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. 1988 Aug;8(8):3267–3271. doi: 10.1128/mcb.8.8.3267

Fidelity of DNA synthesis in a mammalian in vitro replication system.

J Hauser 1, A S Levine 1, K Dixon 1
PMCID: PMC363559  PMID: 2850485

Abstract

We have used the simian virus 40 (SV40)-based shuttle vector pZ189 in a forward-mutation assay to determine the fidelity of DNA replication in the in vitro DNA replication system developed by J.J. Li and T.J. Kelly (Proc. Natl. Acad. Sci. USA 81:6973-6977, 1984). We find that very few base substitution errors (approximately 1/180,000 bases incorporated) are made during in vitro replication of the pZ189 vector in a system derived from CV-1 monkey cells. This replication is completely dependent on added SV40 T antigen and presumably reflects synthesis that is initiated at the SV40 replication origin. The observed level of fidelity is far greater than that reported for in vitro replication of DNA by conventionally purified eucaryotic DNA polymerases alpha and beta. Thus, there must be additional cellular factors in the crude in vitro system that serve to enhance the fidelity of 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. 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]
  2. Cotterill S. M., Reyland M. E., Loeb L. A., Lehman I. R. A cryptic proofreading 3'----5' exonuclease associated with the polymerase subunit of the DNA polymerase-primase from Drosophila melanogaster. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5635–5639. doi: 10.1073/pnas.84.16.5635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Drake J. W. Comparative rates of spontaneous mutation. Nature. 1969 Mar 22;221(5186):1132–1132. doi: 10.1038/2211132a0. [DOI] [PubMed] [Google Scholar]
  6. Dresler S. L., Frattini M. G. DNA replication and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase alpha to inhibition by butylphenyl-deoxyguanosine triphosphate. Nucleic Acids Res. 1986 Sep 11;14(17):7093–7102. doi: 10.1093/nar/14.17.7093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Glazer P. M., Sarkar S. N., Chisholm G. E., Summers W. C. DNA mismatch repair detected in human cell extracts. Mol Cell Biol. 1987 Jan;7(1):218–224. doi: 10.1128/mcb.7.1.218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hare J. T., Taylor J. H. One role for DNA methylation in vertebrate cells is strand discrimination in mismatch repair. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7350–7354. doi: 10.1073/pnas.82.21.7350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hauser J., Levine A. S., Dixon K. Unique pattern of point mutations arising after gene transfer into mammalian cells. EMBO J. 1987 Jan;6(1):63–67. doi: 10.1002/j.1460-2075.1987.tb04719.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hauser J., Seidman M. M., Sidur K., Dixon K. Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells. Mol Cell Biol. 1986 Jan;6(1):277–285. doi: 10.1128/mcb.6.1.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kunkel T. A., Alexander P. S. The base substitution fidelity of eucaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation. J Biol Chem. 1986 Jan 5;261(1):160–166. [PubMed] [Google Scholar]
  12. Kunkel T. A., Sabatino R. D., Bambara R. A. Exonucleolytic proofreading by calf thymus DNA polymerase delta. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4865–4869. doi: 10.1073/pnas.84.14.4865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kunkel T. A. The mutational specificity of DNA polymerases-alpha and -gamma during in vitro DNA synthesis. J Biol Chem. 1985 Oct 15;260(23):12866–12874. [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Loeb L. A., Kunkel T. A. Fidelity of DNA synthesis. Annu Rev Biochem. 1982;51:429–457. doi: 10.1146/annurev.bi.51.070182.002241. [DOI] [PubMed] [Google Scholar]
  17. Munson P. J., Hauser J., Levine A. S., Dixon K. Test of models for the sequence specificity of UV-induced mutations in mammalian cells. Mutat Res. 1987 Jul;179(1):103–114. doi: 10.1016/0027-5107(87)90045-5. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Radman M., Wagner R. Mismatch repair in Escherichia coli. Annu Rev Genet. 1986;20:523–538. doi: 10.1146/annurev.ge.20.120186.002515. [DOI] [PubMed] [Google Scholar]
  20. Reyland M. E., Loeb L. A. On the fidelity of DNA replication. Isolation of high fidelity DNA polymerase-primase complexes by immunoaffinity chromatography. J Biol Chem. 1987 Aug 5;262(22):10824–10830. [PubMed] [Google Scholar]
  21. Robb J. A., Huebner K. Effect of cell chromosome number on simian virus 40 replication. Exp Cell Res. 1973 Sep;81(1):120–126. doi: 10.1016/0014-4827(73)90118-3. [DOI] [PubMed] [Google Scholar]
  22. Seidman M. M., Dixon K., Razzaque A., Zagursky R. J., Berman M. L. A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells. Gene. 1985;38(1-3):233–237. doi: 10.1016/0378-1119(85)90222-7. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Wahl A. F., Crute J. J., Sabatino R. D., Bodner J. B., Marraccino R. L., Harwell L. W., Lord E. M., Bambara R. A. Properties of two forms of DNA polymerase delta from calf thymus. Biochemistry. 1986 Dec 2;25(24):7821–7827. doi: 10.1021/bi00372a006. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. 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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