Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Nov;71(11):8743–8749. doi: 10.1128/jvi.71.11.8743-8749.1997

The N-terminal side of the origin-binding domain of simian virus 40 large T antigen is involved in A/T untwisting.

L Chen 1, W S Joo 1, P A Bullock 1, D T Simmons 1
PMCID: PMC192339  PMID: 9343233

Abstract

We investigated the role of the N-terminal side of simian virus 40 (SV40) large T antigen's origin-binding domain in the initiation of virus DNA replication by analyzing the biochemical activities of mutants containing single point substitutions or deletions in this region. Four mutants with substitutions at residues between 121 and 135 were partially defective in untwisting the A/T-rich track on the late side of the origin but were normal in melting the imperfect palindrome (IP) region on the early side. Deletion of the N-terminal 109 amino acids had no effect on either activity, whereas a longer deletion, up to residue 123, greatly reduced A/T untwisting but not IP melting. These results indicate that the region from residue 121 to 135 is important for A/T untwisting but not for IP melting and demonstrate that these activities are separable. Two point substitution mutants (126PS and 135PL) were characterized further by testing them for origin DNA binding, origin unwinding, oligomerization, and helicase activity. These two mutants were completely defective in origin (form U(R)) unwinding but normal in the other activities. Our results demonstrate that a failure to normally untwist the A/T track is correlated with a defect in origin unwinding. Further, they indicate that some mutants with substitutions in the region from residue 121 to 135 interact with origin DNA incorrectly, perhaps by failing to make appropriate contacts with the A/T-rich DNA.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Arthur A. K., Höss A., Fanning E. Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids. J Virol. 1988 Jun;62(6):1999–2006. doi: 10.1128/jvi.62.6.1999-2006.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Borowiec J. A., Dean F. B., Hurwitz J. Differential induction of structural changes in the simian virus 40 origin of replication by T antigen. J Virol. 1991 Mar;65(3):1228–1235. doi: 10.1128/jvi.65.3.1228-1235.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Borowiec J. A. Inhibition of structural changes in the simian virus 40 core origin of replication by mutation of essential origin sequences. J Virol. 1992 Sep;66(9):5248–5255. doi: 10.1128/jvi.66.9.5248-5255.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bullock P. A., Joo W. S., Sreekumar K. R., Mello C. Initiation of SV40 DNA replication in vitro: analysis of the role played by sequences flanking the core origin on initial synthesis events. Virology. 1997 Jan 20;227(2):460–473. doi: 10.1006/viro.1996.8347. [DOI] [PubMed] [Google Scholar]
  6. Bullock P. A., Seo Y. S., Hurwitz J. Initiation of simian virus 40 DNA synthesis in vitro. Mol Cell Biol. 1991 May;11(5):2350–2361. doi: 10.1128/mcb.11.5.2350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cegielska A., Moarefi I., Fanning E., Virshup D. M. T-antigen kinase inhibits simian virus 40 DNA replication by phosphorylation of intact T antigen on serines 120 and 123. J Virol. 1994 Jan;68(1):269–275. doi: 10.1128/jvi.68.1.269-275.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Collins K. L., Kelly T. J. Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase. Mol Cell Biol. 1991 Apr;11(4):2108–2115. doi: 10.1128/mcb.11.4.2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DeLucia A. L., Deb S., Partin K., Tegtmeyer P. Functional interactions of the simian virus 40 core origin of replication with flanking regulatory sequences. J Virol. 1986 Jan;57(1):138–144. doi: 10.1128/jvi.57.1.138-144.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dean F. B., Borowiec J. A., Ishimi Y., Deb S., Tegtmeyer P., Hurwitz J. Simian virus 40 large tumor antigen requires three core replication origin domains for DNA unwinding and replication in vitro. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8267–8271. doi: 10.1073/pnas.84.23.8267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Dean F. B., Hurwitz J. Simian virus 40 large T antigen untwists DNA at the origin of DNA replication. J Biol Chem. 1991 Mar 15;266(8):5062–5071. [PubMed] [Google Scholar]
  13. Deb S., DeLucia A. L., Baur C. P., Koff A., Tegtmeyer P. Domain structure of the simian virus 40 core origin of replication. Mol Cell Biol. 1986 May;6(5):1663–1670. doi: 10.1128/mcb.6.5.1663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Deb S., Tsui S., Koff A., DeLucia A. L., Parsons R., Tegtmeyer P. The T-antigen-binding domain of the simian virus 40 core origin of replication. J Virol. 1987 Jul;61(7):2143–2149. doi: 10.1128/jvi.61.7.2143-2149.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Denis D., Bullock P. A. Primer-DNA formation during simian virus 40 DNA replication in vitro. Mol Cell Biol. 1993 May;13(5):2882–2890. doi: 10.1128/mcb.13.5.2882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dodson M., Dean F. B., Bullock P., Echols H., Hurwitz J. Unwinding of duplex DNA from the SV40 origin of replication by T antigen. Science. 1987 Nov 13;238(4829):964–967. doi: 10.1126/science.2823389. [DOI] [PubMed] [Google Scholar]
  18. Dornreiter I., Copeland W. C., Wang T. S. Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen. Mol Cell Biol. 1993 Feb;13(2):809–820. doi: 10.1128/mcb.13.2.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Dornreiter I., Höss A., Arthur A. K., Fanning E. SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha. EMBO J. 1990 Oct;9(10):3329–3336. doi: 10.1002/j.1460-2075.1990.tb07533.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Eki T., Matsumoto T., Murakami Y., Hurwitz J. The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems. J Biol Chem. 1992 Apr 15;267(11):7284–7294. [PubMed] [Google Scholar]
  22. 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]
  23. Gralla J. D. Rapid "footprinting" on supercoiled DNA. Proc Natl Acad Sci U S A. 1985 May;82(10):3078–3081. doi: 10.1073/pnas.82.10.3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Grässer F. A., Mann K., Walter G. Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding. J Virol. 1987 Nov;61(11):3373–3380. doi: 10.1128/jvi.61.11.3373-3380.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gurney E. G., Harrison R. O., Fenno J. Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct sublcasses of large T antigen and for similarities among nonviral T antigens. J Virol. 1980 Jun;34(3):752–763. doi: 10.1128/jvi.34.3.752-763.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gurney E. G., Tamowski S., Deppert W. Antigenic binding sites of monoclonal antibodies specific for simian virus 40 large T antigen. J Virol. 1986 Mar;57(3):1168–1172. doi: 10.1128/jvi.57.3.1168-1172.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Han F. X., Hurley L. H. A model for the T-antigen-induced structural alteration of the SV40 replication origin based upon experiments with specific probes for bent, straight, and unwound DNA. Biochemistry. 1996 Jun 18;35(24):7993–8001. doi: 10.1021/bi960251d. [DOI] [PubMed] [Google Scholar]
  28. Harlow E., Crawford L. V., Pim D. C., Williamson N. M. Monoclonal antibodies specific for simian virus 40 tumor antigens. J Virol. 1981 Sep;39(3):861–869. doi: 10.1128/jvi.39.3.861-869.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kalderon D., Smith A. E. In vitro mutagenesis of a putative DNA binding domain of SV40 large-T. Virology. 1984 Nov;139(1):109–137. doi: 10.1016/0042-6822(84)90334-9. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Lin H. J., Upson R. H., Simmons D. T. Nonspecific DNA binding activity of simian virus 40 large T antigen: evidence for the cooperation of two regions for full activity. J Virol. 1992 Sep;66(9):5443–5452. doi: 10.1128/jvi.66.9.5443-5452.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Loeber G., Parsons R., Tegtmeyer P. The zinc finger region of simian virus 40 large T antigen. J Virol. 1989 Jan;63(1):94–100. doi: 10.1128/jvi.63.1.94-100.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Luo X., Sanford D. G., Bullock P. A., Bachovchin W. W. Solution structure of the origin DNA-binding domain of SV40 T-antigen. Nat Struct Biol. 1996 Dec;3(12):1034–1039. doi: 10.1038/nsb1296-1034. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. McVey D., Brizuela L., Mohr I., Marshak D. R., Gluzman Y., Beach D. Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication. Nature. 1989 Oct 12;341(6242):503–507. doi: 10.1038/341503a0. [DOI] [PubMed] [Google Scholar]
  36. McVey D., Ray S., Gluzman Y., Berger L., Wildeman A. G., Marshak D. R., Tegtmeyer P. cdc2 phosphorylation of threonine 124 activates the origin-unwinding functions of simian virus 40 T antigen. J Virol. 1993 Sep;67(9):5206–5215. doi: 10.1128/jvi.67.9.5206-5215.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. McVey D., Woelker B., Tegtmeyer P. Mechanisms of simian virus 40 T-antigen activation by phosphorylation of threonine 124. J Virol. 1996 Jun;70(6):3887–3893. doi: 10.1128/jvi.70.6.3887-3893.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Melendy T., Stillman B. An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication. J Biol Chem. 1993 Feb 15;268(5):3389–3395. [PubMed] [Google Scholar]
  39. Moarefi I. F., Small D., Gilbert I., Höpfner M., Randall S. K., Schneider C., Russo A. A., Ramsperger U., Arthur A. K., Stahl H. Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding. J Virol. 1993 Aug;67(8):4992–5002. doi: 10.1128/jvi.67.8.4992-5002.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mohr I. J., Stillman B., Gluzman Y. Regulation of SV40 DNA replication by phosphorylation of T antigen. EMBO J. 1987 Jan;6(1):153–160. doi: 10.1002/j.1460-2075.1987.tb04733.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Murakami Y., Hurwitz J. DNA polymerase alpha stimulates the ATP-dependent binding of simian virus tumor T antigen to the SV40 origin of replication. J Biol Chem. 1993 May 25;268(15):11018–11027. [PubMed] [Google Scholar]
  42. Nethanel T., Kaufmann G. Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40. J Virol. 1990 Dec;64(12):5912–5918. doi: 10.1128/jvi.64.12.5912-5918.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Parsons R. E., Stenger J. E., Ray S., Welker R., Anderson M. E., Tegtmeyer P. Cooperative assembly of simian virus 40 T-antigen hexamers on functional halves of the replication origin. J Virol. 1991 Jun;65(6):2798–2806. doi: 10.1128/jvi.65.6.2798-2806.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Roberts J. M. Simian virus 40 (SV40) large tumor antigen causes stepwise changes in SV40 origin structure during initiation of DNA replication. Proc Natl Acad Sci U S A. 1989 Jun;86(11):3939–3943. doi: 10.1073/pnas.86.11.3939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Scheffner M., Wessel R., Stahl H. Sequence independent duplex DNA opening reaction catalysed by SV40 large tumor antigen. Nucleic Acids Res. 1989 Jan 11;17(1):93–106. doi: 10.1093/nar/17.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schneider J., Fanning E. Mutations in the phosphorylation sites of simian virus 40 (SV40) T antigen alter its origin DNA-binding specificity for sites I or II and affect SV40 DNA replication activity. J Virol. 1988 May;62(5):1598–1605. doi: 10.1128/jvi.62.5.1598-1605.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Simmons D. T., Chou W., Rodgers K. Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen. J Virol. 1986 Dec;60(3):888–894. doi: 10.1128/jvi.60.3.888-894.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Simmons D. T. DNA-binding region of the simian virus 40 tumor antigen. J Virol. 1986 Mar;57(3):776–785. doi: 10.1128/jvi.57.3.776-785.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Simmons D. T., Loeber G., Tegtmeyer P. Four major sequence elements of simian virus 40 large T antigen coordinate its specific and nonspecific DNA binding. J Virol. 1990 May;64(5):1973–1983. doi: 10.1128/jvi.64.5.1973-1983.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Simmons D. T., Upson R., Wun-Kim K., Young W. Biochemical analysis of mutants with changes in the origin-binding domain of simian virus 40 tumor antigen. J Virol. 1993 Jul;67(7):4227–4236. doi: 10.1128/jvi.67.7.4227-4236.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Simmons D. T., Wun-Kim K., Young W. Identification of simian virus 40 T-antigen residues important for specific and nonspecific binding to DNA and for helicase activity. J Virol. 1990 Oct;64(10):4858–4865. doi: 10.1128/jvi.64.10.4858-4865.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Stillman B. W., Gluzman Y. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol Cell Biol. 1985 Aug;5(8):2051–2060. doi: 10.1128/mcb.5.8.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Strauss M., Argani P., Mohr I. J., Gluzman Y. Studies on the origin-specific DNA-binding domain of simian virus 40 large T antigen. J Virol. 1987 Oct;61(10):3326–3330. doi: 10.1128/jvi.61.10.3326-3330.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Tsurimoto T., Fairman M. P., Stillman B. Simian virus 40 DNA replication in vitro: identification of multiple stages of initiation. Mol Cell Biol. 1989 Sep;9(9):3839–3849. doi: 10.1128/mcb.9.9.3839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Tsurimoto T., Stillman B. Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis. J Biol Chem. 1991 Jan 25;266(3):1961–1968. [PubMed] [Google Scholar]
  59. Virshup D. M., Russo A. A., Kelly T. J. Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A. Mol Cell Biol. 1992 Nov;12(11):4883–4895. doi: 10.1128/mcb.12.11.4883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Waga S., Stillman B. Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nature. 1994 May 19;369(6477):207–212. doi: 10.1038/369207a0. [DOI] [PubMed] [Google Scholar]
  61. Weinberg D. H., Collins K. L., Simancek P., Russo A., Wold M. S., Virshup D. M., Kelly T. J. Reconstitution of simian virus 40 DNA replication with purified proteins. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8692–8696. doi: 10.1073/pnas.87.22.8692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. 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]
  63. Wiekowski M., Dröge P., Stahl H. Monoclonal antibodies as probes for a function of large T antigen during the elongation process of simian virus 40 DNA replication. J Virol. 1987 Feb;61(2):411–418. doi: 10.1128/jvi.61.2.411-418.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. 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]
  65. 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]
  66. 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]
  67. Wun-Kim K., Simmons D. T. Mapping of helicase and helicase substrate-binding domains on simian virus 40 large T antigen. J Virol. 1990 May;64(5):2014–2020. doi: 10.1128/jvi.64.5.2014-2020.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wun-Kim K., Upson R., Young W., Melendy T., Stillman B., Simmons D. T. The DNA-binding domain of simian virus 40 tumor antigen has multiple functions. J Virol. 1993 Dec;67(12):7608–7611. doi: 10.1128/jvi.67.12.7608-7611.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES