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. 1993 Apr 25;21(8):1689–1695. doi: 10.1093/nar/21.8.1689

Topoisomerase activity associated with SV40 large tumor antigen.

A Marton 1, D Jean 1, L Delbecchi 1, D T Simmons 1, P Bourgaux 1
PMCID: PMC309402  PMID: 8388093

Abstract

Purified preparations of simian virus 40 (SV40) large tumor antigen (LT) from three different sources, including LT expressed from a recombinant baculovirus, were found to relax negatively supercoiled cyclic DNA molecules, whether or not they contained SV40 sequences. Relaxation was stimulated by MgCl2 but not by ATP, and inhibited by camptothecin, suggesting the involvement of an enzymatic activity similar to that of topoisomerase I (topo I). However, the pH requirements for relaxation by respectively LT and topo I are different. Also, antibodies reacting with LT inhibited relaxation by preparations of LT but not topo I, whereas antibodies inhibiting relaxation by topo I had no effect on relaxation by LT. Reconstruction experiments suggested that both procedures used to purify LT, immunoaffinity chromatography and DEAE-Sepharose chromatography, separate topo I from LT. Finally, relaxing activity was found in over 40 preparations of LT, and in the few instances where activity could not be found, it probably had been lost during storage, rather than absent from the start. Whereas these results seem to exclude that the activity being detected is that of a contaminant of LT, they would be consistent with this activity being that of a stable topo-LT complex, or else intrinsic to LT itself.

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Selected References

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

  1. Alwine J. C. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins. Mol Cell Biol. 1985 May;5(5):1034–1042. doi: 10.1128/mcb.5.5.1034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Basilico C., Gattoni S., Zouzias D., Valle G. D. Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function. Cell. 1979 Jul;17(3):645–659. doi: 10.1016/0092-8674(79)90272-1. [DOI] [PubMed] [Google Scholar]
  3. Bauer W., Vinograd J. The interaction of closed circular DNA with intercalative dyes. I. The superhelix density of SV40 DNA in the presence and absence of dye. J Mol Biol. 1968 Apr 14;33(1):141–171. doi: 10.1016/0022-2836(68)90286-6. [DOI] [PubMed] [Google Scholar]
  4. Botchan M., Stringer J., Mitchison T., Sambrook J. Integration and excision of SV40 DNA from the chromosome of a transformed cell. Cell. 1980 May;20(1):143–152. doi: 10.1016/0092-8674(80)90242-1. [DOI] [PubMed] [Google Scholar]
  5. Botchan M., Topp W., Sambrook J. Studies on simian virus 40 excision from cellular chromosomes. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):709–719. doi: 10.1101/sqb.1979.043.01.079. [DOI] [PubMed] [Google Scholar]
  6. Brady J., Bolen J. B., Radonovich M., Salzman N., Khoury G. Stimulation of simian virus 40 late gene expression by simian virus 40 tumor antigen. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2040–2044. doi: 10.1073/pnas.81.7.2040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown E. H., Basilico C. Induction of sister chromatid exchange by polyoma large viral tumor antigen in transformed rat fibroblasts. Cancer Res. 1982 May;42(5):1909–1912. [PubMed] [Google Scholar]
  8. Carroll R. B., Hager L., Dulbecco R. Simian virus 40 T antigen binds to DNA. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3754–3757. doi: 10.1073/pnas.71.9.3754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chia W., Rigby P. W. Fate of viral DNA in nonpermissive cells infected with simian virus 40. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6638–6642. doi: 10.1073/pnas.78.11.6638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Colantuoni V., Dailey L., Valle G. D., Basilico C. Requirements for excision and amplification of integrated viral DNA molecules in polyoma virus-transformed cells. J Virol. 1982 Aug;43(2):617–628. doi: 10.1128/jvi.43.2.617-628.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DeCaprio J. A., Ludlow J. W., Figge J., Shew J. Y., Huang C. M., Lee W. H., Marsilio E., Paucha E., Livingston D. M. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell. 1988 Jul 15;54(2):275–283. doi: 10.1016/0092-8674(88)90559-4. [DOI] [PubMed] [Google Scholar]
  13. Della Valle G., Fenton R. G., Basilico C. Polyoma large T antigen regulates the integration of viral DNA sequences into the genome of transformed cells. Cell. 1981 Feb;23(2):347–355. doi: 10.1016/0092-8674(81)90130-6. [DOI] [PubMed] [Google Scholar]
  14. DiMaio D., Nathans D. Regulatory mutants of simian virus 40. Effect of mutations at a T antigen binding site on DNA replication and expression of viral genes. J Mol Biol. 1982 Apr 15;156(3):531–548. doi: 10.1016/0022-2836(82)90265-0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Drlica K., Franco R. J. Inhibitors of DNA topoisomerases. Biochemistry. 1988 Apr 5;27(7):2253–2259. doi: 10.1021/bi00407a001. [DOI] [PubMed] [Google Scholar]
  18. GRACE T. D. Establishment of four strains of cells from insect tissues grown in vitro. Nature. 1962 Aug 25;195:788–789. doi: 10.1038/195788a0. [DOI] [PubMed] [Google Scholar]
  19. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  20. Guerry P., LeBlanc D. J., Falkow S. General method for the isolation of plasmid deoxyribonucleic acid. J Bacteriol. 1973 Nov;116(2):1064–1066. doi: 10.1128/jb.116.2.1064-1066.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Hsiang Y. H., Hertzberg R., Hecht S., Liu L. F. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 1985 Nov 25;260(27):14873–14878. [PubMed] [Google Scholar]
  23. Javaherian K., Tse Y. C., Vega J. Drosophila topoisomerase I: isolation, purification and characterization. Nucleic Acids Res. 1982 Nov 11;10(21):6945–6955. doi: 10.1093/nar/10.21.6945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Keller J. M., Alwine J. C. Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication. Cell. 1984 Feb;36(2):381–389. doi: 10.1016/0092-8674(84)90231-9. [DOI] [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Lane D. P., Crawford L. V. T antigen is bound to a host protein in SV40-transformed cells. Nature. 1979 Mar 15;278(5701):261–263. doi: 10.1038/278261a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Linzer D. I., Levine A. J. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell. 1979 May;17(1):43–52. doi: 10.1016/0092-8674(79)90293-9. [DOI] [PubMed] [Google Scholar]
  29. MacArthur H., Walter G. Monoclonal antibodies specific for the carboxy terminus of simian virus 40 large T antigen. J Virol. 1984 Nov;52(2):483–491. doi: 10.1128/jvi.52.2.483-491.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Miller J., Bullock P., Botchan M. Simian virus 40 T antigen is required for viral excision from chromosomes. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7534–7538. doi: 10.1073/pnas.81.23.7534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nichols W. W., Bradt C. I., Toji L. H., Godley M., Segawa M. Induction of sister chromatid exchanges by transformation with simian virus 40. Cancer Res. 1978 Apr;38(4):960–964. [PubMed] [Google Scholar]
  32. Norkin L. C., Steinberg V. I., Kosz-Vnenchak M. Human glioblastoma cells persistently infected with simian virus 40 carry nondefective episomal viral DNA and acquire the transformed phenotype and numerous chromosomal abnormalities. J Virol. 1985 Feb;53(2):658–666. doi: 10.1128/jvi.53.2.658-666.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. O'Reilly D. R., Miller L. K. Expression and complex formation of simian virus 40 large T antigen and mouse p53 in insect cells. J Virol. 1988 Sep;62(9):3109–3119. doi: 10.1128/jvi.62.9.3109-3119.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Piccinini G., Cardellini E., Reimer G., Arnett F. C., Durban E. An antigenic region of topoisomerase I in DNA polymerase chain reaction-generated fragments recognized by autoantibodies of scleroderma patients. Mol Immunol. 1991 Apr-May;28(4-5):333–339. doi: 10.1016/0161-5890(91)90145-a. [DOI] [PubMed] [Google Scholar]
  35. Piché A., Bourgaux P. Resolution of a polyomavirus-mouse hybrid replicon: viral function required for recombination. J Virol. 1987 Mar;61(3):845–850. doi: 10.1128/jvi.61.3.845-850.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Sadowski P. Site-specific recombinases: changing partners and doing the twist. J Bacteriol. 1986 Feb;165(2):341–347. doi: 10.1128/jb.165.2.341-347.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Scheffner M., Knippers R., Stahl H. RNA unwinding activity of SV40 large T antigen. Cell. 1989 Jun 16;57(6):955–963. doi: 10.1016/0092-8674(89)90334-6. [DOI] [PubMed] [Google Scholar]
  39. Schiedner G., Wessel R., Scheffner M., Stahl H. Renaturation and DNA looping promoted by the SV40 large tumour antigen. EMBO J. 1990 Sep;9(9):2937–2943. doi: 10.1002/j.1460-2075.1990.tb07485.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schneider C., Newman R. A., Sutherland D. R., Asser U., Greaves M. F. A one-step purification of membrane proteins using a high efficiency immunomatrix. J Biol Chem. 1982 Sep 25;257(18):10766–10769. [PubMed] [Google Scholar]
  41. Shalloway D., Kleinberger T., Livingston D. M. Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III digestion. Cell. 1980 Jun;20(2):411–422. doi: 10.1016/0092-8674(80)90627-3. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. 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]
  45. 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]
  46. Tjian R. The binding site on SV40 DNA for a T antigen-related protein. Cell. 1978 Jan;13(1):165–179. doi: 10.1016/0092-8674(78)90147-2. [DOI] [PubMed] [Google Scholar]
  47. Twigg A. J., Sherratt D. Trans-complementable copy-number mutants of plasmid ColE1. Nature. 1980 Jan 10;283(5743):216–218. doi: 10.1038/283216a0. [DOI] [PubMed] [Google Scholar]
  48. Vu H. K., Delbecchi L., Bourgaux-Ramoisy D., Bourgaux P. The same mammalian replicon yields distinct recombination products in different cell lines. J Biol Chem. 1991 May 15;266(14):9320–9326. [PubMed] [Google Scholar]
  49. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]

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