Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 1997 Apr;145(4):891–902. doi: 10.1093/genetics/145.4.891

Roles of Replication Protein-a Subunits 2 and 3 in DNA Replication Fork Movement in Saccharomyces Cerevisiae

H S Maniar 1, R Wilson 1, S J Brill 1
PMCID: PMC1207894  PMID: 9093844

Abstract

Replication Protein-A, the eukaryotic SSB, consists of a large subunit (RPA1) with strong ssDNA binding activity and two smaller subunits (RPA2 and 3) that may cooperate with RPA1 to bind ssDNA in a higher-order mode. To determine the in vivo function of the two smaller subunits and the potential role of higher-order ssDNA binding, we isolated an assortment of heat-lethal mutations in the genes encoding RPA2 and RPA3. At the permissive temperature, the mutants show a range of effects on DNA replication fidelity and sensitivities to UV and MMS. At the nonpermissive temperature, four out of five RPA2 mutants show a fast-stop DNA synthesis phenotype typical of a replication fork block. In contrast, the fifth RPA2 mutant and all RPA3 mutants are able to complete at least one round of DNA replication at the nonpermissive temperature. The effect of these mutations on the stability of the RPA complex was tested using a coprecipitation assay. At the nonpermissive temperature, we find that RPA1 and RPA2 are dissociated in the fast-stop mutants, but not in the slow-stop mutants. Thus, replication fork movement in vivo requires the association of at least two subunits of RPA. This result is consistent with the hypothesis that RPA functions in vivo by binding ssDNA in a higher-order mode.

Full Text

The Full Text of this article is available as a PDF (5.4 MB).

Selected References

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

  1. 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]
  2. Brush G. S., Anderson C. W., Kelly T. J. The DNA-activated protein kinase is required for the phosphorylation of replication protein A during simian virus 40 DNA replication. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12520–12524. doi: 10.1073/pnas.91.26.12520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bujalowski W., Overman L. B., Lohman T. M. Binding mode transitions of Escherichia coli single strand binding protein-single-stranded DNA complexes. Cation, anion, pH, and binding density effects. J Biol Chem. 1988 Apr 5;263(10):4629–4640. [PubMed] [Google Scholar]
  4. 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]
  5. Chrysogelos S., Griffith J. Escherichia coli single-strand binding protein organizes single-stranded DNA in nucleosome-like units. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5803–5807. doi: 10.1073/pnas.79.19.5803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coverley D., Kenny M. K., Lane D. P., Wood R. D. A role for the human single-stranded DNA binding protein HSSB/RPA in an early stage of nucleotide excision repair. Nucleic Acids Res. 1992 Aug 11;20(15):3873–3880. doi: 10.1093/nar/20.15.3873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. Henricksen L. A., Carter T., Dutta A., Wold M. S. Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication. Nucleic Acids Res. 1996 Aug 1;24(15):3107–3112. doi: 10.1093/nar/24.15.3107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heyer W. D., Kolodner R. D. Purification and characterization of a protein from Saccharomyces cerevisiae that binds tightly to single-stranded DNA and stimulates a cognate strand exchange protein. Biochemistry. 1989 Apr 4;28(7):2856–2862. doi: 10.1021/bi00433a017. [DOI] [PubMed] [Google Scholar]
  14. Kim C., Snyder R. O., Wold M. S. Binding properties of replication protein A from human and yeast cells. Mol Cell Biol. 1992 Jul;12(7):3050–3059. doi: 10.1128/mcb.12.7.3050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lee S. H., Kim D. K., Drissi R. Human xeroderma pigmentosum group A protein interacts with human replication protein A and inhibits DNA replication. J Biol Chem. 1995 Sep 15;270(37):21800–21805. doi: 10.1074/jbc.270.37.21800. [DOI] [PubMed] [Google Scholar]
  16. Lee S. H., Kim D. K. The role of the 34-kDa subunit of human replication protein A in simian virus 40 DNA replication in vitro. J Biol Chem. 1995 May 26;270(21):12801–12807. doi: 10.1074/jbc.270.21.12801. [DOI] [PubMed] [Google Scholar]
  17. Li L., Lu X., Peterson C. A., Legerski R. J. An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair. Mol Cell Biol. 1995 Oct;15(10):5396–5402. doi: 10.1128/mcb.15.10.5396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lohman T. M., Ferrari M. E. Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities. Annu Rev Biochem. 1994;63:527–570. doi: 10.1146/annurev.bi.63.070194.002523. [DOI] [PubMed] [Google Scholar]
  19. Lohman T. M., Overman L. B. Two binding modes in Escherichia coli single strand binding protein-single stranded DNA complexes. Modulation by NaCl concentration. J Biol Chem. 1985 Mar 25;260(6):3594–3603. [PubMed] [Google Scholar]
  20. 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]
  21. Pan Z. Q., Park C. H., Amin A. A., Hurwitz J., Sancar A. Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4636–4640. doi: 10.1073/pnas.92.10.4636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Sikorski R. S., 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. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thomas B. J., Rothstein R. Elevated recombination rates in transcriptionally active DNA. Cell. 1989 Feb 24;56(4):619–630. doi: 10.1016/0092-8674(89)90584-9. [DOI] [PubMed] [Google Scholar]
  25. Treuner K., Ramsperger U., Knippers R. Replication protein A induces the unwinding of long double-stranded DNA regions. J Mol Biol. 1996 May 31;259(1):104–112. doi: 10.1006/jmbi.1996.0305. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Zakian V. A., Brewer B. J., Fangman W. L. Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase. Cell. 1979 Aug;17(4):923–934. doi: 10.1016/0092-8674(79)90332-5. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES