Abstract
To identify in vivo pathways that compensate for impaired proliferating cell nuclear antigen (PCNA or Pol30p in yeast) activity, we performed a synthetic lethal screen with the yeast pol30-104 mutation. We identified nine mutations that display synthetic lethality with pol30-104; three mutations affected the structural gene for the large subunit of replication factor C (rfc1), which loads PCNA onto DNA, and six mutations affected three members of the RAD52 epistasis group for DNA recombinational repair (rad50, rad52 and rad57). We also found that pol30-104 displayed synthetic lethality with mutations in other members of the RAD52 epistasis group (rad51 and rad54), but not with mutations in members of the RAD3 nor the RAD6 epistasis group. Analysis of nine different pol30 mutations shows that the requirement for the RAD52 pathway is correlated with a DNA replication defect but not with the relative DNA repair defect caused by pol30 mutations. In addition, mutants that require RAD52 for viability (pol30-100, pol30-104, rfc1-1 and rth1delta) accumulate small single-stranded DNA fragments during DNA replication in vivo. Taken together, these data suggest that the RAD52 pathway is required when there are defects in the maturation of Okazaki fragments.
Full Text
The Full Text of this article is available as a PDF (339.4 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Amin N. S., Holm C. In vivo analysis reveals that the interdomain region of the yeast proliferating cell nuclear antigen is important for DNA replication and DNA repair. Genetics. 1996 Oct;144(2):479–493. doi: 10.1093/genetics/144.2.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Asai T., Bates D. B., Kogoma T. DNA replication triggered by double-stranded breaks in E. coli: dependence on homologous recombination functions. Cell. 1994 Sep 23;78(6):1051–1061. doi: 10.1016/0092-8674(94)90279-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ayyagari R., Impellizzeri K. J., Yoder B. L., Gary S. L., Burgers P. M. A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair. Mol Cell Biol. 1995 Aug;15(8):4420–4429. doi: 10.1128/mcb.15.8.4420. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Budd M. E., Campbell J. L. DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Jan;13(1):496–505. doi: 10.1128/mcb.13.1.496-505.1993. [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.9.2.365-376.1989. [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]
- Burgers P. M., Yoder B. L. ATP-independent loading of the proliferating cell nuclear antigen requires DNA ends. J Biol Chem. 1993 Sep 25;268(27):19923–19926. [PubMed] [Google Scholar]
- Cao Y., Kogoma T. The mechanism of recA polA lethality: suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli. Genetics. 1995 Apr;139(4):1483–1494. doi: 10.1093/genetics/139.4.1483. [DOI] [PMC free article] [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.12.1.155-163.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gross J. D., Grunstein J., Witkin E. M. Inviability of recA- derivatives of the DNA polymerase mutant of De Lucia and Cairns. J Mol Biol. 1971 Jun 14;58(2):631–634. doi: 10.1016/0022-2836(71)90377-9. [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]
- Hartwell L. H., Smith D. Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S. cerevisiae. Genetics. 1985 Jul;110(3):381–395. doi: 10.1093/genetics/110.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ishimi Y., Claude A., Bullock P., Hurwitz J. Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J Biol Chem. 1988 Dec 25;263(36):19723–19733. [PubMed] [Google Scholar]
- Johnson R. E., Kovvali G. K., Guzder S. N., Amin N. S., Holm C., Habraken Y., Sung P., Prakash L., Prakash S. Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair. J Biol Chem. 1996 Nov 8;271(45):27987–27990. doi: 10.1074/jbc.271.45.27987. [DOI] [PubMed] [Google Scholar]
- Johnson R. E., Kovvali G. K., Prakash L., Prakash S. Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA. Science. 1995 Jul 14;269(5221):238–240. doi: 10.1126/science.7618086. [DOI] [PubMed] [Google Scholar]
- Johnston L. H., Nasmyth K. A. Saccharomyces cerevisiae cell cycle mutant cdc9 is defective in DNA ligase. Nature. 1978 Aug 31;274(5674):891–893. doi: 10.1038/274891a0. [DOI] [PubMed] [Google Scholar]
- Johnston L. H. The cdc9 ligase joins completed replicons in baker's yeast. Mol Gen Genet. 1983;190(2):315–317. doi: 10.1007/BF00330657. [DOI] [PubMed] [Google Scholar]
- Johnston L. H., Williamson D. H. An alkaline sucrose gradient analysis of the mechanism of nuclear DNA synthesis in the yeast Saccharomyces cerevisiae. Mol Gen Genet. 1978 Aug 17;164(2):217–225. doi: 10.1007/BF00267387. [DOI] [PubMed] [Google Scholar]
- Kranz J. E., Holm C. Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6629–6633. doi: 10.1073/pnas.87.17.6629. [DOI] [PMC free article] [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]
- 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., Pan Z. Q., Kwong A. D., Burgers P. M., Hurwitz J. Synthesis of DNA by DNA polymerase epsilon in vitro. J Biol Chem. 1991 Nov 25;266(33):22707–22717. [PubMed] [Google Scholar]
- Levin D., Hutchinson F. Relation between single-strand DNA mass and sedimentation distance in alkaline sucrose gradients. J Mol Biol. 1973 Apr 15;75(3):495–502. doi: 10.1016/0022-2836(73)90456-7. [DOI] [PubMed] [Google Scholar]
- Li X., Li J., Harrington J., Lieber M. R., Burgers P. M. Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen. J Biol Chem. 1995 Sep 22;270(38):22109–22112. doi: 10.1074/jbc.270.38.22109. [DOI] [PubMed] [Google Scholar]
- Malkova A., Ivanov E. L., Haber J. E. Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication. Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):7131–7136. doi: 10.1073/pnas.93.14.7131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Malone R. E., Hoekstra M. F. Relationships between a hyper-rec mutation (REM1) and other recombination and repair genes in yeast. Genetics. 1984 May;107(1):33–48. doi: 10.1093/genetics/107.1.33. [DOI] [PMC free article] [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.14.9.6187-6197.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McAlear M. A., Tuffo K. M., Holm C. The large subunit of replication factor C (Rfc1p/Cdc44p) is required for DNA replication and DNA repair in Saccharomyces cerevisiae. Genetics. 1996 Jan;142(1):65–78. doi: 10.1093/genetics/142.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Monk M., Kinross J. Conditional lethality of recA and recB derivatives of a strain of Escherichia coli K-12 with a temperature-sensitive deoxyribonucleic acid polymerase I. J Bacteriol. 1972 Mar;109(3):971–978. doi: 10.1128/jb.109.3.971-978.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Montelone B. A., Hoekstra M. F., Malone R. E. Spontaneous mitotic recombination in yeast: the hyper-recombinational rem1 mutations are alleles of the RAD3 gene. Genetics. 1988 Jun;119(2):289–301. doi: 10.1093/genetics/119.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Montelone B. A., Prakash S., Prakash L. Spontaneous mitotic recombination in mms8-1, an allele of the CDC9 gene of Saccharomyces cerevisiae. J Bacteriol. 1981 Aug;147(2):517–525. doi: 10.1128/jb.147.2.517-525.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nichols A. F., Sancar A. Purification of PCNA as a nucleotide excision repair protein. Nucleic Acids Res. 1992 Jul 11;20(13):2441–2446. doi: 10.1093/nar/20.10.2441. [DOI] [PMC free article] [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]
- Podust V. N., Georgaki A., Strack B., Hübscher U. Calf thymus RF-C as an essential component for DNA polymerase delta and epsilon holoenzymes function. Nucleic Acids Res. 1992 Aug 25;20(16):4159–4165. doi: 10.1093/nar/20.16.4159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robzyk K., Kassir Y. A simple and highly efficient procedure for rescuing autonomous plasmids from yeast. Nucleic Acids Res. 1992 Jul 25;20(14):3790–3790. doi: 10.1093/nar/20.14.3790. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
- Saparbaev M., Prakash L., Prakash S. Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae. Genetics. 1996 Mar;142(3):727–736. doi: 10.1093/genetics/142.3.727. [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]
- Tishkoff D. X., Filosi N., Gaida G. M., Kolodner R. D. A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair. Cell. 1997 Jan 24;88(2):253–263. doi: 10.1016/s0092-8674(00)81846-2. [DOI] [PubMed] [Google Scholar]
- Torres-Ramos C. A., Yoder B. L., Burgers P. M., Prakash S., Prakash L. Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9676–9681. doi: 10.1073/pnas.93.18.9676. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tran H. T., Degtyareva N. P., Koloteva N. N., Sugino A., Masumoto H., Gordenin D. A., Resnick M. A. Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes. Mol Cell Biol. 1995 Oct;15(10):5607–5617. doi: 10.1128/mcb.15.10.5607. [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]
- Turchi J. J., Huang L., Murante R. S., Kim Y., Bambara R. A. Enzymatic completion of mammalian lagging-strand DNA replication. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9803–9807. doi: 10.1073/pnas.91.21.9803. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Umar A., Buermeyer A. B., Simon J. A., Thomas D. C., Clark A. B., Liskay R. M., Kunkel T. A. Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis. Cell. 1996 Oct 4;87(1):65–73. doi: 10.1016/s0092-8674(00)81323-9. [DOI] [PubMed] [Google Scholar]
- Wu X., Li J., Li X., Hsieh C. L., Burgers P. M., Lieber M. R. Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA. Nucleic Acids Res. 1996 Jun 1;24(11):2036–2043. doi: 10.1093/nar/24.11.2036. [DOI] [PMC free article] [PubMed] [Google Scholar]
