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. 1998 Aug 15;26(16):3729–3738. doi: 10.1093/nar/26.16.3729

DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination.

L Islas 1, C F Fairley 1, W F Morgan 1
PMCID: PMC147762  PMID: 9685489

Abstract

DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the discontinuous DNA synthesis was attributable to DNA polymerase alpha. Additionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase alpha activity. To test the relative efficiency of each nuclear DNA polymerase for discontinuous synthesis, equal amounts (as measured by DNA polymerase activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and straightepsilon (+/- PCNA) were used in the discontinuous DNA synthesis assay. DNA polymerase alpha showed the most discontinuous DNA synthesis activity, although small but detectable levels were seen for DNA polymerases delta (+PCNA) and straightepsilon (- PCNA). Klenow fragment and DNA polymerase beta showed no discontinuous DNA synthesis, although at much higher amounts of each enzyme, discontinuous synthesis was seen for both. Discontinuous DNA synthesis by DNA polymerase alpha was seen with substrates containing 3 and 4 bp single-strand stretches of complementarity; however, little synthesis was seen with blunt substrates or with 1 bp stretches. The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryotic cells. These data suggest that DNA polymerase alpha may be able to rejoin double-strand breaks in vivo during replication.

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Selected References

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  1. Bambara R. A., Jessee C. B. Properties of DNA polymerases delta and epsilon, and their roles in eukaryotic DNA replication. Biochim Biophys Acta. 1991 Jan 17;1088(1):11–24. doi: 10.1016/0167-4781(91)90147-e. [DOI] [PubMed] [Google Scholar]
  2. Breimer L. H., Nalbantoglu J., Meuth M. Structure and sequence of mutations induced by ionizing radiation at selectable loci in Chinese hamster ovary cells. J Mol Biol. 1986 Dec 5;192(3):669–674. doi: 10.1016/0022-2836(86)90284-6. [DOI] [PubMed] [Google Scholar]
  3. Catalano C. E., Benkovic S. J. Inactivation of DNA polymerase I (Klenow fragment) by adenosine 2',3'-epoxide 5'-triphosphate: evidence for the formation of a tight-binding inhibitor. Biochemistry. 1989 May 16;28(10):4374–4382. doi: 10.1021/bi00436a038. [DOI] [PubMed] [Google Scholar]
  4. Chui G., Linn S. Further characterization of HeLa DNA polymerase epsilon. J Biol Chem. 1995 Apr 7;270(14):7799–7808. doi: 10.1074/jbc.270.14.7799. [DOI] [PubMed] [Google Scholar]
  5. Clark J. M. DNA synthesis on discontinuous templates by DNA polymerase I of Escherichia coli. Gene. 1991 Jul 31;104(1):75–80. doi: 10.1016/0378-1119(91)90467-p. [DOI] [PubMed] [Google Scholar]
  6. Clark J. M., Joyce C. M., Beardsley G. P. Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli. J Mol Biol. 1987 Nov 5;198(1):123–127. doi: 10.1016/0022-2836(87)90462-1. [DOI] [PubMed] [Google Scholar]
  7. Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grosovsky A. J., de Boer J. G., de Jong P. J., Drobetsky E. A., Glickman B. W. Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells. Proc Natl Acad Sci U S A. 1988 Jan;85(1):185–188. doi: 10.1073/pnas.85.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Henderson G., Simons J. P. Processing of DNA prior to illegitimate recombination in mouse cells. Mol Cell Biol. 1997 Jul;17(7):3779–3785. doi: 10.1128/mcb.17.7.3779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. King J. S., Fairley C. F., Morgan W. F. Bridging the gap. Joining of nonhomologous ends by DNA polymerases. J Biol Chem. 1994 May 6;269(18):13061–13064. [PubMed] [Google Scholar]
  11. King J. S., Fairley C. F., Morgan W. F. DNA end joining by the Klenow fragment of DNA polymerase I. J Biol Chem. 1996 Aug 23;271(34):20450–20457. doi: 10.1074/jbc.271.34.20450. [DOI] [PubMed] [Google Scholar]
  12. Kowalczykowski S. C., Dixon D. A., Eggleston A. K., Lauder S. D., Rehrauer W. M. Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev. 1994 Sep;58(3):401–465. doi: 10.1128/mr.58.3.401-465.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Miles C., Meuth M. DNA sequence determination of gamma-radiation-induced mutations of the hamster aprt locus. Mutat Res. 1989 Oct;227(2):97–102. doi: 10.1016/0165-7992(89)90004-3. [DOI] [PubMed] [Google Scholar]
  14. Nishida C., Reinhard P., Linn S. DNA repair synthesis in human fibroblasts requires DNA polymerase delta. J Biol Chem. 1988 Jan 5;263(1):501–510. [PubMed] [Google Scholar]
  15. Nudler E., Avetissova E., Markovtsov V., Goldfarb A. Transcription processivity: protein-DNA interactions holding together the elongation complex. Science. 1996 Jul 12;273(5272):211–217. doi: 10.1126/science.273.5272.211. [DOI] [PubMed] [Google Scholar]
  16. Pergola F., Zdzienicka M. Z., Lieber M. R. V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair. Mol Cell Biol. 1993 Jun;13(6):3464–3471. doi: 10.1128/mcb.13.6.3464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pfeiffer P., Thode S., Hancke J., Vielmetter W. Mechanisms of overlap formation in nonhomologous DNA end joining. Mol Cell Biol. 1994 Feb;14(2):888–895. doi: 10.1128/mcb.14.2.888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pfeiffer P., Vielmetter W. Joining of nonhomologous DNA double strand breaks in vitro. Nucleic Acids Res. 1988 Feb 11;16(3):907–924. doi: 10.1093/nar/16.3.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Phillips J. W., Morgan W. F. Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome. Mol Cell Biol. 1994 Sep;14(9):5794–5803. doi: 10.1128/mcb.14.9.5794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Roth D. B., Porter T. N., Wilson J. H. Mechanisms of nonhomologous recombination in mammalian cells. Mol Cell Biol. 1985 Oct;5(10):2599–2607. doi: 10.1128/mcb.5.10.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Roth D. B., Wilson J. H. Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction. Mol Cell Biol. 1986 Dec;6(12):4295–4304. doi: 10.1128/mcb.6.12.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sankaranarayanan K. Ionizing radiation and genetic risks. III. Nature of spontaneous and radiation-induced mutations in mammalian in vitro systems and mechanisms of induction of mutations by radiation. Mutat Res. 1991 Jul;258(1):75–97. doi: 10.1016/0165-1110(91)90029-u. [DOI] [PubMed] [Google Scholar]
  23. Syväoja J., Suomensaari S., Nishida C., Goldsmith J. S., Chui G. S., Jain S., Linn S. DNA polymerases alpha, delta, and epsilon: three distinct enzymes from HeLa cells. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6664–6668. doi: 10.1073/pnas.87.17.6664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Taccioli G. E., Rathbun G., Oltz E., Stamato T., Jeggo P. A., Alt F. W. Impairment of V(D)J recombination in double-strand break repair mutants. Science. 1993 Apr 9;260(5105):207–210. doi: 10.1126/science.8469973. [DOI] [PubMed] [Google Scholar]
  25. Tanaka S., Hu S. Z., Wang T. S., Korn D. Preparation and preliminary characterization of monoclonal antibodies against human DNA polymerase alpha. J Biol Chem. 1982 Jul 25;257(14):8386–8390. [PubMed] [Google Scholar]
  26. Thode S., Schäfer A., Pfeiffer P., Vielmetter W. A novel pathway of DNA end-to-end joining. Cell. 1990 Mar 23;60(6):921–928. doi: 10.1016/0092-8674(90)90340-k. [DOI] [PubMed] [Google Scholar]
  27. Wang T. S. Eukaryotic DNA polymerases. Annu Rev Biochem. 1991;60:513–552. doi: 10.1146/annurev.bi.60.070191.002501. [DOI] [PubMed] [Google Scholar]
  28. Ward J. F. DNA damage produced by ionizing radiation in mammalian cells: identities, mechanisms of formation, and reparability. Prog Nucleic Acid Res Mol Biol. 1988;35:95–125. doi: 10.1016/s0079-6603(08)60611-x. [DOI] [PubMed] [Google Scholar]
  29. Weiser T., Gassmann M., Thömmes P., Ferrari E., Hafkemeyer P., Hübscher U. Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus. J Biol Chem. 1991 Jun 5;266(16):10420–10428. [PubMed] [Google Scholar]

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