1999 Feb 1;13(3):307–319. doi: 10.1101/gad.13.3.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Stemmann O., Lechner J. The Saccharomyces cerevisiae kinetochore contains a cyclin-CDK complexing homologue, as identified by in vitro reconstitution. EMBO J. 1996 Jul 15;15(14):3611–3620. [PMC free article] [PubMed] [Google Scholar]
- Stoler S., Keith K. C., Curnick K. E., Fitzgerald-Hayes M. A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis. Genes Dev. 1995 Mar 1;9(5):573–586. doi: 10.1101/gad.9.5.573. [DOI] [PubMed] [Google Scholar]
- 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]
- Strunnikov A. V., Hogan E., Koshland D. SMC2, a Saccharomyces cerevisiae gene essential for chromosome segregation and condensation, defines a subgroup within the SMC family. Genes Dev. 1995 Mar 1;9(5):587–599. doi: 10.1101/gad.9.5.587. [DOI] [PubMed] [Google Scholar]
- Takahashi Y., Iwase M., Konishi M., Tanaka M., Toh-e A., Kikuchi Y. Smt3, a SUMO-1 homolog, is conjugated to Cdc3, a component of septin rings at the mother-bud neck in budding yeast. Biochem Biophys Res Commun. 1999 Jun 16;259(3):582–587. doi: 10.1006/bbrc.1999.0821. [DOI] [PubMed] [Google Scholar]
- Tóth A., Ciosk R., Uhlmann F., Galova M., Schleiffer A., Nasmyth K. Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication. Genes Dev. 1999 Feb 1;13(3):320–333. doi: 10.1101/gad.13.3.320. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uhlmann F., Lottspeich F., Nasmyth K. Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature. 1999 Jul 1;400(6739):37–42. doi: 10.1038/21831. [DOI] [PubMed] [Google Scholar]
- Uhlmann F., Wernic D., Poupart M. A., Koonin E. V., Nasmyth K. Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell. 2000 Oct 27;103(3):375–386. doi: 10.1016/s0092-8674(00)00130-6. [DOI] [PubMed] [Google Scholar]
- Winzeler E. A., Shoemaker D. D., Astromoff A., Liang H., Anderson K., Andre B., Bangham R., Benito R., Boeke J. D., Bussey H. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science. 1999 Aug 6;285(5429):901–906. doi: 10.1126/science.285.5429.901. [DOI] [PubMed] [Google Scholar]