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. 1997 Jul;17(7):3876–3883. doi: 10.1128/mcb.17.7.3876

Denaturation of the simian virus 40 origin of replication mediated by human replication protein A.

C Iftode 1, J A Borowiec 1
PMCID: PMC232240  PMID: 9199322

Abstract

The initiation of simian virus 40 (SV40) replication requires recognition of the viral origin of replication (ori) by SV40 T antigen, followed by denaturation of ori in a reaction dependent upon human replication protein A (hRPA). To understand how origin denaturation is achieved, we constructed a 48-bp SV40 "pseudo-origin" with a central 8-nucleotide (nt) bubble flanked by viral sequences, mimicking a DNA structure found within the SV40 T antigen-ori complex. hRPA bound the pseudo-origin with similar stoichiometry and an approximately fivefold reduced affinity compared to the binding of a 48-nt single-stranded DNA molecule. The presence of hRPA not only distorted the duplex DNA flanking the bubble but also resulted in denaturation of the pseudo-origin substrate in an ATP-independent reaction. Pseudo-origin denaturation occurred in 7 mM MgCl2, distinguishing this reaction from Mg2+-independent DNA-unwinding activities previously reported for hRPA. Tests of other single-stranded DNA-binding proteins (SSBs) revealed that pseudo-origin binding correlates with the known ability of these SSBs to support the T-antigen-dependent origin unwinding activity. Our results suggest that hRPA binding to the T antigen-ori complex induces the denaturation of ori including T-antigen recognition sequences, thus releasing T antigen from ori to unwind the viral DNA. The denaturation activity of hRPA has the potential to play a significant role in other aspects of DNA metabolism, including DNA repair.

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Selected References

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  1. Auborn K. J., Markowitz R. B., Wang E., Yu Y. T., Prives C. Simian virus 40 (SV40) T antigen binds specifically to double-stranded DNA but not to single-stranded DNA or DNA/RNA hybrids containing the SV40 regulatory sequences. J Virol. 1988 Jun;62(6):2204–2208. doi: 10.1128/jvi.62.6.2204-2208.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blackwell L. J., Borowiec J. A. Human replication protein A binds single-stranded DNA in two distinct complexes. Mol Cell Biol. 1994 Jun;14(6):3993–4001. doi: 10.1128/mcb.14.6.3993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blackwell L. J., Borowiec J. A., Mastrangelo I. A. Single-stranded-DNA binding alters human replication protein A structure and facilitates interaction with DNA-dependent protein kinase. Mol Cell Biol. 1996 Sep;16(9):4798–4807. doi: 10.1128/mcb.16.9.4798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boehmer P. E., Lehman I. R. Herpes simplex virus type 1 ICP8: helix-destabilizing properties. J Virol. 1993 Feb;67(2):711–715. doi: 10.1128/jvi.67.2.711-715.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. Fanning E., Knippers R. Structure and function of simian virus 40 large tumor antigen. Annu Rev Biochem. 1992;61:55–85. doi: 10.1146/annurev.bi.61.070192.000415. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Goetz G. S., Dean F. B., Hurwitz J., Matson S. W. The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity. J Biol Chem. 1988 Jan 5;263(1):383–392. [PubMed] [Google Scholar]
  15. Gomes X. V., Henricksen L. A., Wold M. S. Proteolytic mapping of human replication protein A: evidence for multiple structural domains and a conformational change upon interaction with single-stranded DNA. Biochemistry. 1996 Apr 30;35(17):5586–5595. doi: 10.1021/bi9526995. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Huang J. C., Svoboda D. L., Reardon J. T., Sancar A. Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5' and the 6th phosphodiester bond 3' to the photodimer. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3664–3668. doi: 10.1073/pnas.89.8.3664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hurwitz J., Dean F. B., Kwong A. D., Lee S. H. The in vitro replication of DNA containing the SV40 origin. J Biol Chem. 1990 Oct 25;265(30):18043–18046. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Kim C., Paulus B. F., Wold M. S. Interactions of human replication protein A with oligonucleotides. Biochemistry. 1994 Nov 29;33(47):14197–14206. doi: 10.1021/bi00251a031. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Luche R. M., Smart W. C., Marion T., Tillman M., Sumrada R. A., Cooper T. G. Saccharomyces cerevisiae BUF protein binds to sequences participating in DNA replication in addition to those mediating transcriptional repression (URS1) and activation. Mol Cell Biol. 1993 Sep;13(9):5749–5761. doi: 10.1128/mcb.13.9.5749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Matsumoto K., Ishimi Y. Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region. Mol Cell Biol. 1994 Jul;14(7):4624–4632. doi: 10.1128/mcb.14.7.4624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. McEntee K., Weinstock G. M., Lehman I. R. recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein. Proc Natl Acad Sci U S A. 1980 Feb;77(2):857–861. doi: 10.1073/pnas.77.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Monaghan A., Webster A., Hay R. T. Adenovirus DNA binding protein: helix destabilising properties. Nucleic Acids Res. 1994 Mar 11;22(5):742–748. doi: 10.1093/nar/22.5.742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Murti K. G., He D. C., Brinkley B. R., Scott R., Lee S. H. Dynamics of human replication protein A subunit distribution and partitioning in the cell cycle. Exp Cell Res. 1996 Mar 15;223(2):279–289. doi: 10.1006/excr.1996.0083. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. SenGupta D. J., Borowiec J. A. Strand and face: the topography of interactions between the SV40 origin of replication and T-antigen during the initiation of replication. EMBO J. 1994 Feb 15;13(4):982–992. doi: 10.1002/j.1460-2075.1994.tb06343.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sigman D. S., Kuwabara M. D., Chen C. H., Bruice T. W. Nuclease activity of 1,10-phenanthroline-copper in study of protein-DNA interactions. Methods Enzymol. 1991;208:414–433. doi: 10.1016/0076-6879(91)08022-a. [DOI] [PubMed] [Google Scholar]
  35. Singh K. K., Samson L. Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4907–4911. doi: 10.1073/pnas.92.11.4907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stillman B. Smart machines at the DNA replication fork. Cell. 1994 Sep 9;78(5):725–728. doi: 10.1016/s0092-8674(94)90362-x. [DOI] [PubMed] [Google Scholar]
  38. Tang C. M., Tomkinson A. E., Lane W. S., Wold M. S., Seto E. Replication protein A is a component of a complex that binds the human metallothionein IIA gene transcription start site. J Biol Chem. 1996 Aug 30;271(35):21637–21644. doi: 10.1074/jbc.271.35.21637. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Wessel R., Schweizer J., Stahl H. Simian virus 40 T-antigen DNA helicase is a hexamer which forms a binary complex during bidirectional unwinding from the viral origin of DNA replication. J Virol. 1992 Feb;66(2):804–815. doi: 10.1128/jvi.66.2.804-815.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wiekowski M., Schwarz M. W., Stahl H. Simian virus 40 large T antigen DNA helicase. Characterization of the ATPase-dependent DNA unwinding activity and its substrate requirements. J Biol Chem. 1988 Jan 5;263(1):436–442. [PubMed] [Google Scholar]
  42. Wobbe C. R., Dean F., Weissbach L., Hurwitz J. In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5710–5714. doi: 10.1073/pnas.82.17.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. Wood R. D., Robins P., Lindahl T. Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts. Cell. 1988 Apr 8;53(1):97–106. doi: 10.1016/0092-8674(88)90491-6. [DOI] [PubMed] [Google Scholar]
  47. Yoon C., Kuwabara M. D., Law R., Wall R., Sigman D. S. Sequence-dependent variability of DNA structure. Influence of flanking sequences and fragment length on digestion by conformationally sensitive nucleases. J Biol Chem. 1988 Jun 15;263(17):8458–8463. [PubMed] [Google Scholar]
  48. Zijderveld D. C., van der Vliet P. C. Helix-destabilizing properties of the adenovirus DNA-binding protein. J Virol. 1994 Feb;68(2):1158–1164. doi: 10.1128/jvi.68.2.1158-1164.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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