Abstract
We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.
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- Aboussekhra A., Biggerstaff M., Shivji M. K., Vilpo J. A., Moncollin V., Podust V. N., Protić M., Hübscher U., Egly J. M., Wood R. D. Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell. 1995 Mar 24;80(6):859–868. doi: 10.1016/0092-8674(95)90289-9. [DOI] [PubMed] [Google Scholar]
- Bennett C. B., Lewis A. L., Baldwin K. K., Resnick M. A. Lethality induced by a single site-specific double-strand break in a dispensable yeast plasmid. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5613–5617. doi: 10.1073/pnas.90.12.5613. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blank A., Kim B., Loeb L. A. DNA polymerase delta is required for base excision repair of DNA methylation damage in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):9047–9051. doi: 10.1073/pnas.91.19.9047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bohr V. A. Gene specific DNA repair. Carcinogenesis. 1991 Nov;12(11):1983–1992. doi: 10.1093/carcin/12.11.1983. [DOI] [PubMed] [Google Scholar]
- Budd M. E., Campbell J. L. DNA polymerases required for repair of UV-induced damage in Saccharomyces cerevisiae. Mol Cell Biol. 1995 Apr;15(4):2173–2179. doi: 10.1128/mcb.15.4.2173. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Budd M. E., Wittrup K. D., Bailey J. E., Campbell J. L. DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):365–376. doi: 10.1128/mcb.9.2.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burgers P. M. Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon. J Biol Chem. 1991 Nov 25;266(33):22698–22706. [PubMed] [Google Scholar]
- Carr A. M., Hoekstra M. F. The cellular responses to DNA damage. Trends Cell Biol. 1995 Jan;5(1):32–40. doi: 10.1016/s0962-8924(00)88934-5. [DOI] [PubMed] [Google Scholar]
- Cullmann G., Fien K., Kobayashi R., Stillman B. Characterization of the five replication factor C genes of Saccharomyces cerevisiae. Mol Cell Biol. 1995 Sep;15(9):4661–4671. doi: 10.1128/mcb.15.9.4661. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Datta A., Jinks-Robertson S. Association of increased spontaneous mutation rates with high levels of transcription in yeast. Science. 1995 Jun 16;268(5217):1616–1619. doi: 10.1126/science.7777859. [DOI] [PubMed] [Google Scholar]
- Der C. J., Pan B. T., Cooper G. M. rasH mutants deficient in GTP binding. Mol Cell Biol. 1986 Sep;6(9):3291–3294. doi: 10.1128/mcb.6.9.3291. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eki T., Matsumoto T., Murakami Y., Hurwitz J. The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems. J Biol Chem. 1992 Apr 15;267(11):7284–7294. [PubMed] [Google Scholar]
- Fien K., Stillman B. Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex. Mol Cell Biol. 1992 Jan;12(1):155–163. doi: 10.1128/mcb.12.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Freifelder D. Molecular weights of coliphages and coliphage DNA. IV. Molecular weights of DNA from bacteriophages T4, T5 and T7 and the general problem of determination of M. J Mol Biol. 1970 Dec 28;54(3):567–577. doi: 10.1016/0022-2836(70)90127-0. [DOI] [PubMed] [Google Scholar]
- Green M. H., Medcalf A. S., Arlett C. F., Harcourt S. A., Lehmann A. R. DNA strand breakage caused by dichlorvos, methyl methanesulphonate and iodoacetamide in Escherichia coli and cultured Chinese hamster cells. Mutat Res. 1974 Sep;24(3):365–378. doi: 10.1016/0027-5107(74)90181-x. [DOI] [PubMed] [Google Scholar]
- Hartwell L. H., Mortimer R. K., Culotti J., Culotti M. Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants. Genetics. 1973 Jun;74(2):267–286. doi: 10.1093/genetics/74.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Henkin T. M., Sonenshein A. L. Mutations of the Escherichia coli lacUV5 promoter resulting in increased expression in Bacillus subtilis. Mol Gen Genet. 1987 Oct;209(3):467–474. doi: 10.1007/BF00331151. [DOI] [PubMed] [Google Scholar]
- Holm C., Goto T., Wang J. C., Botstein D. DNA topoisomerase II is required at the time of mitosis in yeast. Cell. 1985 Jun;41(2):553–563. doi: 10.1016/s0092-8674(85)80028-3. [DOI] [PubMed] [Google Scholar]
- Howell E. A., McAlear M. A., Rose D., Holm C. CDC44: a putative nucleotide-binding protein required for cell cycle progression that has homology to subunits of replication factor C. Mol Cell Biol. 1994 Jan;14(1):255–267. doi: 10.1128/mcb.14.1.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hutter K. J., Eipel H. E. Microbial determinations by flow cytometry. J Gen Microbiol. 1979 Aug;113(2):369–375. doi: 10.1099/00221287-113-2-369. [DOI] [PubMed] [Google Scholar]
- 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]
- Jarvik J., Botstein D. A genetic method for determining the order of events in a biological pathway. Proc Natl Acad Sci U S A. 1973 Jul;70(7):2046–2050. doi: 10.1073/pnas.70.7.2046. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones J. S., Prakash L. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers. Yeast. 1990 Sep-Oct;6(5):363–366. doi: 10.1002/yea.320060502. [DOI] [PubMed] [Google Scholar]
- Kong X. P., Onrust R., O'Donnell M., Kuriyan J. Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell. 1992 May 1;69(3):425–437. doi: 10.1016/0092-8674(92)90445-i. [DOI] [PubMed] [Google Scholar]
- Krishna T. S., Kong X. P., Gary S., Burgers P. M., Kuriyan J. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell. 1994 Dec 30;79(7):1233–1243. doi: 10.1016/0092-8674(94)90014-0. [DOI] [PubMed] [Google Scholar]
- Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- Kunz B. A., Peters M. G., Kohalmi S. E., Armstrong J. D., Glattke M., Badiani K. Disruption of the RAD52 gene alters the spectrum of spontaneous SUP4-o mutations in Saccharomyces cerevisiae. Genetics. 1989 Jul;122(3):535–542. doi: 10.1093/genetics/122.3.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee S. H., Hurwitz J. Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5672–5676. doi: 10.1073/pnas.87.15.5672. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee S. H., Ishimi Y., Kenny M. K., Bullock P., Dean F. B., Hurwitz J. An inhibitor of the in vitro elongation reaction of simian virus 40 DNA replication is overcome by proliferating-cell nuclear antigen. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9469–9473. doi: 10.1073/pnas.85.24.9469. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee S. H., Kwong A. D., Pan Z. Q., Hurwitz J. Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta. J Biol Chem. 1991 Jan 5;266(1):594–602. [PubMed] [Google Scholar]
- Li R., Waga S., Hannon G. J., Beach D., Stillman B. Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature. 1994 Oct 6;371(6497):534–537. doi: 10.1038/371534a0. [DOI] [PubMed] [Google Scholar]
- Loeb L. A., Preston B. D. Mutagenesis by apurinic/apyrimidinic sites. Annu Rev Genet. 1986;20:201–230. doi: 10.1146/annurev.ge.20.120186.001221. [DOI] [PubMed] [Google Scholar]
- Matsumoto Y., Kim K., Bogenhagen D. F. Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair. Mol Cell Biol. 1994 Sep;14(9):6187–6197. doi: 10.1128/mcb.14.9.6187. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McAlear M. A., Howell E. A., Espenshade K. K., Holm C. Proliferating cell nuclear antigen (pol30) mutations suppress cdc44 mutations and identify potential regions of interaction between the two encoded proteins. Mol Cell Biol. 1994 Jul;14(7):4390–4397. doi: 10.1128/mcb.14.7.4390. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morrison A., Sugino A. The 3'-->5' exonucleases of both DNA polymerases delta and epsilon participate in correcting errors of DNA replication in Saccharomyces cerevisiae. Mol Gen Genet. 1994 Feb;242(3):289–296. doi: 10.1007/BF00280418. [DOI] [PubMed] [Google Scholar]
- Podust L. M., Podust V. N., Sogo J. M., Hübscher U. Mammalian DNA polymerase auxiliary proteins: analysis of replication factor C-catalyzed proliferating cell nuclear antigen loading onto circular double-stranded DNA. Mol Cell Biol. 1995 Jun;15(6):3072–3081. doi: 10.1128/mcb.15.6.3072. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ramotar D., Popoff S. C., Gralla E. B., Demple B. Cellular role of yeast Apn1 apurinic endonuclease/3'-diesterase: repair of oxidative and alkylation DNA damage and control of spontaneous mutation. Mol Cell Biol. 1991 Sep;11(9):4537–4544. doi: 10.1128/mcb.11.9.4537. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shivji K. K., Kenny M. K., Wood R. D. Proliferating cell nuclear antigen is required for DNA excision repair. Cell. 1992 Apr 17;69(2):367–374. doi: 10.1016/0092-8674(92)90416-a. [DOI] [PubMed] [Google Scholar]
- Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
- Spell R. M., Holm C. Nature and distribution of chromosomal intertwinings in Saccharomyces cerevisiae. Mol Cell Biol. 1994 Feb;14(2):1465–1476. doi: 10.1128/mcb.14.2.1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsurimoto T., Melendy T., Stillman B. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature. 1990 Aug 9;346(6284):534–539. doi: 10.1038/346534a0. [DOI] [PubMed] [Google Scholar]
- Tsurimoto T., Stillman B. Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro. Mol Cell Biol. 1989 Feb;9(2):609–619. doi: 10.1128/mcb.9.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsurimoto T., Stillman B. Replication factors required for SV40 DNA replication in vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins. J Biol Chem. 1991 Jan 25;266(3):1950–1960. [PubMed] [Google Scholar]
- Valencia A., Chardin P., Wittinghofer A., Sander C. The ras protein family: evolutionary tree and role of conserved amino acids. Biochemistry. 1991 May 14;30(19):4637–4648. doi: 10.1021/bi00233a001. [DOI] [PubMed] [Google Scholar]
- Xiao W., Samson L. In vivo evidence for endogenous DNA alkylation damage as a source of spontaneous mutation in eukaryotic cells. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2117–2121. doi: 10.1073/pnas.90.6.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yoder B. L., Burgers P. M. Saccharomyces cerevisiae replication factor C. I. Purification and characterization of its ATPase activity. J Biol Chem. 1991 Nov 25;266(33):22689–22697. [PubMed] [Google Scholar]