Abstract
Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.
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Selected References
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- 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]
- 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]
- Atrazhev A., Zhang S., Grosse F. Single-stranded DNA binding protein from calf thymus. Purification, properties, and stimulation of the homologous DNA-polymerase-alpha-primase complex. Eur J Biochem. 1992 Dec 15;210(3):855–865. doi: 10.1111/j.1432-1033.1992.tb17489.x. [DOI] [PubMed] [Google Scholar]
- Borowiec J. A., Dean F. B., Bullock P. A., Hurwitz J. Binding and unwinding--how T antigen engages the SV40 origin of DNA replication. Cell. 1990 Jan 26;60(2):181–184. doi: 10.1016/0092-8674(90)90730-3. [DOI] [PubMed] [Google Scholar]
- Borowiec J. A., Hurwitz J. ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication. Proc Natl Acad Sci U S A. 1988 Jan;85(1):64–68. doi: 10.1073/pnas.85.1.64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Borowiec J. A., Hurwitz J. Localized melting and structural changes in the SV40 origin of replication induced by T-antigen. EMBO J. 1988 Oct;7(10):3149–3158. doi: 10.1002/j.1460-2075.1988.tb03182.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brill S. J., Stillman B. Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase. Genes Dev. 1991 Sep;5(9):1589–1600. doi: 10.1101/gad.5.9.1589. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Bulsink H., Wijnaendts van Resandt R. W., Harmsen B. J., Hilbers C. W. Different DNA-binding modes and cooperativities for bacteriophage M13 gene-5 protein revealed by means of fluorescence depolarisation studies. Eur J Biochem. 1986 Jun 2;157(2):329–334. doi: 10.1111/j.1432-1033.1986.tb09672.x. [DOI] [PubMed] [Google Scholar]
- Carpenter M. L., Kneale G. G. Circular dichroism and fluorescence analysis of the interaction of Pf1 gene 5 protein with poly(dT). J Mol Biol. 1991 Feb 20;217(4):681–689. doi: 10.1016/0022-2836(91)90525-b. [DOI] [PubMed] [Google Scholar]
- Chase J. W., Williams K. R. Single-stranded DNA binding proteins required for DNA replication. Annu Rev Biochem. 1986;55:103–136. doi: 10.1146/annurev.bi.55.070186.000535. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Dean F. B., Bullock P., Murakami Y., Wobbe C. R., Weissbach L., Hurwitz J. Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication. Proc Natl Acad Sci U S A. 1987 Jan;84(1):16–20. doi: 10.1073/pnas.84.1.16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dean F. B., Dodson M., Echols H., Hurwitz J. ATP-dependent formation of a specialized nucleoprotein structure by simian virus 40 (SV40) large tumor antigen at the SV40 replication origin. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8981–8985. doi: 10.1073/pnas.84.24.8981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Deb S. P., Tegtmeyer P. ATP enhances the binding of simian virus 40 large T antigen to the origin of replication. J Virol. 1987 Dec;61(12):3649–3654. doi: 10.1128/jvi.61.12.3649-3654.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Dutta A., Ruppert J. M., Aster J. C., Winchester E. Inhibition of DNA replication factor RPA by p53. Nature. 1993 Sep 2;365(6441):79–82. doi: 10.1038/365079a0. [DOI] [PubMed] [Google Scholar]
- Dutta A., Stillman B. cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication. EMBO J. 1992 Jun;11(6):2189–2199. doi: 10.1002/j.1460-2075.1992.tb05278.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Erdile L. F., Wold M. S., Kelly T. J. The primary structure of the 32-kDa subunit of human replication protein A. J Biol Chem. 1990 Feb 25;265(6):3177–3182. [PubMed] [Google Scholar]
- 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]
- 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]
- Georgaki A., Strack B., Podust V., Hübscher U. DNA unwinding activity of replication protein A. FEBS Lett. 1992 Aug 24;308(3):240–244. doi: 10.1016/0014-5793(92)81283-r. [DOI] [PubMed] [Google Scholar]
- He Z., Brinton B. T., Greenblatt J., Hassell J. A., Ingles C. J. The transactivator proteins VP16 and GAL4 bind replication factor A. Cell. 1993 Jun 18;73(6):1223–1232. doi: 10.1016/0092-8674(93)90650-f. [DOI] [PubMed] [Google Scholar]
- Heyer W. D., Rao M. R., Erdile L. F., Kelly T. J., Kolodner R. D. An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A. EMBO J. 1990 Jul;9(7):2321–2329. doi: 10.1002/j.1460-2075.1990.tb07404.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kansy J. W., Clack B. A., Gray D. M. The binding of fd gene 5 protein to polydeoxynucleotides: evidence from CD measurements for two binding modes. J Biomol Struct Dyn. 1986 Jun;3(6):1079–1110. doi: 10.1080/07391102.1986.10508487. [DOI] [PubMed] [Google Scholar]
- Kelly R. C., Jensen D. E., von Hippel P. H. DNA "melting" proteins. IV. Fluorescence measurements of binding parameters for bacteriophage T4 gene 32-protein to mono-, oligo-, and polynucleotides. J Biol Chem. 1976 Nov 25;251(22):7240–7250. [PubMed] [Google Scholar]
- Kenny M. K., Lee S. H., Hurwitz J. Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9757–9761. doi: 10.1073/pnas.86.24.9757. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Li R., Botchan M. R. The acidic transcriptional activation domains of VP16 and p53 bind the cellular replication protein A and stimulate in vitro BPV-1 DNA replication. Cell. 1993 Jun 18;73(6):1207–1221. doi: 10.1016/0092-8674(93)90649-b. [DOI] [PubMed] [Google Scholar]
- Mastrangelo I. A., Hough P. V., Wall J. S., Dodson M., Dean F. B., Hurwitz J. ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication. Nature. 1989 Apr 20;338(6217):658–662. doi: 10.1038/338658a0. [DOI] [PubMed] [Google Scholar]
- Parsons R., Anderson M. E., Tegtmeyer P. Three domains in the simian virus 40 core origin orchestrate the binding, melting, and DNA helicase activities of T antigen. J Virol. 1990 Feb;64(2):509–518. doi: 10.1128/jvi.64.2.509-518.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rodriguez A. T., Colmenarejo G., Montero F. Thermal denaturation profiles of deoxypolynucleotide-destabilizer ligand complexes: semiempirical studies. Arch Biochem Biophys. 1991 Oct;290(1):133–142. doi: 10.1016/0003-9861(91)90599-e. [DOI] [PubMed] [Google Scholar]
- Siegel L. M., Monty K. J. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta. 1966 Feb 7;112(2):346–362. doi: 10.1016/0926-6585(66)90333-5. [DOI] [PubMed] [Google Scholar]
- Umbricht C. B., Erdile L. F., Jabs E. W., Kelly T. J. Cloning, overexpression, and genomic mapping of the 14-kDa subunit of human replication protein A. J Biol Chem. 1993 Mar 25;268(9):6131–6138. [PubMed] [Google Scholar]
- 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]
- Wold M. S., Kelly T. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2523–2527. doi: 10.1073/pnas.85.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]