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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Nov;87(22):8692–8696. doi: 10.1073/pnas.87.22.8692

Reconstitution of simian virus 40 DNA replication with purified proteins.

D H Weinberg 1, K L Collins 1, P Simancek 1, A Russo 1, M S Wold 1, D M Virshup 1, T J Kelly 1
PMCID: PMC55025  PMID: 2174160

Abstract

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.

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Selected References

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

  1. 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]
  2. 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]
  3. Challberg M. D., Kelly T. J. Animal virus DNA replication. Annu Rev Biochem. 1989;58:671–717. doi: 10.1146/annurev.bi.58.070189.003323. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Fairman M. P., Stillman B. Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J. 1988 Apr;7(4):1211–1218. doi: 10.1002/j.1460-2075.1988.tb02933.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Ishimi Y., Claude A., Bullock P., Hurwitz J. Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J Biol Chem. 1988 Dec 25;263(36):19723–19733. [PubMed] [Google Scholar]
  9. Lee M. Y., Tan C. K., Downey K. M., So A. G. Further studies on calf thymus DNA polymerase delta purified to homogeneity by a new procedure. Biochemistry. 1984 Apr 24;23(9):1906–1913. doi: 10.1021/bi00304a003. [DOI] [PubMed] [Google Scholar]
  10. Lee S. H., Eki T., Hurwitz J. Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7361–7365. doi: 10.1073/pnas.86.19.7361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6973–6977. doi: 10.1073/pnas.81.22.6973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication. Mol Cell Biol. 1985 Jun;5(6):1238–1246. doi: 10.1128/mcb.5.6.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Prelich G., Kostura M., Marshak D. R., Mathews M. B., Stillman B. The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vitro. Nature. 1987 Apr 2;326(6112):471–475. doi: 10.1038/326471a0. [DOI] [PubMed] [Google Scholar]
  17. Prelich G., Stillman B. Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA. Cell. 1988 Apr 8;53(1):117–126. doi: 10.1016/0092-8674(88)90493-x. [DOI] [PubMed] [Google Scholar]
  18. Prelich G., Tan C. K., Kostura M., Mathews M. B., So A. G., Downey K. M., Stillman B. Functional identity of proliferating cell nuclear antigen and a DNA polymerase-delta auxiliary protein. Nature. 1987 Apr 2;326(6112):517–520. doi: 10.1038/326517a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Rosenfeld P. J., Kelly T. J. Purification of nuclear factor I by DNA recognition site affinity chromatography. J Biol Chem. 1986 Jan 25;261(3):1398–1408. [PubMed] [Google Scholar]
  21. 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]
  22. Stillman B. Initiation of eukaryotic DNA replication in vitro. Annu Rev Cell Biol. 1989;5:197–245. doi: 10.1146/annurev.cb.05.110189.001213. [DOI] [PubMed] [Google Scholar]
  23. Tsurimoto T., Stillman B. Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta. EMBO J. 1989 Dec 1;8(12):3883–3889. doi: 10.1002/j.1460-2075.1989.tb08567.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tsurimoto T., Stillman B. Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro. Mol Cell Biol. 1989 Feb;9(2):609–619. doi: 10.1128/mcb.9.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Virshup D. M., Kauffman M. G., Kelly T. J. Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A. EMBO J. 1989 Dec 1;8(12):3891–3898. doi: 10.1002/j.1460-2075.1989.tb08568.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Virshup D. M., Kelly T. J. Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1989 May;86(10):3584–3588. doi: 10.1073/pnas.86.10.3584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weinberg D. H., Kelly T. J. Requirement for two DNA polymerases in the replication of simian virus 40 DNA in vitro. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9742–9746. doi: 10.1073/pnas.86.24.9742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Wobbe C. R., Weissbach L., Borowiec J. A., Dean F. B., Murakami Y., Bullock P., Hurwitz J. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1834–1838. doi: 10.1073/pnas.84.7.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wold M. S., Kelly T. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2523–2527. doi: 10.1073/pnas.85.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Wold M. S., Weinberg D. H., Virshup D. M., Li J. J., Kelly T. J. Identification of cellular proteins required for simian virus 40 DNA replication. J Biol Chem. 1989 Feb 15;264(5):2801–2809. [PubMed] [Google Scholar]
  34. Yang L., Wold M. S., Li J. J., Kelly T. J., Liu L. F. Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1987 Feb;84(4):950–954. doi: 10.1073/pnas.84.4.950. [DOI] [PMC free article] [PubMed] [Google Scholar]

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