Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1987 Oct 12;15(19):7877–7888. doi: 10.1093/nar/15.19.7877

Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains.

J Taljanidisz 1, R S Decker 1, Z S Guo 1, M L DePamphilis 1, N Sarkar 1
PMCID: PMC306314  PMID: 2444924

Abstract

Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA.

Full text

PDF
7877

Images in this article

7882Image
on p.7882
7883Image
on p.7883

Selected References

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

  1. Anderson S., Kaufman G., DePamphilis M. L. RNA primers in SV40 DNA replication: identification of transient RNA-DNA covalent linkages in replicating DNA. Biochemistry. 1977 Nov 15;16(23):4990–4998. doi: 10.1021/bi00642a009. [DOI] [PubMed] [Google Scholar]
  2. Ariga H., Sugano S. Initiation of simian virus 40 DNA replication in vitro. J Virol. 1983 Nov;48(2):481–491. doi: 10.1128/jvi.48.2.481-491.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banfalvi G., Bhattacharya S., Sarkar N. Selective isolation of mercurated DNA by affinity chromatography on thiol matrices. Anal Biochem. 1985 Apr;146(1):64–70. doi: 10.1016/0003-2697(85)90396-3. [DOI] [PubMed] [Google Scholar]
  4. Banfalvi G., Sarkar N. Analysis of the 5'-termini of nascent DNA chains synthesized in permeable cells of Bacillus subtilis. J Mol Biol. 1983 Jan 15;163(2):147–169. doi: 10.1016/0022-2836(83)90001-3. [DOI] [PubMed] [Google Scholar]
  5. Banfalvi G., Sarkar N. Origin and degradation of the RNA primers at the 5' termini of nascent DNA chains in Bacillus subtilis. J Mol Biol. 1985 Nov 20;186(2):275–282. doi: 10.1016/0022-2836(85)90104-4. [DOI] [PubMed] [Google Scholar]
  6. Banfalvi G., Sooki-Toth A., Sarkar N., Csuzi S., Antoni F. Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes. Eur J Biochem. 1984 Mar 15;139(3):553–559. doi: 10.1111/j.1432-1033.1984.tb08041.x. [DOI] [PubMed] [Google Scholar]
  7. Bhattacharya S., Sarkar N. Study of deoxyribonucleic acid replication in permeable cells of Bacillus subtilis using mercurated nucleotide substrates. Biochemistry. 1981 May 26;20(11):3029–3034. doi: 10.1021/bi00514a007. [DOI] [PubMed] [Google Scholar]
  8. Bhattacharya S., Sarkar N. Transforming activity of mercury-substituted DNA synthesized in vitro by permeable cells of Bacillus subtilis. J Biol Chem. 1982 Feb 25;257(4):1610–1612. [PubMed] [Google Scholar]
  9. Cory J. G., Mansell M. M., Whitford T. W., Jr Control of ribonucleotide reductase in mammalian cells. Adv Enzyme Regul. 1976;14:45–62. doi: 10.1016/0065-2571(76)90007-8. [DOI] [PubMed] [Google Scholar]
  10. Decker R. S., Yamaguchi M., Possenti R., Bradley M. K., DePamphilis M. L. In vitro initiation of DNA replication in simian virus 40 chromosomes. J Biol Chem. 1987 Aug 5;262(22):10863–10872. [PubMed] [Google Scholar]
  11. Decker R. S., Yamaguchi M., Possenti R., DePamphilis M. L. Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis. Mol Cell Biol. 1986 Nov;6(11):3815–3825. doi: 10.1128/mcb.6.11.3815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eliasson R., Reichard P. Replication of polyoma DNA in isolated nuclei. VII. Initiator RNA synthesis during nucleotide depletion. J Mol Biol. 1979 Apr 15;129(3):393–409. doi: 10.1016/0022-2836(79)90503-5. [DOI] [PubMed] [Google Scholar]
  13. Hay R. T., DePamphilis M. L. Initiation of SV40 DNA replication in vivo: location and structure of 5' ends of DNA synthesized in the ori region. Cell. 1982 Apr;28(4):767–779. doi: 10.1016/0092-8674(82)90056-3. [DOI] [PubMed] [Google Scholar]
  14. Hay R. T., Hendrickson E. A., DePamphilis M. L. Sequence specificity for the initiation of RNA-primed simian virus 40 DNA synthesis in vivo. J Mol Biol. 1984 May 15;175(2):131–157. doi: 10.1016/0022-2836(84)90471-6. [DOI] [PubMed] [Google Scholar]
  15. Hendrickson E. A., Fritze C. E., Folk W. R., DePamphilis M. L. The origin of bidirectional DNA replication in polyoma virus. EMBO J. 1987 Jul;6(7):2011–2018. doi: 10.1002/j.1460-2075.1987.tb02465.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Longacre S. S., Mach B. Purification of specific DNA sequences by sulfhydryl-Sepharose chromatography of mercurated polynucleotides. J Biol Chem. 1978 Oct 25;253(20):7500–7507. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Stillman B. Chromatin assembly during SV40 DNA replication in vitro. Cell. 1986 May 23;45(4):555–565. doi: 10.1016/0092-8674(86)90287-4. [DOI] [PubMed] [Google Scholar]
  21. Su R. T., DePamphilis M. L. In vitro replication of simian virus 40 DNA in a nucleoprotein complex. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3466–3470. doi: 10.1073/pnas.73.10.3466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Su R. T., DePamphilis M. L. Simian virus 40 DNA replication in isolated replicating viral chromosomes. J Virol. 1978 Oct;28(1):53–65. doi: 10.1128/jvi.28.1.53-65.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Taljanidisz J., Karnik P., Sarkar N. Messenger ribonucleic acid for the lipoprotein of the Escherichia coli outer membrane is polyadenylated. J Mol Biol. 1987 Feb 5;193(3):507–515. doi: 10.1016/0022-2836(87)90263-4. [DOI] [PubMed] [Google Scholar]
  24. Tapper D. P., Anderson S., DePamphilis M. L. Distribution of replicating simian virus 40 DNA in intact cells and its maturation in isolated nuclei. J Virol. 1982 Mar;41(3):877–892. doi: 10.1128/jvi.41.3.877-892.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Vishwanatha J. K., Yamaguchi M., DePamphilis M. L., Baril E. F. Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex. Nucleic Acids Res. 1986 Sep 25;14(18):7305–7323. doi: 10.1093/nar/14.18.7305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wobbe C. R., Dean F. B., Murakami Y., Weissbach L., Hurwitz J. Simian virus 40 DNA replication in vitro: study of events preceding elongation of chains. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4612–4616. doi: 10.1073/pnas.83.13.4612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Yamaguchi M., DePamphilis M. L. DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1646–1650. doi: 10.1073/pnas.83.6.1646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yamaguchi M., Hendrickson E. A., DePamphilis M. L. DNA primase-DNA polymerase alpha from simian cells. Modulation of RNA primer synthesis by ribonucleoside triphosphates. J Biol Chem. 1985 May 25;260(10):6254–6263. [PubMed] [Google Scholar]
  30. Yamaguchi M., Hendrickson E. A., DePamphilis M. L. DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA. Mol Cell Biol. 1985 May;5(5):1170–1183. doi: 10.1128/mcb.5.5.1170. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES