Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Jul;85(14):5046–5050. doi: 10.1073/pnas.85.14.5046

Bypass and termination at apurinic sites during replication of single-stranded DNA in vitro: a model for apurinic site mutagenesis.

D Hevroni 1, Z Livneh 1
PMCID: PMC281684  PMID: 3293048

Abstract

Mutations produced in Escherichia coli by apurinic sites are believed to arise via SOS-assisted translesion replication. Analysis of replication products synthesized on depurinated single-stranded DNA by DNA polymerase III holoenzyme revealed that apurinic sites frequently blocked in vitro replication. Bypass frequency of an apurinic site was estimated to be 10-15%. Direct evidence for replicative bypass was obtained in a complete single-stranded----replicative form replication system containing DNA polymerase III holoenzyme, single-stranded DNA binding protein, DNA polymerase I, and DNa ligase, by demonstrating the sensitivity of fully replicated products to the apurinic endonuclease activity of E. coli exonuclease III. Termination at apurinic sites, like termination at pyrimidine photodimers, involved dissociation of the polymerase from the blocked termini, followed by initiations at available primer templates. When no regular primer templates were available, the polymerase underwent repeated cycles of dissociation and rebinding at the blocked termini and, while bound, carried out multiple polymerization-excision reactions opposite the apurinic sites, leading to turnover of dNTPs into dNMPs. From the in vitro turnover rates, we could predict with striking accuracy the specificity of apurinic site mutagenesis, as determined in vivo in depurinated single-stranded DNA from an M13-lac hybrid phage. This finding is consistent with the view that DNA polymerase III holoenzyme carries out the mutagenic "misinsertion" step during apurinic site mutagenesis in vivo and that the specificity of the process is determined primarily by the polymerase. SOS-induced proteins such as UmuD/C might act as processivity-like factors to stabilize the polymerase-DNA complex, thus increasing the efficiency of the next stage of past-lesion polymerization required to complete the bypass reaction.

Full text

PDF
5046

Images in this article

Selected References

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

  1. Bridges B. A., Mottershead R. P. Mutagenic DNA repair in Escherichia coli. III. Requirement for a function of DNA polymerase III in ultraviolet-light mutagenesis. Mol Gen Genet. 1976 Feb 27;144(1):53–58. doi: 10.1007/BF00277304. [DOI] [PubMed] [Google Scholar]
  2. Bridges B. A., Woodgate R. Mutagenic repair in Escherichia coli: products of the recA gene and of the umuD and umuC genes act at different steps in UV-induced mutagenesis. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4193–4197. doi: 10.1073/pnas.82.12.4193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brotcorne-Lannoye A., Maenhaut-Michel G., Radman M. Involvement of DNA polymerase III in UV-induced mutagenesis of bacteriophage lambda. Mol Gen Genet. 1985;199(1):64–69. doi: 10.1007/BF00327511. [DOI] [PubMed] [Google Scholar]
  4. Burgers P. M., Kornberg A. The cycling of Escherichia coli DNA polymerase III holoenzyme in replication. J Biol Chem. 1983 Jun 25;258(12):7669–7675. [PubMed] [Google Scholar]
  5. Chase J. W., Whittier R. F., Auerbach J., Sancar A., Rupp W. D. Amplification of single-strand DNA binding protein in Escherichia coli. Nucleic Acids Res. 1980 Jul 25;8(14):3215–3227. doi: 10.1093/nar/8.14.3215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fersht A. R., Knill-Jones J. W. Contribution of 3' leads to 5' exonuclease activity of DNA polymerase III holoenzyme from Escherichia coli to specificity. J Mol Biol. 1983 Apr 25;165(4):669–682. doi: 10.1016/s0022-2836(83)80273-3. [DOI] [PubMed] [Google Scholar]
  7. Fersht A. R., Knill-Jones J. W., Tsui W. C. Kinetic basis of spontaneous mutation. Misinsertion frequencies, proofreading specificities and cost of proofreading by DNA polymerases of Escherichia coli. J Mol Biol. 1982 Mar 25;156(1):37–51. doi: 10.1016/0022-2836(82)90457-0. [DOI] [PubMed] [Google Scholar]
  8. Hagensee M. E., Timme T. L., Bryan S. K., Moses R. E. DNA polymerase III of Escherichia coli is required for UV and ethyl methanesulfonate mutagenesis. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4195–4199. doi: 10.1073/pnas.84.12.4195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kunkel T. A. Mutational specificity of depurination. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1494–1498. doi: 10.1073/pnas.81.5.1494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kunkel T. A., Schaaper R. M., Loeb L. A. Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro. Biochemistry. 1983 May 10;22(10):2378–2384. doi: 10.1021/bi00279a012. [DOI] [PubMed] [Google Scholar]
  11. Lindahl T., Andersson A. Rate of chain breakage at apurinic sites in double-stranded deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3618–3623. doi: 10.1021/bi00769a019. [DOI] [PubMed] [Google Scholar]
  12. Lindahl T. DNA glycosylases, endonucleases for apurinic/apyrimidinic sites, and base excision-repair. Prog Nucleic Acid Res Mol Biol. 1979;22:135–192. doi: 10.1016/s0079-6603(08)60800-4. [DOI] [PubMed] [Google Scholar]
  13. Lindahl T., Karlström O. Heat-induced depyrimidination of deoxyribonucleic acid in neutral solution. Biochemistry. 1973 Dec 4;12(25):5151–5154. doi: 10.1021/bi00749a020. [DOI] [PubMed] [Google Scholar]
  14. Lindahl T., Nyberg B. Rate of depurination of native deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3610–3618. doi: 10.1021/bi00769a018. [DOI] [PubMed] [Google Scholar]
  15. Livneh Z. Mechanism of replication of ultraviolet-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli. Implications for SOS mutagenesis. J Biol Chem. 1986 Jul 15;261(20):9526–9533. [PubMed] [Google Scholar]
  16. Livneh Z. Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4599–4603. doi: 10.1073/pnas.83.13.4599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Loeb L. A. Apurinic sites as mutagenic intermediates. Cell. 1985 Mar;40(3):483–484. doi: 10.1016/0092-8674(85)90191-6. [DOI] [PubMed] [Google Scholar]
  18. Loeb L. A., Preston B. D. Mutagenesis by apurinic/apyrimidinic sites. Annu Rev Genet. 1986;20:201–230. doi: 10.1146/annurev.ge.20.120186.001221. [DOI] [PubMed] [Google Scholar]
  19. Lu C., Scheuermann R. H., Echols H. Capacity of RecA protein to bind preferentially to UV lesions and inhibit the editing subunit (epsilon) of DNA polymerase III: a possible mechanism for SOS-induced targeted mutagenesis. Proc Natl Acad Sci U S A. 1986 Feb;83(3):619–623. doi: 10.1073/pnas.83.3.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McHenry C., Kornberg A. DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits. J Biol Chem. 1977 Sep 25;252(18):6478–6484. [PubMed] [Google Scholar]
  21. Moore P. D., Bose K. K., Rabkin S. D., Strauss B. S. Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases. Proc Natl Acad Sci U S A. 1981 Jan;78(1):110–114. doi: 10.1073/pnas.78.1.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Randall S. K., Eritja R., Kaplan B. E., Petruska J., Goodman M. F. Nucleotide insertion kinetics opposite abasic lesions in DNA. J Biol Chem. 1987 May 15;262(14):6864–6870. [PubMed] [Google Scholar]
  23. Sagher D., Strauss B. Insertion of nucleotides opposite apurinic/apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides. Biochemistry. 1983 Sep 13;22(19):4518–4526. doi: 10.1021/bi00288a026. [DOI] [PubMed] [Google Scholar]
  24. Schaaper R. M., Glickman B. W., Loeb L. A. Mutagenesis resulting from depurination is an SOS process. Mutat Res. 1982 Nov;106(1):1–9. doi: 10.1016/0027-5107(82)90186-5. [DOI] [PubMed] [Google Scholar]
  25. Schaaper R. M., Loeb L. A. Depurination causes mutations in SOS-induced cells. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1773–1777. doi: 10.1073/pnas.78.3.1773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shwartz H., Livneh Z. Dynamics of termination during in vitro replication of ultraviolet-irradiated DNA with DNA polymerase III holoenzyme of Escherichia coli. J Biol Chem. 1987 Aug 5;262(22):10518–10523. [PubMed] [Google Scholar]
  27. Takeshita M., Chang C. N., Johnson F., Will S., Grollman A. P. Oligodeoxynucleotides containing synthetic abasic sites. Model substrates for DNA polymerases and apurinic/apyrimidinic endonucleases. J Biol Chem. 1987 Jul 25;262(21):10171–10179. [PubMed] [Google Scholar]
  28. Van Den Hondel C. A., Weijers A., Konings R. N., Schoenmakers J. G. Studies on bacteriophage M13 DNA. 2. The gene order of the M13 genome. Eur J Biochem. 1975 May 6;53(2):559–567. doi: 10.1111/j.1432-1033.1975.tb04099.x. [DOI] [PubMed] [Google Scholar]
  29. Villani G., Boiteux S., Radman M. Mechanism of ultraviolet-induced mutagenesis: extent and fidelity of in vitro DNA synthesis on irradiated templates. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3037–3041. doi: 10.1073/pnas.75.7.3037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Weiner J. H., Bertsch L. L., Kornberg A. The deoxyribonucleic acid unwinding protein of Escherichia coli. Properties and functions in replication. J Biol Chem. 1975 Mar 25;250(6):1972–1980. [PubMed] [Google Scholar]
  32. Witkin E. M. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev. 1976 Dec;40(4):869–907. doi: 10.1128/br.40.4.869-907.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES