Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1984 Feb;3(2):303–307. doi: 10.1002/j.1460-2075.1984.tb01800.x

Salt-stable binding of the large T protein to DNA in polyoma virus chromatin.

P Clertant, P Gaudray, F Cuzin
PMCID: PMC557338  PMID: 6325165

Abstract

Nucleoprotein complexes extracted from the nuclei of mouse cells lytically infected with polyoma virus contain an ATPase activity which appears to correspond to that of the viral large T protein, as it exhibits the same characteristic properties; in particular, the activity is extensively inhibited by polyclonal antibodies from animals bearing polyoma tumors (anti-T antigen antibodies) and by monoclonal antibodies against large T. Significant amounts of DNA were immunoprecipitated by adding these antibodies to the nucleoprotein complex, suggesting that the protein is tightly bound to DNA in the viral chromatin. Since one of the monoclonal antibodies quantitatively immunoprecipitated the pulse-labeled replicative intermediates, we conclude that some large T protein remains physically associated with the DNA throughout its replication cycle. After exposure to salt concentrations higher than 1 M KCl, about half of the large T-specific ATPase activity was still observed to co-sediment with 21S form I viral DNA. The observations that the sedimentation coefficient of the salt-stable complexes was shifted to 16S after a limited endonucleolytic digestion, and that both the viral DNA and the ATPase activity were co-precipitated in the presence of polyethylene glycol at high ionic strength, further demonstrated that the protein is engaged in an unusually stable complex with DNA in the viral chromatin.

Full text

PDF
303

Selected References

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

  1. Bourgaux P., Bourgaux-Ramoisy D., Seiler P. The replication of the ring-shaped DNA of polyoma virus. II. Identification of molecules at various stages of replication. J Mol Biol. 1971 Jul 14;59(1):195–206. doi: 10.1016/0022-2836(71)90421-9. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Clertant P., Cuzin F. Initiation of polyoma virus DNA replication in vitro and its dependence on the viral gene A protein. Nucleic Acids Res. 1980 Oct 10;8(19):4377–4392. doi: 10.1093/nar/8.19.4377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cogen B. Virus-specific early RNA in 3T6 cells infected by a tsA mutant of polyoma virus. Virology. 1978 Mar;85(1):222–230. doi: 10.1016/0042-6822(78)90426-9. [DOI] [PubMed] [Google Scholar]
  6. Dilworth S. M., Griffin B. E. Monoclonal antibodies against polyoma virus tumor antigens. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1059–1063. doi: 10.1073/pnas.79.4.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dilworth S. M. Protein kinase activities associated with distinct antigenic forms of polyoma virus middle T-antigen. EMBO J. 1982;1(11):1319–1328. doi: 10.1002/j.1460-2075.1982.tb01317.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fanning E., Nowak B., Burger C. Detection and characterization of multiple forms of simian virus 40 large T antigen. J Virol. 1981 Jan;37(1):92–102. doi: 10.1128/jvi.37.1.92-102.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Francke B., Eckhart W. Polyoma gene function required for viral DNA synthesis. Virology. 1973 Sep;55(1):127–135. doi: 10.1016/s0042-6822(73)81014-1. [DOI] [PubMed] [Google Scholar]
  10. Gache C., Rossi B., Lazdunski M. (Na+, K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis. Eur J Biochem. 1976 May 17;65(1):293–306. doi: 10.1111/j.1432-1033.1976.tb10417.x. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Gaudray P., Tyndall C., Kamen R., Cuzin F. The high affinity binding site on polyoma virus DNA for the viral large-T protein. Nucleic Acids Res. 1981 Nov 11;9(21):5697–5710. doi: 10.1093/nar/9.21.5697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Magnusson G., Winnacker E. L., Eliasson R., Reichard P. Replication of polyoma DNA in isolated nuclei. II. Evidence for semi-conservative replication. J Mol Biol. 1972 Dec 30;72(3):539–552. doi: 10.1016/0022-2836(72)90173-8. [DOI] [PubMed] [Google Scholar]
  14. Mann K., Hunter T. Association of simian virus 40 T antigen with simian virus 40 nucleoprotein complexes. J Virol. 1979 Jan;29(1):232–241. doi: 10.1128/jvi.29.1.232-241.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mastromei G., Eliasson R., Reichard P. Stimulation of polyoma DNA replication in isolated nucleoprotein complexes by factors from Drosophila embryos. J Mol Biol. 1981 Oct 5;151(4):627–643. doi: 10.1016/0022-2836(81)90427-7. [DOI] [PubMed] [Google Scholar]
  16. Noguchi H., Prem veer Reddy G., Pardee A. B. Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei. Cell. 1983 Feb;32(2):443–451. doi: 10.1016/0092-8674(83)90464-6. [DOI] [PubMed] [Google Scholar]
  17. Rassoulzadegan M., Cowie A., Carr A., Glaichenhaus N., Kamen R., Cuzin F. The roles of individual polyoma virus early proteins in oncogenic transformation. Nature. 1982 Dec 23;300(5894):713–718. doi: 10.1038/300713a0. [DOI] [PubMed] [Google Scholar]
  18. Reiser J., Renart J., Crawford L. V., Stark G. R. Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin. J Virol. 1980 Jan;33(1):78–87. doi: 10.1128/jvi.33.1.78-87.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rudolph K., Mann K. Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes. J Virol. 1983 Aug;47(2):276–286. doi: 10.1128/jvi.47.2.276-286.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Segawa M., Sugano S., Yamaguchi N. Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40. J Virol. 1980 Aug;35(2):320–330. doi: 10.1128/jvi.35.2.320-330.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Waldeck W., Spaeren U., Mastromei G., Eliasson R., Reichard P. Replication of polyoma DNA in nuclear extracts and nucleoprotein complexes. J Mol Biol. 1979 Dec 15;135(3):675–689. doi: 10.1016/0022-2836(79)90171-2. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES