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. 1989 Dec;9(12):5525–5536. doi: 10.1128/mcb.9.12.5525

Properties of the DNA-binding domain of the simian virus 40 large T antigen.

D McVey 1, M Strauss 1, Y Gluzman 1
PMCID: PMC363723  PMID: 2555700

Abstract

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.

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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. 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]
  3. Auborn K., Guo M., Prives C. Helicase, DNA-binding, and immunological properties of replication-defective simian virus 40 mutant T antigens. J Virol. 1989 Feb;63(2):912–918. doi: 10.1128/jvi.63.2.912-918.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  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. Bradley M. K., Hudson J., Villanueva M. S., Livingston D. M. Specific in vitro adenylylation of the simian virus 40 large tumor antigen. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6574–6578. doi: 10.1073/pnas.81.21.6574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Burger C., Fanning E. Specific DNA binding activity of T antigen subclasses varies among different SV40-transformed cell lines. Virology. 1983 Apr 15;126(1):19–31. doi: 10.1016/0042-6822(83)90459-2. [DOI] [PubMed] [Google Scholar]
  10. Clark R., Peden K., Pipas J. M., Nathans D., Tjian R. Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants. Mol Cell Biol. 1983 Feb;3(2):220–228. doi: 10.1128/mcb.3.2.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeLucia A. L., Lewton B. A., Tjian R., Tegtmeyer P. Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication. J Virol. 1983 Apr;46(1):143–150. doi: 10.1128/jvi.46.1.143-150.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. Dixon R. A., Nathans D. Purification of simian virus 40 large T antigen by immunoaffinity chromatography. J Virol. 1985 Mar;53(3):1001–1004. doi: 10.1128/jvi.53.3.1001-1004.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Evans R. M., Hollenberg S. M. Zinc fingers: gilt by association. Cell. 1988 Jan 15;52(1):1–3. doi: 10.1016/0092-8674(88)90522-3. [DOI] [PubMed] [Google Scholar]
  18. Fanning E., Westphal K. H., Brauer D., Cörlin D. Subclasses of simian virus 40 large T antigen: differential binding of two subclasses of T antigen from productively infected cells to viral and cellular DNA. EMBO J. 1982;1(9):1023–1028. doi: 10.1002/j.1460-2075.1982.tb01290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Gish W. R., Botchan M. R. Simian virus 40-transformed human cells that express large T antigens defective for viral DNA replication. J Virol. 1987 Sep;61(9):2864–2876. doi: 10.1128/jvi.61.9.2864-2876.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gluzman Y., Frisque R. J., Sambrook J. Origin-defective mutants of SV40. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):293–300. doi: 10.1101/sqb.1980.044.01.033. [DOI] [PubMed] [Google Scholar]
  22. Goldman N., Brown M., Khoury G. Modification of SV40 T antigen by poly ADP-ribosylation. Cell. 1981 May;24(2):567–572. doi: 10.1016/0092-8674(81)90347-0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Hanas J. S., Hazuda D. J., Bogenhagen D. F., Wu F. Y., Wu C. W. Xenopus transcription factor A requires zinc for binding to the 5 S RNA gene. J Biol Chem. 1983 Dec 10;258(23):14120–14125. [PubMed] [Google Scholar]
  25. Hansen U., Tenen D. G., Livingston D. M., Sharp P. A. T antigen repression of SV40 early transcription from two promoters. Cell. 1981 Dec;27(3 Pt 2):603–613. doi: 10.1016/0092-8674(81)90402-5. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Hoey T., Warrior R., Manak J., Levine M. DNA-binding activities of the Drosophila melanogaster even-skipped protein are mediated by its homeo domain and influenced by protein context. Mol Cell Biol. 1988 Nov;8(11):4598–4607. doi: 10.1128/mcb.8.11.4598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jarvis D. L., Butel J. S. Modification of simian virus 40 large tumor antigen by glycosylation. Virology. 1985 Mar;141(2):173–189. doi: 10.1016/0042-6822(85)90250-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jones K. A., Tjian R. Essential contact residues within SV40 large T antigen binding sites I and II identified by alkylation-interference. Cell. 1984 Jan;36(1):155–162. doi: 10.1016/0092-8674(84)90084-9. [DOI] [PubMed] [Google Scholar]
  30. Kadonaga J. T., Carner K. R., Masiarz F. R., Tjian R. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell. 1987 Dec 24;51(6):1079–1090. doi: 10.1016/0092-8674(87)90594-0. [DOI] [PubMed] [Google Scholar]
  31. Kadonaga J. T., Courey A. J., Ladika J., Tjian R. Distinct regions of Sp1 modulate DNA binding and transcriptional activation. Science. 1988 Dec 16;242(4885):1566–1570. doi: 10.1126/science.3059495. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Klausing K., Scheffner M., Scheidtmann K. H., Stahl H., Knippers R. DNA binding properties and replication activity of the T antigen related D2 phosphoprotein. Biochemistry. 1989 Mar 7;28(5):2238–2244. doi: 10.1021/bi00431a040. [DOI] [PubMed] [Google Scholar]
  34. Klausing K., Scheidtmann K. H., Baumann E. A., Knippers R. Effects of in vitro dephosphorylation on DNA-binding and DNA helicase activities of simian virus 40 large tumor antigen. J Virol. 1988 Apr;62(4):1258–1265. doi: 10.1128/jvi.62.4.1258-1265.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Klockmann U., Deppert W. Acylated simian virus 40 large T-antigen: a new subclass associated with a detergent-resistant lamina of the plasma membrane. EMBO J. 1983;2(7):1151–1157. doi: 10.1002/j.1460-2075.1983.tb01560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Laughon A., Scott M. P. Sequence of a Drosophila segmentation gene: protein structure homology with DNA-binding proteins. Nature. 1984 Jul 5;310(5972):25–31. doi: 10.1038/310025a0. [DOI] [PubMed] [Google Scholar]
  37. Li J. J., Peden K. W., Dixon R. A., Kelly T. Functional organization of the simian virus 40 origin of DNA replication. Mol Cell Biol. 1986 Apr;6(4):1117–1128. doi: 10.1128/mcb.6.4.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Margolskee R. F., Nathans D. Simian virus 40 mutant T antigens with relaxed specificity for the nucleotide sequence at the viral DNA origin of replication. J Virol. 1984 Feb;49(2):386–393. doi: 10.1128/jvi.49.2.386-393.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. McKay R. D. Binding of a simian virus 40 T antigen-related protein to DNA. J Mol Biol. 1981 Jan 25;145(3):471–488. doi: 10.1016/0022-2836(81)90540-4. [DOI] [PubMed] [Google Scholar]
  43. Mohr I. J., Fairman M. P., Stillman B., Gluzman Y. Large T-antigen mutants define multiple steps in the initiation of simian virus 40 DNA replication. J Virol. 1989 Oct;63(10):4181–4188. doi: 10.1128/jvi.63.10.4181-4188.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Mohr I. J., Gluzman Y., Fairman M. P., Strauss M., McVey D., Stillman B., Gerard R. D. Production of simian virus 40 large tumor antigen in bacteria: altered DNA-binding specificity and dna-replication activity of underphosphorylated large tumor antigen. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6479–6483. doi: 10.1073/pnas.86.17.6479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Mole S. E., Gannon J. V., Ford M. J., Lane D. P. Structure and function of SV40 large-T antigen. Philos Trans R Soc Lond B Biol Sci. 1987 Dec 15;317(1187):455–469. doi: 10.1098/rstb.1987.0072. [DOI] [PubMed] [Google Scholar]
  47. Newport J. W., Lonberg N., Kowalczykowski S. C., von Hippel P. H. Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids. II. Specificity of binding to DNA and RNA. J Mol Biol. 1981 Jan 5;145(1):105–121. doi: 10.1016/0022-2836(81)90336-3. [DOI] [PubMed] [Google Scholar]
  48. Otting G., Qian Y. Q., Müller M., Affolter M., Gehring W., Wüthrich K. Secondary structure determination for the Antennapedia homeodomain by nuclear magnetic resonance and evidence for a helix-turn-helix motif. EMBO J. 1988 Dec 20;7(13):4305–4309. doi: 10.1002/j.1460-2075.1988.tb03329.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Prives C., Covey L., Scheller A., Gluzman Y. DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication. Mol Cell Biol. 1983 Nov;3(11):1958–1966. doi: 10.1128/mcb.3.11.1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rio D. C., Tjian R. SV40 T antigen binding site mutations that affect autoregulation. Cell. 1983 Apr;32(4):1227–1240. doi: 10.1016/0092-8674(83)90305-7. [DOI] [PubMed] [Google Scholar]
  51. Rio D., Robbins A., Myers R., Tjian R. Regulation of simian virus 40 early transcription in vitro by a purified tumor antigen. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5706–5710. doi: 10.1073/pnas.77.10.5706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Ryder K., Silver S., DeLucia A. L., Fanning E., Tegtmeyer P. An altered DNA conformation in origin region I is a determinant for the binding of SV40 large T antigen. Cell. 1986 Mar 14;44(5):719–725. doi: 10.1016/0092-8674(86)90838-x. [DOI] [PubMed] [Google Scholar]
  53. Ryder K., Vakalopoulou E., Mertz R., Mastrangelo I., Hough P., Tegtmeyer P., Fanning E. Seventeen base pairs of region I encode a novel tripartite binding signal for SV40 T antigen. Cell. 1985 Sep;42(2):539–548. doi: 10.1016/0092-8674(85)90111-4. [DOI] [PubMed] [Google Scholar]
  54. Sauer R. T., Yocum R. R., Doolittle R. F., Lewis M., Pabo C. O. Homology among DNA-binding proteins suggests use of a conserved super-secondary structure. Nature. 1982 Jul 29;298(5873):447–451. doi: 10.1038/298447a0. [DOI] [PubMed] [Google Scholar]
  55. Scheidtmann K. H., Echle B., Walter G. Simian virus 40 large T antigen is phosphorylated at multiple sites clustered in two separate regions. J Virol. 1982 Oct;44(1):116–133. doi: 10.1128/jvi.44.1.116-133.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Scheidtmann K. H., Hardung M., Echle B., Walter G. DNA-binding activity of simian virus 40 large T antigen correlates with a distinct phosphorylation state. J Virol. 1984 Apr;50(1):1–12. doi: 10.1128/jvi.50.1.1-12.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Shaw S. B., Tegtmeyer P. Binding of dephosphorylated A protein to SV40 DNA. Virology. 1981 Nov;115(1):88–96. doi: 10.1016/0042-6822(81)90091-x. [DOI] [PubMed] [Google Scholar]
  58. Shortle D. R., Margolskee R. F., Nathans D. Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6128–6131. doi: 10.1073/pnas.76.12.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Simanis V., Lane D. P. An immunoaffinity purification procedure for SV40 large T antigen. Virology. 1985 Jul 15;144(1):88–100. doi: 10.1016/0042-6822(85)90308-3. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. 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]
  62. Simmons D. T. Geometry of the simian virus 40 large tumor antigen-DNA complex as probed by protease digestion. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2086–2090. doi: 10.1073/pnas.85.7.2086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Spillman T., Giacherio D., Hager L. P. Single strand DNA binding of simian virus 40 tumor antigen. J Biol Chem. 1979 Apr 25;254(8):3100–3104. [PubMed] [Google Scholar]
  64. 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]
  65. Steitz T. A., Ohlendorf D. H., McKay D. B., Anderson W. F., Matthews B. W. Structural similarity in the DNA-binding domains of catabolite gene activator and cro repressor proteins. Proc Natl Acad Sci U S A. 1982 May;79(10):3097–3100. doi: 10.1073/pnas.79.10.3097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Stillman B., Gerard R. D., Guggenheimer R. A., Gluzman Y. T antigen and template requirements for SV40 DNA replication in vitro. EMBO J. 1985 Nov;4(11):2933–2939. doi: 10.1002/j.1460-2075.1985.tb04026.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. 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]
  68. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  69. Tegtmeyer P., Schwartz M., Collins J. K., Rundell K. Regulation of tumor antigen synthesis by simain virus 40 gene A. J Virol. 1975 Jul;16(1):168–178. doi: 10.1128/jvi.16.1.168-178.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Tegtmeyer P. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J Virol. 1972 Oct;10(4):591–598. doi: 10.1128/jvi.10.4.591-598.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Wilson V. G., Tevethia M. J., Lewton B. A., Tegtmeyer P. DNA binding properties of simian virus 40 temperature-sensitive A proteins. J Virol. 1982 Nov;44(2):458–466. doi: 10.1128/jvi.44.2.458-466.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. 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]

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