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. 1989 Oct;86(19):7301–7305. doi: 10.1073/pnas.86.19.7301

UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD'.

R Woodgate 1, M Rajagopalan 1, C Lu 1, H Echols 1
PMCID: PMC298049  PMID: 2552436

Abstract

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli produces a marked elevation in mutation rate. The mutation pathway is a component of the induced, multigene SOS response. SOS mutagenesis is a tightly regulated process dependent on two RecA-mediated proteolytic events: cleavage of the LexA repressor to induce the UmuC and UmuD mutagenesis proteins, and cleavage of UmuD to UmuD' to activate the mutation pathway. To investigate the protein-protein interactions responsible for SOS mutagenesis, we have studied the interaction of UmuC, UmuD, and UmuD'. To probe intracellular interaction, we have used immunoprecipitation techniques with antibodies against UmuC or UmuD and UmuD'. We have found that antibody to UmuC precipitates UmuD' from cell extracts, and antibody to UmuD and UmuD' precipitates UmuC. Thus we conclude that UmuC probably associates tightly with UmuD' in cells. For biochemical studies, we have purified the UmuC and UmuD' proteins to use with the previously purified UmuD. UmuC associates strongly with an affinity column of UmuD and UmuD', eluting only under strongly dissociating conditions (2 M urea or 1.5 M KSCN). UmuC also associates efficiently with UmuD or UmuD' in solution, as judged by velocity sedimentation in a glycerol gradient. The likely stoichiometry is one UmuC with a dimeric UmuD or UmuD'. From these experiments and previous work, we infer that SOS mutagenesis depends on the action of the UmuC-UmuD' complex and probably RecA to rescue a stalled DNA polymerase III holoenzyme at the DNA lesion.

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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. 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]
  3. 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]
  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. Bridges B. A., Woodgate R. The two-step model of bacterial UV mutagenesis. Mutat Res. 1985 Jun-Jul;150(1-2):133–139. doi: 10.1016/0027-5107(85)90110-1. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Burckhardt S. E., Woodgate R., Scheuermann R. H., Echols H. UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1811–1815. doi: 10.1073/pnas.85.6.1811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Crowl R. Expression of human interferon genes in E. coli with the lambda PL promoter. Methods Enzymol. 1986;119:376–383. doi: 10.1016/0076-6879(86)19057-4. [DOI] [PubMed] [Google Scholar]
  10. Dutreix M., Moreau P. L., Bailone A., Galibert F., Battista J. R., Walker G. C., Devoret R. New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis. J Bacteriol. 1989 May;171(5):2415–2423. doi: 10.1128/jb.171.5.2415-2423.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Formosa T., Burke R. L., Alberts B. M. Affinity purification of bacteriophage T4 proteins essential for DNA replication and genetic recombination. Proc Natl Acad Sci U S A. 1983 May;80(9):2442–2446. doi: 10.1073/pnas.80.9.2442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Friedman D. I., Imperiale M. J., Adhya S. L. RNA 3' end formation in the control of gene expression. Annu Rev Genet. 1987;21:453–488. doi: 10.1146/annurev.ge.21.120187.002321. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Hevroni D., Livneh Z. Bypass and termination at apurinic sites during replication of single-stranded DNA in vitro: a model for apurinic site mutagenesis. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5046–5050. doi: 10.1073/pnas.85.14.5046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hoyt M. A., Knight D. M., Das A., Miller H. I., Echols H. Control of phage lambda development by stability and synthesis of cII protein: role of the viral cIII and host hflA, himA and himD genes. Cell. 1982 Dec;31(3 Pt 2):565–573. doi: 10.1016/0092-8674(82)90312-9. [DOI] [PubMed] [Google Scholar]
  18. Jonczyk P., Fijalkowska I., Ciesla Z. Overproduction of the epsilon subunit of DNA polymerase III counteracts the SOS mutagenic response of Escherichia coli. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9124–9127. doi: 10.1073/pnas.85.23.9124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Khidhir M. A., Casaregola S., Holland I. B. Mechanism of transient inhibition of DNA synthesis in ultraviolet-irradiated E. coli: inhibition is independent of recA whilst recovery requires RecA protein itself and an additional, inducible SOS function. Mol Gen Genet. 1985;199(1):133–140. doi: 10.1007/BF00327522. [DOI] [PubMed] [Google Scholar]
  21. Kitagawa Y., Akaboshi E., Shinagawa H., Horii T., Ogawa H., Kato T. Structural analysis of the umu operon required for inducible mutagenesis in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4336–4340. doi: 10.1073/pnas.82.13.4336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Lu C., Echols H. RecA protein and SOS. Correlation of mutagenesis phenotype with binding of mutant RecA proteins to duplex DNA and LexA cleavage. J Mol Biol. 1987 Aug 5;196(3):497–504. doi: 10.1016/0022-2836(87)90027-1. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  28. 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]
  29. Nohmi T., Battista J. R., Dodson L. A., Walker G. C. RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1816–1820. doi: 10.1073/pnas.85.6.1816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Perry K. L., Elledge S. J., Mitchell B. B., Marsh L., Walker G. C. umuDC and mucAB operons whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4331–4335. doi: 10.1073/pnas.82.13.4331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rupp W. D., Wilde C. E., 3rd, Reno D. L., Howard-Flanders P. Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli. J Mol Biol. 1971 Oct 14;61(1):25–44. doi: 10.1016/0022-2836(71)90204-x. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Shinagawa H., Iwasaki H., Kato T., Nakata A. RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1806–1810. doi: 10.1073/pnas.85.6.1806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Slilaty S. N., Little J. W. Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism. Proc Natl Acad Sci U S A. 1987 Jun;84(12):3987–3991. doi: 10.1073/pnas.84.12.3987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Slilaty S. N., Rupley J. A., Little J. W. Intramolecular cleavage of LexA and phage lambda repressors: dependence of kinetics on repressor concentration, pH, temperature, and solvent. Biochemistry. 1986 Nov 4;25(22):6866–6875. doi: 10.1021/bi00370a020. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. 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]
  39. 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]
  40. Witkin E. M., Roegner-Maniscalco V., Sweasy J. B., McCall J. O. Recovery from ultraviolet light-induced inhibition of DNA synthesis requires umuDC gene products in recA718 mutant strains but not in recA+ strains of Escherichia coli. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6805–6809. doi: 10.1073/pnas.84.19.6805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]

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