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Reduced dosage of a single fission yeast MCM protein causes genetic instability and S phase delay
D.T. Liang, J.A. Hodson, S.L. Forsburg
Journal of Cell Science 1999 112: 559-567;

Summary

MCM proteins are a conserved family of eukaryotic replication factors implicated in the initiation of DNA replication and in the discrimination between replicated and unreplicated chromatin. However, most mcm mutants in yeast arrest the cell cycle after bulk DNA synthesis has occurred. We investigated the basis for this late S phase arrest by analyzing the effects of a temperature-sensitive mutation in fission yeast cdc19(+)(mcm2(+)). cdc19-P1 cells show a dramatic loss of viability at the restrictive temperature, which is not typical of all S phase mutants. The cdc19-P1 cell cycle arrest requires an intact damage-response checkpoint and is accompanied by increased rates of chromosome loss and mitotic recombination. Chromosomes from cdc19-P1 cells migrate aberrantly in pulsed-field gels, typical of strains arrested with unresolved replication intermediates. The cdc19-P1 mutation reduces the level of the Cdc19 protein at all temperatures. We compared the effects of disruptions of cdc19(+)(mcm2(+)), cdc21(+)(mcm4(+)), nda4(+)(mcm5(+)) and mis5(+)(mcm6(+)); in all cases, the null mutants underwent delayed S phase but were unable to proceed through the cell cycle. Examination of protein levels suggests that this delayed S phase reflects limiting, but not absent, MCM proteins. Thus, reduced dosage of MCM proteins allows replication initiation, but is insufficient for completion of S phase and cell cycle progression.

REFERENCES

    1. Aparicio O. M.,
    2. Weinstein D. M. and
    3. Bell S. P.
    (1997). Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91, 59–69
    OpenUrlCrossRefPubMedWeb of Science
    1. Bodi Z.,
    2. Gysler-Junker A. and
    3. Kohli J.
    (1991). A quantitative assay to measure chromosome stability in Schizosaccharomyces pombe. Mol. Gen. Genet 229, 77–80
    OpenUrlPubMed
    1. Caligiuri M. and
    2. Beach D.
    (1993). Sct1 functions in partnership with Cdc10 in a transcription complex that activates cell cycle START and inhibits differentiation. Cell 72, 607–619
    OpenUrlCrossRefPubMedWeb of Science
    1. Carpenter P. B.,
    2. Mueller P. R. and
    3. Dunphy W. G.
    (1996). Role for a Xenopus Orc2-related protein in controlling DNA replication. Nature 379, 357–360
    OpenUrlCrossRefPubMed
    1. Chong J. P. J.,
    2. Mahbubani H. M.,
    3. Khoo C.-Y. and
    4. Blow J. J.
    (1995). Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature 375, 418–421
    OpenUrlCrossRefPubMed
    1. Chong J. P. J.,
    2. Thommes P. and
    3. Blow J. J.
    (1996). The role of MCM/P1 proteins in the licensing of DNA replication. Trends Biochem. Sci 21, 102–106
    OpenUrlCrossRefPubMedWeb of Science
    1. Chu G.,
    2. Vollrath D. and
    3. Davis R. W.
    (1986). Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234, 1582–1585
    OpenUrlAbstract/FREE Full Text
    1. Coleman T. R.,
    2. Carpenter P. B. and
    3. Dunphy W. G.
    (1996). The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts. Cell 87, 53–64
    OpenUrlCrossRefPubMedWeb of Science
    1. Coxon A.,
    2. Maundrell K. and
    3. Kearsey S. E.
    (1992). Fission yeast cdc21+ belongs to a family of proteins involved in an early step of chromosome replication. Nucl. Acids Res 20, 5571–5577
    OpenUrlAbstract/FREE Full Text
    1. D'Urso G.,
    2. Grallert B. and
    3. Nurse P.
    (1995). 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 108, 3109–3118
    OpenUrlAbstract/FREE Full Text
    1. D'Urso G. and
    2. Nurse P.
    (1997). Schizosaccharomyces pombe cdc20+ encodes DNA polymerase epsilon and is required for chromosomal replication but not for the S phase checkpoint. Proc. Natl. Acad. Sci. USA 94, 12491–12496
    OpenUrlAbstract/FREE Full Text
    1. Donovan S.,
    2. Harwood J.,
    3. Drury L. S. and
    4. Diffley J. F. X.
    (1997). Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc. Natl. Acad. Sci. USA 94, 5611–5616
    OpenUrlAbstract/FREE Full Text
    1. Ekwall K.,
    2. Javerzat J. P.,
    3. Lorentz A.,
    4. Schmidt H.,
    5. Cranston G. and
    6. Allshire R.
    (1995). The chromodomain protein Swi6—a key component at fission yeast centromeres. Science 269, 1429–1431
    OpenUrlAbstract/FREE Full Text
    1. Enoch T. and
    2. Nurse P.
    (1990). Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication. Cell 60, 665–673
    OpenUrlCrossRefPubMedWeb of Science
    1. Fernandez Sarabia M.-J.,
    2. McInerny C.,
    3. Harris P.,
    4. Gordon C. and
    5. Fantes P.
    (1993). The cell cycle genes cdc22 +and suc22 +of the fission yeast Schizosaccharomyces pombe encode the large and small subunits of ribonucleotide reductase. Mol. Gen. Genet 238, 241–251
    OpenUrlPubMedWeb of Science
    1. Forsburg S. L. and
    2. Nurse P.
    (1994). The fission yeast cdc19 +gene encodes a member of the MCM family of replication proteins. J. Cell Sci 107, 2779–2788
    OpenUrlAbstract/FREE Full Text
    1. Forsburg S. L.,
    2. Sherman D. A.,
    3. Ottilie S.,
    4. Yasuda J. R. and
    5. Hodson J. A.
    (1997). Mutational analysis of Cdc19p, a Schizosaccharomyces pombe MCM protein. Genetics 147, 1025–1041
    OpenUrlAbstract/FREE Full Text
    1. Francesconi S.,
    2. De Recondo A. M. and
    3. Baldacci G.
    (1995). DNA polymerase delta is required for the replication feedback control of cell cycle progression in Schizosaccharomyces pombe. Mol. Gen. Genet 246, 561–569
    OpenUrlCrossRefPubMedWeb of Science
    1. Francesconi S.,
    2. Park H. and
    3. Wang T. S. F.
    (1993). Fission yeast with DNA polymerase delta temperature sensitive alleles exhibits cell division cycle phenotype. Nucl. Acids Res 21, 3821–3828
    OpenUrlAbstract/FREE Full Text
    1. Gould K. L.,
    2. Burns C. G.,
    3. Feoktistova A.,
    4. Hu C.-P.,
    5. Pasion S. G. and
    6. Forsburg S. L.
    (1998). Fission yeast cdc24+ encodes a novel replication factor required for chromosome integrity. Genetics 149, 1221–1233
    OpenUrlAbstract/FREE Full Text
    1. Grallert B. and
    2. Nurse P.
    (1996). The ORC1 homolog orp1 in fission yeast plays a key role in regulating onset of S phase. Genes Dev 10, 2644–2654
    OpenUrlAbstract/FREE Full Text
    1. Hennessy K. M.,
    2. Lee A.,
    3. Chen E. and
    4. Botstein D.
    (1991). A group of interacting yeast DNA replication genes. Genes Dev 5, 958–969
    OpenUrlAbstract/FREE Full Text
    1. Iino Y. and
    2. Yamamoto M.
    (1997). The Schizosaccharomyces pombe cdc6 gene encodes the catalytic subunit of DNA polymerase delta. Mol. Gen. Genet 254, 93–97
    OpenUrlCrossRefPubMedWeb of Science
    1. Johnston L.,
    2. Barker D. and
    3. Nurse P.
    (1986). Molecular cloning of the Schizosaccharomyces pombe DNA ligase gene cdc17 and an associated sequence promoting high frequency plasmid transformation. Gene 41, 321–325
    OpenUrlCrossRefPubMedWeb of Science
    1. Kearsey S. E. and
    2. Labib K.
    (1998). MCM proteins: evolution, properties, and role in DNA replication. Biochim Biophys Acta 1398, 113–136
    OpenUrlPubMed
    1. Kelly T. J.,
    2. Martin G. S.,
    3. Forsburg S. L.,
    4. Stephen R. J.,
    5. Russo A. and
    6. Nurse P.
    (1993). The fission yeast cdc18 +gene product couples S phase to START and mitosis. Cell 74, 371–382
    OpenUrlCrossRefPubMedWeb of Science
    1. Kubota Y.,
    2. Mimura S.,
    3. Nishimoto S.-I.,
    4. Takisawa H. and
    5. Nojima H.
    (1995). Identification of the yeast MCM3 related protein as a component of Xenopus DNA replication licensing factor. Cell 81, 601–609
    OpenUrlCrossRefPubMedWeb of Science
    1. Lei M.,
    2. Kawasaki Y. and
    3. Tye B. K.
    (1996). Physical interactions among MCM proteins and effects of MCM dosage on DNA replication in Saccharomyces cerevisiae. Mol. Cell. Biol 16, 5081–5090
    OpenUrlAbstract/FREE Full Text
    1. Lindsay H. D.,
    2. Griffiths D. J. F.,
    3. Edwards R. J.,
    4. Christensen P. U.,
    5. Murray J. M.,
    6. Osman F.,
    7. Walworth N. and
    8. Carr A. M.
    (1998). S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe. Genes Dev 12, 382–395
    OpenUrlAbstract/FREE Full Text
    1. Lorentz A.,
    2. Ostermann K.,
    3. Fleck O. and
    4. Schmidt H.
    (1994). Switching gene swi6, involved in repression of silent mating-type loci in fission yeast, encodes a homolog of chromatin-associated proteins from drosophila and mammals. Gene 143, 139–143
    OpenUrlCrossRefPubMedWeb of Science
    1. Madine M. A.,
    2. Khoo C.-Y.,
    3. Mills A. D. and
    4. Laskey R. A.
    (1995). MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells. Nature 375, 421–424
    OpenUrlCrossRefPubMed
    1. Maiorano D.,
    2. Blom van Assendelft G. and
    3. Kearsey S. E.
    (1996). Fission yeast cdc21, a member of the MCM protein family, is required for onset ofS phase and located in the nucleus throughout the cell cycle. EMBO J 15, 861–872
    OpenUrlPubMedWeb of Science
    1. Miyake S.,
    2. Okishio N.,
    3. Samejima I.,
    4. Hiraoka Y.,
    5. Toda T.,
    6. Saitoh I. and
    7. Yanagida M.
    (1993). Fission yeast genes nda1 +and nda4 +, mutations of which lead to S-phase block, chromatin alteration and Ca2+suppression, are members of the CDC46 / MCM2 family. Mol. Biol. Cell 4, 1003–1015
    OpenUrlAbstract/FREE Full Text
    1. Moreno S.,
    2. Klar A. and
    3. Nurse P.
    (1991). Molecular genetic analysis of the fission yeast Schizosaccharomyces pombe. Methods Enzymol 194, 795–823
    OpenUrlCrossRefPubMedWeb of Science
    1. Nasmyth K.
    (1977). Temperature-sensitive lethal mutants in the structural gene for DNA ligase in the yeast Schizosaccharomyces pombe. Cell 12, 1109–1120
    OpenUrlCrossRefPubMedWeb of Science
    1. Nasmyth K. and
    2. Nurse P.
    (1981). Cell division cycle mutants altered in DNA replication and mitosis in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet 182, 119–124
    OpenUrlCrossRefPubMedWeb of Science
    1. Nurse P.,
    2. Thuriaux P. and
    3. Nasmyth K.
    (1976). Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet 146, 167–178
    OpenUrlCrossRefPubMedWeb of Science
    1. Ohi R.,
    2. Feoktistova A. and
    3. Gould K. L.
    (1996). Construction of vectors and a genomic library for use with his3 deficient strains of Schizosaccharomyces pombe. Gene 174, 315–318
    OpenUrlCrossRefPubMedWeb of Science
    1. Okishio N.,
    2. Adachi Y. and
    3. Yanagida M.
    (1996). Fission yeast nda1 and nda4, MCM homologs required for DNA replication, are constitutive nuclear proteins. J. Cell Sci 109, 319–326
    OpenUrlAbstract/FREE Full Text
    1. Rhind N. and
    2. Russell P.
    (1998). The Schizosaccharomyces pombe S-phase checkpoint differentiates between different types of DNA damage. Genetics 149, 1729–1737
    OpenUrlAbstract/FREE Full Text
    1. Romanowski P.,
    2. Madine M. A.,
    3. Rowles A.,
    4. Blow J. J. and
    5. Laskey R. A.
    (1996). The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin. Curr. Biol 6, 1416–1425
    OpenUrlCrossRefPubMedWeb of Science
    1. Rowles A. and
    2. Blow J. J.
    (1997). Chromatin proteins involved in the initiation of DNA replication. Curr. Opin. Genet. Dev 7, 152–157
    OpenUrlCrossRefPubMedWeb of Science
    1. Rowles A.,
    2. Chong J. P. J.,
    3. Brown L.,
    4. Howell M.,
    5. Evan G. I. and
    6. Blow J. J.
    (1996). Interaction between the origin recognition complex and the replication licensing system in Xenopus. Cell 87, 287–296
    OpenUrlCrossRefPubMedWeb of Science
    1. Sazer S. and
    2. Sherwood S. W.
    (1990). Mitochondrial growth and DNA synthesis occur in the absence of nuclear DNA replication in fission yeast. J. Cell Sci 97, 509–516
    OpenUrlAbstract/FREE Full Text
    1. Sherman D. A. and
    2. Forsburg S. L.
    (1998). Schizosaccharomyces pombe Mcm3p, an essential nuclear protein, associates tightly with Nda4p (Mcm5p). Nucl. Acids Res 26, 3955–3960
    OpenUrlAbstract/FREE Full Text
    1. Sherman D. A.,
    2. Pasion S. G. and
    3. Forsburg S. L.
    (1998). Fission yeast Cdc19p (MCM2) associates with a core complex containing Cdc21p (MCM4) and Mis5p (MCM6). Mol. Biol. Cell 9, 1833–1845
    OpenUrlAbstract/FREE Full Text
    1. Sinha P.,
    2. Chang V. and
    3. Tye B. K.
    (1986). A mutant that affects the function of autonomously replicating sequences in yeast. J. Mol. Biol 192, 805–814
    OpenUrlCrossRefPubMedWeb of Science
    1. Stewart E.,
    2. Chapman C. R.,
    3. Al-Khodairy F.,
    4. Carr A. M. and
    5. Enoch T.
    (1997). rqh1+, a fission yeast gene related to the Bloom's and Werner's syndrome genes, is required for reversible S phase arrest. EMBO J 16, 2682–2692
    OpenUrlAbstract
    1. Takahashi K.,
    2. Yamada H. and
    3. Yanagida M.
    (1994). Fission yeast minichromosome loss mutants mis cause lethal aneuploidy and replication abnormality. Mol. Biol. Cell 5, 1145–1158
    OpenUrlAbstract/FREE Full Text
    1. Tanaka K.,
    2. Okazaki K.,
    3. Okazaki N.,
    4. Ueda T.,
    5. Sugiyama A.,
    6. Nojima H. and
    7. Okayama H.
    (1992). A new cdc gene required for S phase entry of Schizosaccharomyces pombe encodes a protein similar to the cdc10 +and SWI4 gene products. EMBO J 11, 4923–4932
    OpenUrlPubMedWeb of Science
    1. Tanaka T.,
    2. Knapp D. and
    3. Nasmyth K.
    (1997). Loading of an MCM protein onto DNA replication origins is regulated by Cdc6p and CDKs. Cell 90, 649–660
    OpenUrlCrossRefPubMedWeb of Science
    1. Toda T.,
    2. Nakaseko Y.,
    3. Niwa O. and
    4. Yanagida M.
    (1984). Mapping of rDNA genes by integration of hybrid plasmids in Schizosaccharomyces pombe. Curr. Genet 8, 93–97
    OpenUrlCrossRefPubMed
    1. Waseem N. H.,
    2. Labib K.,
    3. Nurse P. and
    4. Lane D. P.
    (1992). Isolation and analysis of the fission yeast gene encoding polymeraseaccessory protein PCNA. EMBO J 11, 5111–5120
    OpenUrlPubMedWeb of Science
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Journal Articles
Reduced dosage of a single fission yeast MCM protein causes genetic instability and S phase delay
D.T. Liang, J.A. Hodson, S.L. Forsburg
Journal of Cell Science 1999 112: 559-567;
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