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Mol Cell Biol. 1996 Apr; 16(4): 1356–1366.
PMCID: PMC231120

Coordinating DNA replication to produce one copy of the genome requires genes that act in ubiquitin metabolism.

Abstract

We have developed a genetic screen of the yeast Saccharomyces cerevisiae to identify genes that act to coordinate DNA replication so that each part of the genome is copied exactly once per cell cycle. A mutant was recovered in this screen that accumulates aberrantly high DNA contents but does not complete a second round of synthesis. The mutation principally responsible for this phenotype is in the DOA4 gene, which encodes a ubiquitin hydrolase, one of several yeast genes that encode enzymes that can remove the signalling polypeptide ubiquitin hydrolase, one of several yeast genes that encode enzymes that can remove the signaling polypeptide ubiquitin from its covalently linked conjugated forms. DOA4 is nonessential, and deleting this gene causes uncoordinated replication. Overreplication does not occur in cells with limiting amounts of Cdc7 protein kinase, suggesting that entry into S phase is required for this phenotype. The DNA formed in doa4 mutants is not highly unusual in the sense that mitotic recombination rates are normal, implying that a high level of repair is not induced. The temperature sensitivity of doa4 mutations is partially suppressed by extra copies of the polyubiquitin gene UB14, but overreplication still occurs in the presence of this suppressor. Mutations in DOA4 cause loss of the free ubiquitin pool in cells under heat stress conditions, and extra copies of UB14 restore this pool without restoring coordination of replication. We conclude that a ubiquitin-mediated signaling event directly involving the ubiquitin hydrolase encoded by DOA4 is needed in S. cerevisiae to prevent uncoordinated DNA replication.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Baker RT, Tobias JW, Varshavsky A. Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3, and functional analysis of the UBP gene family. J Biol Chem. 1992 Nov 15;267(32):23364–23375. [PubMed]
  • Blow JJ, Laskey RA. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature. 1988 Apr 7;332(6164):546–548. [PubMed]
  • Boeke JD, Trueheart J, Natsoulis G, Fink GR. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. [PubMed]
  • Chen P, Johnson P, Sommer T, Jentsch S, Hochstrasser M. Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MAT alpha 2 repressor. Cell. 1993 Jul 30;74(2):357–369. [PubMed]
  • Chen Y, Hennessy KM, Botstein D, Tye BK. CDC46/MCM5, a yeast protein whose subcellular localization is cell cycle-regulated, is involved in DNA replication at autonomously replicating sequences. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10459–10463. [PMC free article] [PubMed]
  • Chong JP, Mahbubani HM, Khoo CY, Blow JJ. Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature. 1995 Jun 1;375(6530):418–421. [PubMed]
  • Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell. 1994 Oct 7;79(1):13–21. [PubMed]
  • Coverley D, Laskey RA. Regulation of eukaryotic DNA replication. Annu Rev Biochem. 1994;63:745–776. [PubMed]
  • Dahmann C, Diffley JF, Nasmyth KA. 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. [PubMed]
  • Feldmann H, Winnacker EL. A putative homologue of the human autoantigen Ku from Saccharomyces cerevisiae. J Biol Chem. 1993 Jun 15;268(17):12895–12900. [PubMed]
  • Gendimenico GJ, Bouquin PL, Tramposch KM. Diphenylamine-colorimetric method for DNA assay: a shortened procedure by incubating samples at 50 degrees C. Anal Biochem. 1988 Aug 15;173(1):45–48. [PubMed]
  • Gibson SI, Surosky RT, Tye BK. The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication. Mol Cell Biol. 1990 Nov;10(11):5707–5720. [PMC free article] [PubMed]
  • Gietz RD, Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. [PubMed]
  • Glazer AN, Rye HS. Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection. Nature. 1992 Oct 29;359(6398):859–861. [PubMed]
  • Glotzer M, Murray AW, Kirschner MW. Cyclin is degraded by the ubiquitin pathway. Nature. 1991 Jan 10;349(6305):132–138. [PubMed]
  • Greenfeder SA, Newlon CS. A replication map of a 61-kb circular derivative of Saccharomyces cerevisiae chromosome III. Mol Biol Cell. 1992 Sep;3(9):999–1013. [PMC free article] [PubMed]
  • Hand R. Eucaryotic DNA: organization of the genome for replication. Cell. 1978 Oct;15(2):317–325. [PubMed]
  • Handeli S, Weintraub H. The ts41 mutation in Chinese hamster cells leads to successive S phases in the absence of intervening G2, M, and G1. Cell. 1992 Nov 13;71(4):599–611. [PubMed]
  • Hartwell LH. Macromolecule synthesis in temperature-sensitive mutants of yeast. J Bacteriol. 1967 May;93(5):1662–1670. [PMC free article] [PubMed]
  • Hartwell LH, Smith D. Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S. cerevisiae. Genetics. 1985 Jul;110(3):381–395. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Heichman KA, Roberts JM. Rules to replicate by. Cell. 1994 Nov 18;79(4):557–562. [PubMed]
  • Hershko A, Ciechanover A. The ubiquitin system for protein degradation. Annu Rev Biochem. 1992;61:761–807. [PubMed]
  • Hoekstra MF, Seifert HS, Nickoloff J, Heffron F. Shuttle mutagenesis: bacterial transposons for genetic manipulations in yeast. Methods Enzymol. 1991;194:329–342. [PubMed]
  • 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. [PubMed]
  • Jentsch S. The ubiquitin-conjugation system. Annu Rev Genet. 1992;26:179–207. [PubMed]
  • Johnson LM, Snyder M, Chang LM, Davis RW, Campbell JL. Isolation of the gene encoding yeast DNA polymerase I. Cell. 1985 Nov;43(1):369–377. [PubMed]
  • King RW, Peters JM, Tugendreich S, Rolfe M, Hieter P, Kirschner MW. 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. [PubMed]
  • Kubota Y, Mimura S, Nishimoto S, Takisawa H, Nojima H. Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Cell. 1995 May 19;81(4):601–609. [PubMed]
  • Latterich M, Watson MD. Isolation and characterization of osmosensitive vacuolar mutants of Saccharomyces cerevisiae. Mol Microbiol. 1991 Oct;5(10):2417–2426. [PubMed]
  • Lawrence CW. Classical mutagenesis techniques. Methods Enzymol. 1991;194:273–281. [PubMed]
  • Li JJ, 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. [PubMed]
  • Madine MA, Khoo CY, Mills AD, Laskey RA. MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells. Nature. 1995 Jun 1;375(6530):421–424. [PubMed]
  • May GS, McGoldrick CA, Holt CL, Denison SH. The bimB3 mutation of Aspergillus nidulans uncouples DNA replication from the completion of mitosis. J Biol Chem. 1992 Aug 5;267(22):15737–15743. [PubMed]
  • Miles J, Formosa T. Evidence that POB1, a Saccharomyces cerevisiae protein that binds to DNA polymerase alpha, acts in DNA metabolism in vivo. Mol Cell Biol. 1992 Dec;12(12):5724–5735. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Murray A. Cyclin ubiquitination: the destructive end of mitosis. Cell. 1995 Apr 21;81(2):149–152. [PubMed]
  • Newlon CS, Lipchitz LR, Collins I, Deshpande A, Devenish RJ, Green RP, Klein HL, Palzkill TG, Ren RB, Synn S, et al. Analysis of a circular derivative of Saccharomyces cerevisiae chromosome III: a physical map and identification and location of ARS elements. Genetics. 1991 Oct;129(2):343–357. [PMC free article] [PubMed]
  • Nurse P. Ordering S phase and M phase in the cell cycle. Cell. 1994 Nov 18;79(4):547–550. [PubMed]
  • Pagano M, Tam SW, Theodoras AM, Beer-Romero P, Del Sal G, Chau V, Yew PR, Draetta GF, Rolfe M. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science. 1995 Aug 4;269(5224):682–685. [PubMed]
  • Papa FR, Hochstrasser M. The yeast DOA4 gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene. Nature. 1993 Nov 25;366(6453):313–319. [PubMed]
  • Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. [PMC free article] [PubMed]
  • Reynolds AE, McCarroll RM, Newlon CS, Fangman WL. Time of replication of ARS elements along yeast chromosome III. Mol Cell Biol. 1989 Oct;9(10):4488–4494. [PMC free article] [PubMed]
  • Riles L, Dutchik JE, Baktha A, McCauley BK, Thayer EC, Leckie MP, Braden VV, Depke JE, Olson MV. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics. 1993 May;134(1):81–150. [PMC free article] [PubMed]
  • Rivin CJ, Fangman WL. Replication fork rate and origin activation during the S phase of Saccharomyces cerevisiae. J Cell Biol. 1980 Apr;85(1):108–115. [PMC free article] [PubMed]
  • Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. [PubMed]
  • Sclafani RA, Jackson AL. Cdc7 protein kinase for DNA metabolism comes of age. Mol Microbiol. 1994 Mar;11(5):805–810. [PubMed]
  • Shamanski FL, Orr-Weaver TL. The Drosophila plutonium and pan gu genes regulate entry into S phase at fertilization. Cell. 1991 Sep 20;66(6):1289–1300. [PubMed]
  • Shero JH, Koval M, Spencer F, Palmer RE, Hieter P, Koshland D. Analysis of chromosome segregation in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:749–773. [PubMed]
  • Siede W, Friedberg AS, Friedberg EC. RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7985–7989. [PMC free article] [PubMed]
  • Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. [PMC free article] [PubMed]
  • Slonimski PP, Perrodin G, Croft JH. Ethidium bromide induced mutation of yeast mitochondria: complete transformation of cells into respiratory deficient non-chromosomal "petites". Biochem Biophys Res Commun. 1968 Feb 15;30(3):232–239. [PubMed]
  • 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. [PubMed]
  • Tye BK. The MCM2-3-5 proteins: are they replication licensing factors? Trends Cell Biol. 1994 May;4(5):160–166. [PubMed]

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