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
. 1986 Feb;83(3):619–623. doi: 10.1073/pnas.83.3.619

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.

C Lu, R H Scheuermann, H Echols
PMCID: PMC322915  PMID: 3456159

Abstract

The RecA protein of Escherichia coli is required for SOS-induced mutagenesis in addition to its recombinational and regulatory roles. Most SOS-induced mutations probably occur during replication across a DNA lesion (targeted mutagenesis). We have suggested previously that RecA might participate in targeted mutagenesis by binding preferentially to the site of the DNA damage (e.g., pyrimidine dimer) because of its partially unwound character; DNA polymerase III (polIII) will then encounter RecA-coated DNA at the lesion and might replicate across the damaged site with reduced fidelity. In this report, we analyze at a biochemical level two major predictions of this model. With respect to lesion recognition, we show that purified RecA protein binds more efficiently to UV-irradiated double-stranded DNA than to nonirradiated DNA, as judged by filter-binding and gel electrophoresis assays. With respect to replication fidelity, Fersht and Knill-Jones [Fersht, A. R. & Knill-Jones, J. W. (1983) J. Mol. Biol. 165, 669-682] have found that RecA inhibits the 3'----5' exonuclease (editing function) of polIII holoenzyme. We extend this observation by demonstrating that RecA inhibits the exonuclease of the purified editing subunit of polIII, epsilon protein. Thus, we suggest that the activities of RecA required for targeted mutagenesis are lesion-recognition, followed by localized inhibition of the editing capacity of the epsilon subunit of polIII holoenzme. In this proposed mechanism, one activation signal for RecA for mutagenesis is the lesion itself. Because UV-irradiated, double-stranded DNA efficiently activates RecA for cleavage of the LexA repressor, the lesion itself may also often serve as an activation signal for induction of SOS-controlled genes.

Full text

PDF
619

Images in this article

Selected References

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

  1. Bagg A., Kenyon C. J., Walker G. C. Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5749–5753. doi: 10.1073/pnas.78.9.5749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baltimore D. Somatic mutation gains its place among the generators of diversity. Cell. 1981 Nov;26(3 Pt 1):295–296. doi: 10.1016/0092-8674(81)90196-3. [DOI] [PubMed] [Google Scholar]
  3. Blanco M., Herrera G., Collado P., Rebollo J. E., Botella L. M. Influence of RecA protein on induced mutagenesis. Biochimie. 1982 Aug-Sep;64(8-9):633–636. doi: 10.1016/s0300-9084(82)80102-8. [DOI] [PubMed] [Google Scholar]
  4. Bridges B. A., Woodgate R. Mutagenic repair in Escherichia coli. X. The umuC gene product may be required for replication past pyrimidine dimers but not for the coding error in UV-mutagenesis. Mol Gen Genet. 1984;196(2):364–366. doi: 10.1007/BF00328073. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Cox M. M., McEntee K., Lehman I. R. A simple and rapid procedure for the large scale purification of the recA protein of Escherichia coli. J Biol Chem. 1981 May 10;256(9):4676–4678. [PubMed] [Google Scholar]
  7. D'Ari R., Huisman O. DNA replication and indirect induction of the SOS response in Escherichia coli. Biochimie. 1982 Aug-Sep;64(8-9):623–627. doi: 10.1016/s0300-9084(82)80100-4. [DOI] [PubMed] [Google Scholar]
  8. Echols H., Lu C., Burgers P. M. Mutator strains of Escherichia coli, mutD and dnaQ, with defective exonucleolytic editing by DNA polymerase III holoenzyme. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2189–2192. doi: 10.1073/pnas.80.8.2189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Echols H. Mutation rate: some biological and biochemical considerations. Biochimie. 1982 Aug-Sep;64(8-9):571–575. doi: 10.1016/s0300-9084(82)80089-8. [DOI] [PubMed] [Google Scholar]
  10. Echols H. SOS functions, cancer and inducible evolution. Cell. 1981 Jul;25(1):1–2. doi: 10.1016/0092-8674(81)90223-3. [DOI] [PubMed] [Google Scholar]
  11. Ennis D. G., Fisher B., Edmiston S., Mount D. W. Dual role for Escherichia coli RecA protein in SOS mutagenesis. Proc Natl Acad Sci U S A. 1985 May;82(10):3325–3329. doi: 10.1073/pnas.82.10.3325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Kato T., Shinoura Y. Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light. Mol Gen Genet. 1977 Nov 14;156(2):121–131. doi: 10.1007/BF00283484. [DOI] [PubMed] [Google Scholar]
  14. Little J. W. Autodigestion of lexA and phage lambda repressors. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1375–1379. doi: 10.1073/pnas.81.5.1375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Little J. W., Mount D. W. The SOS regulatory system of Escherichia coli. Cell. 1982 May;29(1):11–22. doi: 10.1016/0092-8674(82)90085-x. [DOI] [PubMed] [Google Scholar]
  16. Livneh Z., Lehman I. R. Recombinational bypass of pyrimidine dimers promoted by the recA protein of Escherichia coli. Proc Natl Acad Sci U S A. 1982 May;79(10):3171–3175. doi: 10.1073/pnas.79.10.3171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maki H., Horiuchi T., Kornberg A. The polymerase subunit of DNA polymerase III of Escherichia coli. I. Amplification of the dnaE gene product and polymerase activity of the alpha subunit. J Biol Chem. 1985 Oct 25;260(24):12982–12986. [PubMed] [Google Scholar]
  18. McClintock B. The significance of responses of the genome to challenge. Science. 1984 Nov 16;226(4676):792–801. doi: 10.1126/science.15739260. [DOI] [PubMed] [Google Scholar]
  19. McEntee K., Weinstock G. M., Lehman I. R. Binding of the recA protein of Escherichia coli to single- and double-stranded DNA. J Biol Chem. 1981 Aug 25;256(16):8835–8844. [PubMed] [Google Scholar]
  20. McHenry C. S. DNA polymerase III holoenzyme of Escherichia coli: components and function of a true replicative complex. Mol Cell Biochem. 1985 Feb;66(1):71–85. doi: 10.1007/BF00231826. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. McPartland A., Green L., Echols H. Control of recA gene RNA in E. coli: regulatory and signal genes. Cell. 1980 Jul;20(3):731–737. doi: 10.1016/0092-8674(80)90319-0. [DOI] [PubMed] [Google Scholar]
  23. Miller J. H. Carcinogens induce targeted mutations in Escherichia coli. Cell. 1982 Nov;31(1):5–7. doi: 10.1016/0092-8674(82)90398-1. [DOI] [PubMed] [Google Scholar]
  24. Miller J. H. Mutagenic specificity of ultraviolet light. J Mol Biol. 1985 Mar 5;182(1):45–65. doi: 10.1016/0022-2836(85)90026-9. [DOI] [PubMed] [Google Scholar]
  25. Pearlman D. A., Holbrook S. R., Pirkle D. H., Kim S. H. Molecular models for DNA damaged by photoreaction. Science. 1985 Mar 15;227(4692):1304–1308. doi: 10.1126/science.3975615. [DOI] [PubMed] [Google Scholar]
  26. Phizicky E. M., Roberts J. W. Induction of SOS functions: regulation of proteolytic activity of E. coli RecA protein by interaction with DNA and nucleoside triphosphate. Cell. 1981 Jul;25(1):259–267. doi: 10.1016/0092-8674(81)90251-8. [DOI] [PubMed] [Google Scholar]
  27. Scheuermann R. H., Echols H. A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7747–7751. doi: 10.1073/pnas.81.24.7747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Spanos A., Sedgwick S. G., Yarranton G. T., Hübscher U., Banks G. R. Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Apr 24;9(8):1825–1839. doi: 10.1093/nar/9.8.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Steinborn G. Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis. I. Isolation of uvm mutants and their phenotypical characterization in DNA repair and mutagenesis. Mol Gen Genet. 1978 Sep 20;165(1):87–93. doi: 10.1007/BF00270380. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. WACKER A., DELLWEG H., JACHERTS D. [Thymine dimerization and survival of bacteria]. J Mol Biol. 1962 May;4:410–412. doi: 10.1016/s0022-2836(62)80022-9. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Weigle J. J. Induction of Mutations in a Bacterial Virus. Proc Natl Acad Sci U S A. 1953 Jul;39(7):628–636. doi: 10.1073/pnas.39.7.628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Witkin E. M., Kogoma T. Involvement of the activated form of RecA protein in SOS mutagenesis and stable DNA replication in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7539–7543. doi: 10.1073/pnas.81.23.7539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Witkin E. M., Wermundsen I. E. Targeted and untargeted mutagenesis by various inducers of SOS functions in Escherichia coli. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):881–886. doi: 10.1101/sqb.1979.043.01.095. [DOI] [PubMed] [Google Scholar]
  38. Wood R. D., Hutchinson F. Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light. J Mol Biol. 1984 Mar 5;173(3):293–305. doi: 10.1016/0022-2836(84)90122-0. [DOI] [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