Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1998 Mar;148(3):989–1005. doi: 10.1093/genetics/148.3.989

Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism.

K Umezu 1, N Sugawara 1, C Chen 1, J E Haber 1, R D Kolodner 1
PMCID: PMC1460019  PMID: 9539419

Abstract

Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

Full Text

The Full Text of this article is available as a PDF (432.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adachi Y., Laemmli U. K. Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A. J Cell Biol. 1992 Oct;119(1):1–15. doi: 10.1083/jcb.119.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alani E., Thresher R., Griffith J. D., Kolodner R. D. Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein. J Mol Biol. 1992 Sep 5;227(1):54–71. doi: 10.1016/0022-2836(92)90681-9. [DOI] [PubMed] [Google Scholar]
  3. Bochkarev A., Pfuetzner R. A., Edwards A. M., Frappier L. Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA. Nature. 1997 Jan 9;385(6612):176–181. doi: 10.1038/385176a0. [DOI] [PubMed] [Google Scholar]
  4. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  5. Bonneu M., Crouzet M., Urdaci M., Aigle M. Direct detection of yeast mutants with reduced viability on plates by erythrosine B staining. Anal Biochem. 1991 Mar 2;193(2):225–230. doi: 10.1016/0003-2697(91)90013-j. [DOI] [PubMed] [Google Scholar]
  6. Brill S. J., Stillman B. Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication. Nature. 1989 Nov 2;342(6245):92–95. doi: 10.1038/342092a0. [DOI] [PubMed] [Google Scholar]
  7. Brown G. W., Hines J. C., Fisher P., Ray D. S. Isolation of the genes encoding the 51-kilodalton and 28-kilodalton subunits of Crithidia fasciculata replication protein A. Mol Biochem Parasitol. 1994 Jan;63(1):135–142. doi: 10.1016/0166-6851(94)90016-7. [DOI] [PubMed] [Google Scholar]
  8. Brush G. S., Morrow D. M., Hieter P., Kelly T. J. The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15075–15080. doi: 10.1073/pnas.93.26.15075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burns J. L., Guzder S. N., Sung P., Prakash S., Prakash L. An affinity of human replication protein A for ultraviolet-damaged DNA. J Biol Chem. 1996 May 17;271(20):11607–11610. doi: 10.1074/jbc.271.20.11607. [DOI] [PubMed] [Google Scholar]
  10. Cadwell R. C., Joyce G. F. Mutagenic PCR. PCR Methods Appl. 1994 Jun;3(6):S136–S140. doi: 10.1101/gr.3.6.s136. [DOI] [PubMed] [Google Scholar]
  11. Cardoso M. C., Leonhardt H., Nadal-Ginard B. Reversal of terminal differentiation and control of DNA replication: cyclin A and Cdk2 specifically localize at subnuclear sites of DNA replication. Cell. 1993 Sep 24;74(6):979–992. doi: 10.1016/0092-8674(93)90721-2. [DOI] [PubMed] [Google Scholar]
  12. Carty M. P., Zernik-Kobak M., McGrath S., Dixon K. UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein. EMBO J. 1994 May 1;13(9):2114–2123. doi: 10.1002/j.1460-2075.1994.tb06487.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Connolly B., White C. I., Haber J. E. Physical monitoring of mating type switching in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jun;8(6):2342–2349. doi: 10.1128/mcb.8.6.2342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Coverley D., Kenny M. K., Munn M., Rupp W. D., Lane D. P., Wood R. D. Requirement for the replication protein SSB in human DNA excision repair. Nature. 1991 Feb 7;349(6309):538–541. doi: 10.1038/349538a0. [DOI] [PubMed] [Google Scholar]
  15. Din S., Brill S. J., Fairman M. P., Stillman B. Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev. 1990 Jun;4(6):968–977. doi: 10.1101/gad.4.6.968. [DOI] [PubMed] [Google Scholar]
  16. Dornreiter I., Erdile L. F., Gilbert I. U., von Winkler D., Kelly T. J., Fanning E. Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen. EMBO J. 1992 Feb;11(2):769–776. doi: 10.1002/j.1460-2075.1992.tb05110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Erdile L. F., Heyer W. D., Kolodner R., Kelly T. J. Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication. J Biol Chem. 1991 Jun 25;266(18):12090–12098. [PubMed] [Google Scholar]
  18. Fairman M. P., Stillman B. Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J. 1988 Apr;7(4):1211–1218. doi: 10.1002/j.1460-2075.1988.tb02933.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Firmenich A. A., Elias-Arnanz M., Berg P. A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52. Mol Cell Biol. 1995 Mar;15(3):1620–1631. doi: 10.1128/mcb.15.3.1620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fishman-Lobell J., Rudin N., Haber J. E. Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated. Mol Cell Biol. 1992 Mar;12(3):1292–1303. doi: 10.1128/mcb.12.3.1292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fotedar R., Roberts J. M. Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex. EMBO J. 1992 Jun;11(6):2177–2187. doi: 10.1002/j.1460-2075.1992.tb05277.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Galcheva-Gargova Z., Konstantinov K. N., Wu I. H., Klier F. G., Barrett T., Davis R. J. Binding of zinc finger protein ZPR1 to the epidermal growth factor receptor. Science. 1996 Jun 21;272(5269):1797–1802. doi: 10.1126/science.272.5269.1797. [DOI] [PubMed] [Google Scholar]
  23. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gomes X. V., Wold M. S. Structural analysis of human replication protein A. Mapping functional domains of the 70-kDa subunit. J Biol Chem. 1995 Mar 3;270(9):4534–4543. doi: 10.1074/jbc.270.9.4534. [DOI] [PubMed] [Google Scholar]
  25. He Z., Henricksen L. A., Wold M. S., Ingles C. J. RPA involvement in the damage-recognition and incision steps of nucleotide excision repair. Nature. 1995 Apr 6;374(6522):566–569. doi: 10.1038/374566a0. [DOI] [PubMed] [Google Scholar]
  26. Johnson A. W., Kolodner R. D. Synthetic lethality of sep1 (xrn1) ski2 and sep1 (xrn1) ski3 mutants of Saccharomyces cerevisiae is independent of killer virus and suggests a general role for these genes in translation control. Mol Cell Biol. 1995 May;15(5):2719–2727. doi: 10.1128/mcb.15.5.2719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kenny M. K., Schlegel U., Furneaux H., Hurwitz J. The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. J Biol Chem. 1990 May 5;265(13):7693–7700. [PubMed] [Google Scholar]
  28. Kim D. K., Stigger E., Lee S. H. Role of the 70-kDa subunit of human replication protein A (I). Single-stranded dna binding activity, but not polymerase stimulatory activity, is required for DNA replication. J Biol Chem. 1996 Jun 21;271(25):15124–15129. doi: 10.1074/jbc.271.25.15124. [DOI] [PubMed] [Google Scholar]
  29. Lin Y. L., Chen C., Keshav K. F., Winchester E., Dutta A. Dissection of functional domains of the human DNA replication protein complex replication protein A. J Biol Chem. 1996 Jul 19;271(29):17190–17198. doi: 10.1074/jbc.271.29.17190. [DOI] [PubMed] [Google Scholar]
  30. Lin Y. L., Shivji M. K., Chen C., Kolodner R., Wood R. D., Dutta A. The evolutionarily conserved zinc finger motif in the largest subunit of human replication protein A is required for DNA replication and mismatch repair but not for nucleotide excision repair. J Biol Chem. 1998 Jan 16;273(3):1453–1461. doi: 10.1074/jbc.273.3.1453. [DOI] [PubMed] [Google Scholar]
  31. Liu V. F., Weaver D. T. The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells. Mol Cell Biol. 1993 Dec;13(12):7222–7231. doi: 10.1128/mcb.13.12.7222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Longhese M. P., Neecke H., Paciotti V., Lucchini G., Plevani P. The 70 kDa subunit of replication protein A is required for the G1/S and intra-S DNA damage checkpoints in budding yeast. Nucleic Acids Res. 1996 Sep 15;24(18):3533–3537. doi: 10.1093/nar/24.18.3533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Longhese M. P., Plevani P., Lucchini G. Replication factor A is required in vivo for DNA replication, repair, and recombination. Mol Cell Biol. 1994 Dec;14(12):7884–7890. doi: 10.1128/mcb.14.12.7884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Maniar H. S., Wilson R., Brill S. J. Roles of replication protein-A subunits 2 and 3 in DNA replication fork movement in Saccharomyces cerevisiae. Genetics. 1997 Apr;145(4):891–902. doi: 10.1093/genetics/145.4.891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Matsuda T., Saijo M., Kuraoka I., Kobayashi T., Nakatsu Y., Nagai A., Enjoji T., Masutani C., Sugasawa K., Hanaoka F. DNA repair protein XPA binds replication protein A (RPA). J Biol Chem. 1995 Feb 24;270(8):4152–4157. doi: 10.1074/jbc.270.8.4152. [DOI] [PubMed] [Google Scholar]
  36. Matsunaga T., Park C. H., Bessho T., Mu D., Sancar A. Replication protein A confers structure-specific endonuclease activities to the XPF-ERCC1 and XPG subunits of human DNA repair excision nuclease. J Biol Chem. 1996 May 10;271(19):11047–11050. doi: 10.1074/jbc.271.19.11047. [DOI] [PubMed] [Google Scholar]
  37. Meyer R. R., Laine P. S. The single-stranded DNA-binding protein of Escherichia coli. Microbiol Rev. 1990 Dec;54(4):342–380. doi: 10.1128/mr.54.4.342-380.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Moore S. P., Erdile L., Kelly T., Fishel R. The human homologous pairing protein HPP-1 is specifically stimulated by the cognate single-stranded binding protein hRP-A. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9067–9071. doi: 10.1073/pnas.88.20.9067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Muhlrad D., Hunter R., Parker R. A rapid method for localized mutagenesis of yeast genes. Yeast. 1992 Feb;8(2):79–82. doi: 10.1002/yea.320080202. [DOI] [PubMed] [Google Scholar]
  40. Ogawa T., Shinohara A., Nabetani A., Ikeya T., Yu X., Egelman E. H., Ogawa H. RecA-like recombination proteins in eukaryotes: functions and structures of RAD51 genes. Cold Spring Harb Symp Quant Biol. 1993;58:567–576. doi: 10.1101/sqb.1993.058.01.063. [DOI] [PubMed] [Google Scholar]
  41. Park M. S., Ludwig D. L., Stigger E., Lee S. H. Physical interaction between human RAD52 and RPA is required for homologous recombination in mammalian cells. J Biol Chem. 1996 Aug 2;271(31):18996–19000. doi: 10.1074/jbc.271.31.18996. [DOI] [PubMed] [Google Scholar]
  42. Parker A. E., Clyne R. K., Carr A. M., Kelly T. J. The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A. Mol Cell Biol. 1997 May;17(5):2381–2390. doi: 10.1128/mcb.17.5.2381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Philipova D., Mullen J. R., Maniar H. S., Lu J., Gu C., Brill S. J. A hierarchy of SSB protomers in replication protein A. Genes Dev. 1996 Sep 1;10(17):2222–2233. doi: 10.1101/gad.10.17.2222. [DOI] [PubMed] [Google Scholar]
  44. Rodgers K. K., Bu Z., Fleming K. G., Schatz D. G., Engelman D. M., Coleman J. E. A zinc-binding domain involved in the dimerization of RAG1. J Mol Biol. 1996 Jul 5;260(1):70–84. doi: 10.1006/jmbi.1996.0382. [DOI] [PubMed] [Google Scholar]
  45. Santocanale C., Neecke H., Longhese M. P., Lucchini G., Plevani P. Mutations in the gene encoding the 34 kDa subunit of yeast replication protein A cause defective S phase progression. J Mol Biol. 1995 Dec 8;254(4):595–607. doi: 10.1006/jmbi.1995.0641. [DOI] [PubMed] [Google Scholar]
  46. Shepard D. A., Ehnstrom J. G., Skinner P. J., Schiff L. A. Mutations in the zinc-binding motif of the reovirus capsid protein delta 3 eliminate its ability to associate with capsid protein mu 1. J Virol. 1996 Mar;70(3):2065–2068. doi: 10.1128/jvi.70.3.2065-2068.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shinohara A., Ogawa H., Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell. 1992 May 1;69(3):457–470. doi: 10.1016/0092-8674(92)90447-k. [DOI] [PubMed] [Google Scholar]
  48. Sugawara N., Haber J. E. Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation. Mol Cell Biol. 1992 Feb;12(2):563–575. doi: 10.1128/mcb.12.2.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sugiyama T., Zaitseva E. M., Kowalczykowski S. C. A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. J Biol Chem. 1997 Mar 21;272(12):7940–7945. doi: 10.1074/jbc.272.12.7940. [DOI] [PubMed] [Google Scholar]
  50. Sung P. Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science. 1994 Aug 26;265(5176):1241–1243. doi: 10.1126/science.8066464. [DOI] [PubMed] [Google Scholar]
  51. Sung P. Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev. 1997 May 1;11(9):1111–1121. doi: 10.1101/gad.11.9.1111. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. White C. I., Haber J. E. Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. EMBO J. 1990 Mar;9(3):663–673. doi: 10.1002/j.1460-2075.1990.tb08158.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wobbe C. R., Weissbach L., Borowiec J. A., Dean F. B., Murakami Y., Bullock P., Hurwitz J. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1834–1838. doi: 10.1073/pnas.84.7.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wold M. S., Li J. J., Kelly T. J. Initiation of simian virus 40 DNA replication in vitro: large-tumor-antigen- and origin-dependent unwinding of the template. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3643–3647. doi: 10.1073/pnas.84.11.3643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wold M. S., Weinberg D. H., Virshup D. M., Li J. J., Kelly T. J. Identification of cellular proteins required for simian virus 40 DNA replication. J Biol Chem. 1989 Feb 15;264(5):2801–2809. [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES