A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair (original) (raw)

Abstract

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.

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Selected References

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  1. Alani E., Cao L., Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987 Aug;116(4):541–545. doi: 10.1534/genetics.112.541.test. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauer G. A., Burgers P. M. Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene. Nucleic Acids Res. 1990 Jan 25;18(2):261–265. doi: 10.1093/nar/18.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bauer G. A., Burgers P. M. The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen interacts with mammalian DNA polymerase delta. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7506–7510. doi: 10.1073/pnas.85.20.7506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. Cadwell R. C., Joyce G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992 Aug;2(1):28–33. doi: 10.1101/gr.2.1.28. [DOI] [PubMed] [Google Scholar]
  9. Campbell J. L. Yeast DNA replication. J Biol Chem. 1993 Dec 5;268(34):25261–25264. [PubMed] [Google Scholar]
  10. 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]
  11. Flores-Rozas H., Kelman Z., Dean F. B., Pan Z. Q., Harper J. W., Elledge S. J., O'Donnell M., Hurwitz J. Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8655–8659. doi: 10.1073/pnas.91.18.8655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harrington J. J., Lieber M. R. The characterization of a mammalian DNA structure-specific endonuclease. EMBO J. 1994 Mar 1;13(5):1235–1246. doi: 10.1002/j.1460-2075.1994.tb06373.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Impellizzeri K. J., Anderson B., Burgers P. M. The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair. J Bacteriol. 1991 Nov;173(21):6807–6810. doi: 10.1128/jb.173.21.6807-6810.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kohalmi L., Kunz B. A. In vitro mutagenesis of the yeast SUP4-o gene to identify all substitutions that can be detected in vivo with the SUP4-o system. Environ Mol Mutagen. 1992;19(4):282–287. doi: 10.1002/em.2850190404. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Krishna T. S., Fenyö D., Kong X. P., Gary S., Chait B. T., Burgers P., Kuriyan J. Crystallization of proliferating cell nuclear antigen (PCNA) from Saccharomyces cerevisiae. J Mol Biol. 1994 Aug 12;241(2):265–268. doi: 10.1006/jmbi.1994.1495. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. Luckow B., Bunz F., Stillman B., Lichter P., Schütz G. Cloning, expression, and chromosomal localization of the 140-kilodalton subunit of replication factor C from mice and humans. Mol Cell Biol. 1994 Mar;14(3):1626–1634. doi: 10.1128/mcb.14.3.1626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Morrison A., Christensen R. B., Alley J., Beck A. K., Bernstine E. G., Lemontt J. F., Lawrence C. W. REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase. J Bacteriol. 1989 Oct;171(10):5659–5667. doi: 10.1128/jb.171.10.5659-5667.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ng L., McConnell M., Tan C. K., Downey K. M., Fisher P. A. Interaction of DNA polymerase delta, proliferating cell nuclear antigen, and synthetic oligonucleotide template-primers. Analysis by polyacrylamide gel electrophoresis-band mobility shift assay. J Biol Chem. 1993 Jun 25;268(18):13571–13576. [PubMed] [Google Scholar]
  27. Ng L., Prelich G., Anderson C. W., Stillman B., Fisher P. A. Drosophila proliferating cell nuclear antigen. Structural and functional homology with its mammalian counterpart. J Biol Chem. 1990 Jul 15;265(20):11948–11954. [PubMed] [Google Scholar]
  28. 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]
  29. Percival K. J., Klein M. B., Burgers P. M. Molecular cloning and primary structure of the uracil-DNA-glycosylase gene from Saccharomyces cerevisiae. J Biol Chem. 1989 Feb 15;264(5):2593–2598. [PubMed] [Google Scholar]
  30. 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]
  31. Prelich G., Tan C. K., Kostura M., Mathews M. B., So A. G., Downey K. M., Stillman B. Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein. Nature. 1987 Apr 2;326(6112):517–520. doi: 10.1038/326517a0. [DOI] [PubMed] [Google Scholar]
  32. Reagan M. S., Pittenger C., Siede W., Friedberg E. C. Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene. J Bacteriol. 1995 Jan;177(2):364–371. doi: 10.1128/jb.177.2.364-371.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Sommers C. H., Miller E. J., Dujon B., Prakash S., Prakash L. Conditional lethality of null mutations in RTH1 that encodes the yeast counterpart of a mammalian 5'- to 3'-exonuclease required for lagging strand DNA synthesis in reconstituted systems. J Biol Chem. 1995 Mar 3;270(9):4193–4196. doi: 10.1074/jbc.270.9.4193. [DOI] [PubMed] [Google Scholar]
  35. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tan C. K., Castillo C., So A. G., Downey K. M. An auxiliary protein for DNA polymerase-delta from fetal calf thymus. J Biol Chem. 1986 Sep 15;261(26):12310–12316. [PubMed] [Google Scholar]
  37. Tsurimoto T., Stillman B. Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1023–1027. doi: 10.1073/pnas.87.3.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tsurimoto T., Stillman B. Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta. EMBO J. 1989 Dec 1;8(12):3883–3889. doi: 10.1002/j.1460-2075.1989.tb08567.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Waga S., Hannon G. J., Beach D., Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature. 1994 Jun 16;369(6481):574–578. doi: 10.1038/369574a0. [DOI] [PubMed] [Google Scholar]
  41. Wang Z., Wu X., Friedberg E. C. DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Feb;13(2):1051–1058. doi: 10.1128/mcb.13.2.1051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Waseem N. H., Labib K., Nurse P., Lane D. P. Isolation and analysis of the fission yeast gene encoding polymerase delta accessory protein PCNA. EMBO J. 1992 Dec;11(13):5111–5120. doi: 10.1002/j.1460-2075.1992.tb05618.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wertman K. F., Drubin D. G., Botstein D. Systematic mutational analysis of the yeast ACT1 gene. Genetics. 1992 Oct;132(2):337–350. doi: 10.1093/genetics/132.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]