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. 1991 Feb;65(2):687–699. doi: 10.1128/jvi.65.2.687-699.1991

The DNA-binding properties of polyomavirus large T antigen are altered by ATP and other nucleotides.

H E Lorimer 1, E H Wang 1, C Prives 1
PMCID: PMC239808  PMID: 1846192

Abstract

We have examined the influence of ATP on the DNA-binding properties of polyomavirus large T antigen (Py TAg). Utilizing nitrocellulose filter binding, DNase I footprinting, and gel mobility shift assays, we observed that ATP increased Py TAg binding to DNA fragments containing either all Py TAg-binding sites (whole origin) or those sites within (core origin) or adjacent to (early) the origin of replication. Even nonspecific binding to DNA fragments lacking Py TAg-binding sites was increased somewhat by ATP. Binding to the core origin was increased to a greater extent than binding to other DNA fragments tested. Gel band mobility shift assays revealed that ATP increased the production of core origin-specific Py TAg-DNA complexes of high molecular weight. ATP stimulation depended on the presence of MgCl2. Other nucleotides and nonhydrolyzable ATP analogs also increased Py TAg binding to the core origin but to various degrees: ATP, dATP, 5'-adenylyl imidodiphosphate (AMPPNP) greater than 5'-adenylyl methylenediphosphate (AMPPCP) greater than dCTP greater than UTP greater than TTP. GTP and dGTP did not increase DNA binding by Py TAg. The rates of association and disassociation of Py TAg with all the DNA fragments were altered by the presence of ATP. DNase I footprinting showed that ATP extensively extended the region protected within the core origin and also produced a distinctive DNase I-hypersensitive site on the late strand at nucleotides 5255 to 5262 (TTACTATG).

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Selected References

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  1. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  2. Bergqvist A., Nilsson M., Bondeson K., Magnusson G. Loss of DNA-binding and new transcriptional trans-activation function in polyomavirus large T-antigen with mutation of zinc finger motif. Nucleic Acids Res. 1990 May 11;18(9):2715–2720. doi: 10.1093/nar/18.9.2715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Borowiec J. A., Zhang L., Sasse-Dwight S., Gralla J. D. DNA supercoiling promotes formation of a bent repression loop in lac DNA. J Mol Biol. 1987 Jul 5;196(1):101–111. doi: 10.1016/0022-2836(87)90513-4. [DOI] [PubMed] [Google Scholar]
  6. Bradley M. K., Smith T. F., Lathrop R. H., Livingston D. M., Webster T. A. Consensus topography in the ATP binding site of the simian virus 40 and polyomavirus large tumor antigens. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4026–4030. doi: 10.1073/pnas.84.12.4026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Campbell B. A., Villarreal L. P. Functional analysis of the individual enhancer core sequences of polyomavirus: cell-specific uncoupling of DNA replication from transcription. Mol Cell Biol. 1988 May;8(5):1993–2004. doi: 10.1128/mcb.8.5.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clark R., Lane D. P., Tjian R. Use of monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity. J Biol Chem. 1981 Nov 25;256(22):11854–11858. [PubMed] [Google Scholar]
  9. Clertant P., Gaudray P., May E., Cuzin F. The nucleotide binding site detected by affinity labeling in the large T proteins of polyoma and SV40 viruses is distinct from their ATPase catalytic site. J Biol Chem. 1984 Dec 25;259(24):15196–15203. [PubMed] [Google Scholar]
  10. Cowie A., Kamen R. Guanine nucleotide contacts within viral DNA sequences bound by polyomavirus large T antigen. J Virol. 1986 Feb;57(2):505–514. doi: 10.1128/jvi.57.2.505-514.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cowie A., Kamen R. Multiple binding sites for polyomavirus large T antigen within regulatory sequences of polyomavirus DNA. J Virol. 1984 Dec;52(3):750–760. doi: 10.1128/jvi.52.3.750-760.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cowie A., de Villiers J., Kamen R. Immortalization of rat embryo fibroblasts by mutant polyomavirus large T antigens deficient in DNA binding. Mol Cell Biol. 1986 Dec;6(12):4344–4352. doi: 10.1128/mcb.6.12.4344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dailey L., Basilico C. Sequences in the polyomavirus DNA regulatory region involved in viral DNA replication and early gene expression. J Virol. 1985 Jun;54(3):739–749. doi: 10.1128/jvi.54.3.739-749.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Deb S., DeLucia A. L., Koff A., Tsui S., Tegtmeyer P. The adenine-thymine domain of the simian virus 40 core origin directs DNA bending and coordinately regulates DNA replication. Mol Cell Biol. 1986 Dec;6(12):4578–4584. doi: 10.1128/mcb.6.12.4578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dilworth S. M., Cowie A., Kamen R. I., Griffin B. E. DNA binding activity of polyoma virus large tumor antigen. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1941–1945. doi: 10.1073/pnas.81.7.1941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gaudray P., Clertant P., Cuzin F. ATP phosphohydrolase (ATPase) activity of a polyoma virus T antigen. Eur J Biochem. 1980 Aug;109(2):553–560. doi: 10.1111/j.1432-1033.1980.tb04827.x. [DOI] [PubMed] [Google Scholar]
  20. Gidoni D., Scheller A., Barnet B., Hantzopoulos P., Oren M., Prives C. Different forms of simian virus 40 large tumor antigen varying in their affinities for DNA. J Virol. 1982 May;42(2):456–466. doi: 10.1128/jvi.42.2.456-466.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gottlieb P., Nasoff M. S., Fisher E. F., Walsh A. M., Caruthers M. H. Binding studies of SV40 T-antigen to SV40 binding site II. Nucleic Acids Res. 1985 Sep 25;13(18):6621–6634. doi: 10.1093/nar/13.18.6621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hendrickson E. A., Fritze C. E., Folk W. R., DePamphilis M. L. The origin of bidirectional DNA replication in polyoma virus. EMBO J. 1987 Jul;6(7):2011–2018. doi: 10.1002/j.1460-2075.1987.tb02465.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Katinka M., Yaniv M. DNA replication origin of polyoma virus: early proximal boundary. J Virol. 1983 Jul;47(1):244–248. doi: 10.1128/jvi.47.1.244-248.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kern F. G., Dailey L., Basilico C. Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids. Mol Cell Biol. 1985 Aug;5(8):2070–2079. doi: 10.1128/mcb.5.8.2070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Luthman H., Nilsson M. G., Magnusson G. Non-contiguous segments of the polyoma genome required in cis for DNA replication. J Mol Biol. 1982 Nov 15;161(4):533–550. doi: 10.1016/0022-2836(82)90406-5. [DOI] [PubMed] [Google Scholar]
  26. Luthman H., Osterlund M., Magnusson G. Inhibition of polyoma DNA synthesis by base pair substitutions at the replication origin. Nucleic Acids Res. 1984 Oct 11;12(19):7503–7515. doi: 10.1093/nar/12.19.7503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Mastrangelo I. A., Hough P. V., Wilson V. G., Wall J. S., Hainfeld J. F., Tegtmeyer P. Monomers through trimers of large tumor antigen bind in region I and monomers through tetramers bind in region II of simian virus 40 origin of replication DNA as stable structures in solution. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3626–3630. doi: 10.1073/pnas.82.11.3626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Muller W. J., Mueller C. R., Mes A. M., Hassell J. A. Polyomavirus origin for DNA replication comprises multiple genetic elements. J Virol. 1983 Sep;47(3):586–599. doi: 10.1128/jvi.47.3.586-599.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Murakami Y., Wobbe C. R., Weissbach L., Dean F. B., Hurwitz J. Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1986 May;83(9):2869–2873. doi: 10.1073/pnas.83.9.2869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nilsson S. V., Magnusson G. Activities of polyomavirus large-T-antigen proteins expressed by mutant genes. J Virol. 1984 Sep;51(3):768–775. doi: 10.1128/jvi.51.3.768-775.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pallas D. C., Schley C., Mahoney M., Harlow E., Schaffhausen B. S., Roberts T. M. Polyomavirus small t antigen: overproduction in bacteria, purification, and utilization for monoclonal and polyclonal antibody production. J Virol. 1986 Dec;60(3):1075–1084. doi: 10.1128/jvi.60.3.1075-1084.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Peden K. W., Srinivasan A., Farber J. M., Pipas J. M. Mutants with changes within or near a hydrophobic region of simian virus 40 large tumor antigen are defective for binding cellular protein p53. Virology. 1989 Jan;168(1):13–21. doi: 10.1016/0042-6822(89)90398-x. [DOI] [PubMed] [Google Scholar]
  35. Pomerantz B. J., Hassell J. A. Polyomavirus and simian virus 40 large T antigens bind to common DNA sequences. J Virol. 1984 Mar;49(3):925–937. doi: 10.1128/jvi.49.3.925-937.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Prives C., Murakami Y., Kern F. G., Folk W., Basilico C., Hurwitz J. DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro. Mol Cell Biol. 1987 Oct;7(10):3694–3704. doi: 10.1128/mcb.7.10.3694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rice W. C., Lorimer H. E., Prives C., Miller L. K. Expression of polyomavirus large T antigen by using a baculovirus vector. J Virol. 1987 May;61(5):1712–1716. doi: 10.1128/jvi.61.5.1712-1716.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Riggs A. D., Bourgeois S., Cohn M. The lac repressor-operator interaction. 3. Kinetic studies. J Mol Biol. 1970 Nov 14;53(3):401–417. doi: 10.1016/0022-2836(70)90074-4. [DOI] [PubMed] [Google Scholar]
  39. Runzler R., Thompson S., Fanning E. Oligomerization and origin DNA-binding activity of simian virus 40 large T antigen. J Virol. 1987 Jul;61(7):2076–2083. doi: 10.1128/jvi.61.7.2076-2083.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Scheller A., Prives C. Simian virus 40 and polyomavirus large tumor antigens have different requirements for high-affinity sequence-specific DNA binding. J Virol. 1985 May;54(2):532–545. doi: 10.1128/jvi.54.2.532-545.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schirmbeck R., Deppert W. Analysis of mechanisms controlling the interactions of SV40 large T antigen with the SV40 ORI region. Virology. 1988 Aug;165(2):527–538. doi: 10.1016/0042-6822(88)90597-1. [DOI] [PubMed] [Google Scholar]
  42. Seki M., Enomoto T., Eki T., Miyajima A., Murakami Y., Hanaoka F., Ui M. DNA helicase and nucleoside-5'-triphosphatase activities of polyoma virus large tumor antigen. Biochemistry. 1990 Jan 30;29(4):1003–1009. doi: 10.1021/bi00456a024. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Tang W. J., Berger S. L., Triezenberg S. J., Folk W. R. Nucleotides in the polyomavirus enhancer that control viral transcription and DNA replication. Mol Cell Biol. 1987 May;7(5):1681–1690. doi: 10.1128/mcb.7.5.1681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tang W. J., Folk W. R. Asp-286----Asn-286 in polyomavirus large T antigen relaxes the specificity of binding to the polyomavirus origin. J Virol. 1989 Jan;63(1):242–249. doi: 10.1128/jvi.63.1.242-249.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tyndall C., La Mantia G., Thacker C. M., Favaloro J., Kamen R. A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication. Nucleic Acids Res. 1981 Dec 11;9(23):6231–6250. doi: 10.1093/nar/9.23.6231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Veldman G. M., Lupton S., Kamen R. Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression. Mol Cell Biol. 1985 Apr;5(4):649–658. doi: 10.1128/mcb.5.4.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vogt B., Vakalopoulou E., Fanning E. Allosteric control of simian virus 40 T-antigen binding to viral origin DNA. J Virol. 1986 Jun;58(3):765–772. doi: 10.1128/jvi.58.3.765-772.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wang E. H., Friedman P. N., Prives C. The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen. Cell. 1989 May 5;57(3):379–392. doi: 10.1016/0092-8674(89)90913-6. [DOI] [PubMed] [Google Scholar]
  50. Weichselbraun I., Haider G., Wintersberger E. Optimal replication of plasmids carrying polyomavirus origin regions requires two high-affinity binding sites for large T antigen. J Virol. 1989 Feb;63(2):961–964. doi: 10.1128/jvi.63.2.961-964.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. de Villiers J., Schaffner W., Tyndall C., Lupton S., Kamen R. Polyoma virus DNA replication requires an enhancer. Nature. 1984 Nov 15;312(5991):242–246. doi: 10.1038/312242a0. [DOI] [PubMed] [Google Scholar]

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