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. 1991 Apr;11(4):2108–2115. doi: 10.1128/mcb.11.4.2108

Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

K L Collins 1, T J Kelly 1
PMCID: PMC359898  PMID: 1848671

Abstract

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

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Selected References

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

  1. Alberts B. M. Prokaryotic DNA replication mechanisms. Philos Trans R Soc Lond B Biol Sci. 1987 Dec 15;317(1187):395–420. doi: 10.1098/rstb.1987.0068. [DOI] [PubMed] [Google Scholar]
  2. Arai K., Kornberg A. A general priming system employing only dnaB protein and primase for DNA replication. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4308–4312. doi: 10.1073/pnas.76.9.4308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Braithwaite A. W., Sturzbecher H. W., Addison C., Palmer C., Rudge K., Jenkins J. R. Mouse p53 inhibits SV40 origin-dependent DNA replication. Nature. 1987 Oct 1;329(6138):458–460. doi: 10.1038/329458a0. [DOI] [PubMed] [Google Scholar]
  4. Cha T. A., Alberts B. M. Effects of the bacteriophage T4 gene 41 and gene 32 proteins on RNA primer synthesis: coupling of leading- and lagging-strand DNA synthesis at a replication fork. Biochemistry. 1990 Feb 20;29(7):1791–1798. doi: 10.1021/bi00459a018. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. Gannon J. V., Lane D. P. Interactions between SV40 T antigen and DNA polymerase alpha. New Biol. 1990 Jan;2(1):84–92. [PubMed] [Google Scholar]
  10. Gannon J. V., Lane D. P. p53 and DNA polymerase alpha compete for binding to SV40 T antigen. Nature. 1987 Oct 1;329(6138):456–458. doi: 10.1038/329456a0. [DOI] [PubMed] [Google Scholar]
  11. Hurwitz J., Dean F. B., Kwong A. D., Lee S. H. The in vitro replication of DNA containing the SV40 origin. J Biol Chem. 1990 Oct 25;265(30):18043–18046. [PubMed] [Google Scholar]
  12. 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]
  13. Kelly T. J. SV40 DNA replication. J Biol Chem. 1988 Dec 5;263(34):17889–17892. [PubMed] [Google Scholar]
  14. 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]
  15. Lasken R. S., Kornberg A. The primosomal protein n' of Escherichia coli is a DNA helicase. J Biol Chem. 1988 Apr 25;263(12):5512–5518. [PubMed] [Google Scholar]
  16. LeBowitz J. H., McMacken R. The Escherichia coli dnaB replication protein is a DNA helicase. J Biol Chem. 1986 Apr 5;261(10):4738–4748. [PubMed] [Google Scholar]
  17. Lee M. S., Marians K. J. Escherichia coli replication factor Y, a component of the primosome, can act as a DNA helicase. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8345–8349. doi: 10.1073/pnas.84.23.8345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Lehman I. R., Kaguni L. S. DNA polymerase alpha. J Biol Chem. 1989 Mar 15;264(8):4265–4268. [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. Richardson C. C. Bacteriophage T7: minimal requirements for the replication of a duplex DNA molecule. Cell. 1983 Jun;33(2):315–317. doi: 10.1016/0092-8674(83)90411-7. [DOI] [PubMed] [Google Scholar]
  25. Richardson R. W., Nossal N. G. Trypsin cleavage in the COOH terminus of the bacteriophage T4 gene 41 DNA helicase alters the primase-helicase activities of the T4 replication complex in vitro. J Biol Chem. 1989 Mar 15;264(8):4732–4739. [PubMed] [Google Scholar]
  26. Smale S. T., Tjian R. T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication. Mol Cell Biol. 1986 Nov;6(11):4077–4087. doi: 10.1128/mcb.6.11.4077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Tsurimoto T., Melendy T., Stillman B. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature. 1990 Aug 9;346(6284):534–539. doi: 10.1038/346534a0. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Wang E. H., Friedman P. N., Prives C. The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen. Cell. 1989 May 5;57(3):379–392. doi: 10.1016/0092-8674(89)90913-6. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. 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]
  39. 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]

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