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. 1978 Jun;5(6):1933–1946. doi: 10.1093/nar/5.6.1933

Effect of 2',3'-dideoxythymidine-5'-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase alpha is the only polymerase required for cellular DNA replication.

M A Waqar, M J Evans, J A Huberman
PMCID: PMC342135  PMID: 673840

Abstract

We have studied the effects of the nucleotide analogue, 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100-500 micrometer when the dTTP concentration is maintained at 10 micrometer. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases beta or gamma is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase alpha is very resistant. These observations suggest that DNA polymerase alpha may be the only DNA polymerase required for all aspects of cellular DNA synthesis.

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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. Atkinson M. R., Deutscher M. P., Kornberg A., Russell A. F., Moffatt J. G. Enzymatic synthesis of deoxyribonucleic acid. XXXIV. Termination of chain growth by a 2',3'-dideoxyribonucleotide. Biochemistry. 1969 Dec;8(12):4897–4904. doi: 10.1021/bi00840a037. [DOI] [PubMed] [Google Scholar]
  3. Bolden A., Noy G. P., Weissbach A. DNA polymerase of mitochondria is a gamma-polymerase. J Biol Chem. 1977 May 25;252(10):3351–3356. [PubMed] [Google Scholar]
  4. Byars N., Kidson C. DNA chain termination by 2',3'-dideoxythymidine in replicating mammalian cells. Biochemistry. 1975 Jul 15;14(14):3159–3164. doi: 10.1021/bi00685a019. [DOI] [PubMed] [Google Scholar]
  5. Chang L. M., Bollum F. J. Variation of deoxyribonucleic acid polymerase activities during rat liver regeneration. J Biol Chem. 1972 Dec 25;247(24):7948–7950. [PubMed] [Google Scholar]
  6. Chang L. M., Brown M., Bollum F. J. Induction of DNA polymerase in mouse L cells. J Mol Biol. 1973 Feb 15;74(1):1–8. doi: 10.1016/0022-2836(73)90349-5. [DOI] [PubMed] [Google Scholar]
  7. Chang L. M., Brown M., Bollum F. J. Induction of DNA polymerase in mouse L cells. J Mol Biol. 1973 Feb 15;74(1):1–8. doi: 10.1016/0022-2836(73)90349-5. [DOI] [PubMed] [Google Scholar]
  8. Chiu R. W., Baril E. F. Nuclear DNA polymerases and the HeLa cell cycle. J Biol Chem. 1975 Oct 10;250(19):7951–7957. [PubMed] [Google Scholar]
  9. Edenberg H. J., Huberman J. A. Eukaryotic chromosome replication. Annu Rev Genet. 1975;9:245–284. doi: 10.1146/annurev.ge.09.120175.001333. [DOI] [PubMed] [Google Scholar]
  10. Eliasson R., Reichard P. Primase initiates Okazaki pieces during polyoma DNA synthesis. Nature. 1978 Mar 9;272(5649):184–185. doi: 10.1038/272184a0. [DOI] [PubMed] [Google Scholar]
  11. Evans M. J., Harvey S. R., Plummer M. J., Evans R. T., Laskowski M., Sr Murine DNA polymerases. I. Distinguishing characteristics of two activities separated by phosphocellulose chromatography. Proc Soc Exp Biol Med. 1974 Oct;147(1):35–42. doi: 10.3181/00379727-147-38277. [DOI] [PubMed] [Google Scholar]
  12. Faras A. J., Taylor J. M., Levinson W. E., Goodman H. M., Bishop J. M. RNA-directed DNA polymerase of Rous sarcoma virus: initiation of synthesis with 70 S viral RNA as template. J Mol Biol. 1973 Sep 5;79(1):163–183. doi: 10.1016/0022-2836(73)90277-5. [DOI] [PubMed] [Google Scholar]
  13. Fraser J. M., Huberman J. A. In vitro HeLa cell DNA synthesis similarity to in vivo replication. J Mol Biol. 1977 Nov 25;117(1):249–272. doi: 10.1016/0022-2836(77)90033-x. [DOI] [PubMed] [Google Scholar]
  14. Hunter T., Francke B. In vitro polyoma DNA synthesis: inhibition by 1-beta-d-arabinofuranosyl CTP. J Virol. 1975 Apr;15(4):759–775. doi: 10.1128/jvi.15.4.759-775.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Loeb L. A. Purification and properties of deoxyribonucleic acid polymerase from nuclei of sea urchin embryos. J Biol Chem. 1969 Apr 10;244(7):1672–1681. [PubMed] [Google Scholar]
  16. Loomis L. W., Rossomando E. F., Chang L. M. DNA polymerase of Dictyostelium discoideum. Biochim Biophys Acta. 1976 Apr 2;425(4):469–477. doi: 10.1016/0005-2787(76)90011-3. [DOI] [PubMed] [Google Scholar]
  17. Plummer M. J., Evans M. J. Response of murine DNA polymerases C and N to partial hepatectomy. J Med. 1976;7(1):81–91. [PubMed] [Google Scholar]
  18. Reichard P., Eliasson R., Söderman G. Initiator RNA in discontinuous polyoma DNA synthesis. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4901–4905. doi: 10.1073/pnas.71.12.4901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Smoler D., Molineux I., Baltimore D. Direction of polymerization by the avian myeloblastosis virus deoxyribonucleic acid polymerase. J Biol Chem. 1971 Dec 25;246(24):7697–7700. [PubMed] [Google Scholar]
  20. Spadari S., Weissbach A. HeLa cell R-deoxyribonucleic acid polymerases. Separation and characterization of two enzymatic activities. J Biol Chem. 1974 Sep 25;249(18):5809–5815. [PubMed] [Google Scholar]
  21. Spadari S., Weissbach A. RNA-primed DNA synthesis: specific catalysis by HeLa cell DNA polymerase alpha. Proc Natl Acad Sci U S A. 1975 Feb;72(2):503–507. doi: 10.1073/pnas.72.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Spadari S., Weissbach A. The interrelation between DNA synthesis and various DNA polymerase activities in synchronized HeLa cells. J Mol Biol. 1974 Jun 15;86(1):11–20. doi: 10.1016/s0022-2836(74)80003-3. [DOI] [PubMed] [Google Scholar]
  23. Wang H. F., Popenoe E. A. Variation of deoxyribonucleic acid polymerase activities during avian erythropoiesis. Biochim Biophys Acta. 1977 Jan 3;474(1):98–108. doi: 10.1016/0005-2787(77)90217-9. [DOI] [PubMed] [Google Scholar]
  24. Weissbach A., Baltimore D., Bollum F., Gallo R., Korn D. Nomenclature of eukaryotic DNA polymerases. Science. 1975 Oct 24;190(4212):401–402. doi: 10.1126/science.1179222. [DOI] [PubMed] [Google Scholar]
  25. de Recondo A. M., Abadiedebat J. Regenerating rat liver DNA polymerases: disimilitude or relationship between nuclear and cytoplasmic enzymes? Nucleic Acids Res. 1976 Aug;3(8):1823–1837. doi: 10.1093/nar/3.8.1823. [DOI] [PMC free article] [PubMed] [Google Scholar]

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