Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Nov;174(21):6844–6851. doi: 10.1128/jb.174.21.6844-6851.1992

The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein.

J Hauser 1, A S Levine 1, D G Ennis 1, K M Chumakov 1, R Woodgate 1
PMCID: PMC207361  PMID: 1400235

Abstract

Inducible mutagenesis in Escherichia coli requires the direct action of the chromosomally encoded UmuDC proteins or functional homologs found on certain naturally occurring plasmids. Although structurally similar, the five umu-like operons that have been characterized at the molecular level vary in their ability to enhance cellular and phage mutagenesis; of these operons, the mucAB genes from the N-group plasmid pKM101 are the most efficient at promoting mutagenesis. During the mutagenic process, UmuD is posttranslationally processed to an active form, UmuD'. To explain the more potent mutagenic efficiency of mucAB compared with that of umuDC it has been suggested that unlike UmuD, intact MucA is functional for mutagenesis. To examine this possibility, we have overproduced and purified the MucA protein. Although functionally similar to UmuD, MucA was cleaved much more rapidly both in vitro and in vivo than UmuD. In vivo, restoration of mutagenesis functions to normally nonmutable recA430, recA433, recA435, or recA730 delta(umuDC)595::cat strains by either MucA+ or mutant MucA protein correlated with the appearance of the cleavage product, MucA'. These results suggest that most of the differences in mutagenic phenotype exhibited by MucAB and UmuDC correlate with the efficiency of posttranslational processing of MucA and UmuD rather than an inherent activity of the unprocessed proteins.

Full text

PDF
6844

Images in this article

Selected References

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

  1. Bailone A., Sommer S., Knezević J., Dutreix M., Devoret R. A RecA protein mutant deficient in its interaction with the UmuDC complex. Biochimie. 1991 Apr;73(4):479–484. doi: 10.1016/0300-9084(91)90115-h. [DOI] [PubMed] [Google Scholar]
  2. Balganesh M., Setlow J. K. Genes from plasmid pKM101 in Haemophilus influenzae: separation of functions of mucA and mucB. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7753–7756. doi: 10.1073/pnas.82.22.7753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bates H., Bridges B. A. Mutagenic DNA repair in Escherichia coli. XIX. On the roles of RecA protein in ultraviolet light mutagenesis. Biochimie. 1991 Apr;73(4):485–489. doi: 10.1016/0300-9084(91)90116-i. [DOI] [PubMed] [Google Scholar]
  4. Bernard H. U., Helinski D. R. Use of the lambda phage promoter PL to promote gene expression in hybrid plasmid cloning vehicles. Methods Enzymol. 1979;68:482–492. doi: 10.1016/0076-6879(79)68037-0. [DOI] [PubMed] [Google Scholar]
  5. Blanco M., Herrera G., Aleixandre V. Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli. Mol Gen Genet. 1986 Nov;205(2):234–239. doi: 10.1007/BF00430433. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Blanco M., Rebollo J. E. Plasmid pKM101-dependent repair and mutagenesis in Escherichia coli cells with mutations lexB30 tif and zab-53 in the recA gene. Mutat Res. 1981 May;81(3):265–275. doi: 10.1016/0027-5107(81)90115-9. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Crowl R., Seamans C., Lomedico P., McAndrew S. Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli. Gene. 1985;38(1-3):31–38. doi: 10.1016/0378-1119(85)90200-8. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Ennis D. G., Ossanna N., Mount D. W. Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage. J Bacteriol. 1989 May;171(5):2533–2541. doi: 10.1128/jb.171.5.2533-2541.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gimble F. S., Sauer R. T. Lambda repressor inactivation: properties of purified ind- proteins in the autodigestion and RecA-mediated cleavage reactions. J Mol Biol. 1986 Nov 5;192(1):39–47. doi: 10.1016/0022-2836(86)90462-6. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Lin L. L., Little J. W. Autodigestion and RecA-dependent cleavage of Ind- mutant LexA proteins. J Mol Biol. 1989 Dec 5;210(3):439–452. doi: 10.1016/0022-2836(89)90121-6. [DOI] [PubMed] [Google Scholar]
  19. Lin L. L., Little J. W. Isolation and characterization of noncleavable (Ind-) mutants of the LexA repressor of Escherichia coli K-12. J Bacteriol. 1988 May;170(5):2163–2173. doi: 10.1128/jb.170.5.2163-2173.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Lodwick D., Owen D., Strike P. DNA sequence analysis of the imp UV protection and mutation operon of the plasmid TP110: identification of a third gene. Nucleic Acids Res. 1990 Sep 11;18(17):5045–5050. doi: 10.1093/nar/18.17.5045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Markham B. E., Little J. W., Mount D. W. Nucleotide sequence of the lexA gene of Escherichia coli K-12. Nucleic Acids Res. 1981 Aug 25;9(16):4149–4161. doi: 10.1093/nar/9.16.4149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marston F. A. The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J. 1986 Nov 15;240(1):1–12. doi: 10.1042/bj2400001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McCann J., Spingarn N. E., Kobori J., Ames B. N. Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids. Proc Natl Acad Sci U S A. 1975 Mar;72(3):979–983. doi: 10.1073/pnas.72.3.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McNally K. P., Freitag N. E., Walker G. C. LexA-independent expression of a mutant mucAB operon. J Bacteriol. 1990 Nov;172(11):6223–6231. doi: 10.1128/jb.172.11.6223-6231.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mieschendahl M., Müller-Hill B. F'-coded, temperature-sensitive lambda cI857 repressor gene for easy construction and regulation of lambda promoter-dependent expression systems. J Bacteriol. 1985 Dec;164(3):1366–1369. doi: 10.1128/jb.164.3.1366-1369.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Monti-Bragadin C., Babudri N., Samer L. Expression of the plasmid pKM101--determined DNA repair system in recA- and lex- strains of Escherichia coli. Mol Gen Genet. 1976 Jun 15;145(3):303–306. doi: 10.1007/BF00325827. [DOI] [PubMed] [Google Scholar]
  30. Mortelmans K. E., Stocker B. A. Ultraviolet light protection, enhancement of ultraviolet light mutagenesis, and mutator effect of plasmid R46 in Salmonella typhimurium. J Bacteriol. 1976 Oct;128(1):271–282. doi: 10.1128/jb.128.1.271-282.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Perry K. L., Walker G. C. Identification of plasmid (pKM101)-coded proteins involved in mutagenesis and UV resistance. Nature. 1982 Nov 18;300(5889):278–281. doi: 10.1038/300278a0. [DOI] [PubMed] [Google Scholar]
  34. Rebollo J. E., Moreau P. L., Blanco M., Devoret R. Restoration of RecA protein activity by genetic complementation. Mol Gen Genet. 1984;195(1-2):83–89. doi: 10.1007/BF00332728. [DOI] [PubMed] [Google Scholar]
  35. Rosenberg M., Echols H. Differential recognition of ultraviolet lesions by RecA protein. Possible mechanism for preferential targeting of SOS mutagenesis to (6-4) dipyrimidine sites. J Biol Chem. 1990 Nov 25;265(33):20641–20645. [PubMed] [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Sedgwick S. G., Goodwin P. A. Differences in mutagenic and recombinational DNA repair in enterobacteria. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4172–4176. doi: 10.1073/pnas.82.12.4172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sedgwick S. G., Ho C., Woodgate R. Mutagenic DNA repair in enterobacteria. J Bacteriol. 1991 Sep;173(18):5604–5611. doi: 10.1128/jb.173.18.5604-5611.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shiba T., Iwasaki H., Nakata A., Shinagawa H. Proteolytic processing of MucA protein in SOS mutagenesis: both processed and unprocessed MucA may be active in the mutagenesis. Mol Gen Genet. 1990 Nov;224(2):169–176. doi: 10.1007/BF00271549. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Slater S. C., Maurer R. Requirements for bypass of UV-induced lesions in single-stranded DNA of bacteriophage phi X174 in Salmonella typhimurium. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1251–1255. doi: 10.1073/pnas.88.4.1251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Smith C. M., Koch W. H., Franklin S. B., Foster P. L., Cebula T. A., Eisenstadt E. Sequence analysis and mapping of the Salmonella typhimurium LT2 umuDC operon. J Bacteriol. 1990 Sep;172(9):4964–4978. doi: 10.1128/jb.172.9.4964-4978.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Spikes D., Setlow J. K. A plasmid carrying mucA and mucB genes from pKM101 in Haemophilus influenzae and Escherichia coli. J Bacteriol. 1989 Oct;171(10):5753–5755. doi: 10.1128/jb.171.10.5753-5755.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Strike P., Lodwick D. Plasmid genes affecting DNA repair and mutation. J Cell Sci Suppl. 1987;6:303–321. doi: 10.1242/jcs.1984.supplement_6.20. [DOI] [PubMed] [Google Scholar]
  48. Sweasy J. B., Witkin E. M., Sinha N., Roegner-Maniscalco V. RecA protein of Escherichia coli has a third essential role in SOS mutator activity. J Bacteriol. 1990 Jun;172(6):3030–3036. doi: 10.1128/jb.172.6.3030-3036.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tanooka H., Tanaka K., Shinozaki K. Heterospecific expression of misrepair-enhancing activity of mucAB in Escherichia coli and Bacillus subtilis. J Bacteriol. 1991 May;173(9):2906–2914. doi: 10.1128/jb.173.9.2906-2914.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thomas S. M., Crowne H. M., Pidsley S. C., Sedgwick S. G. Structural characterization of the Salmonella typhimurium LT2 umu operon. J Bacteriol. 1990 Sep;172(9):4979–4987. doi: 10.1128/jb.172.9.4979-4987.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Waleh N. S., Stocker B. A. Effect of host lex, recA, recF, and uvrD genotypes on the ultraviolet light-protecting and related properties of plasmid R46 in Escherichia coli. J Bacteriol. 1979 Feb;137(2):830–838. doi: 10.1128/jb.137.2.830-838.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. 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]
  54. Woodgate R. Construction of a umuDC operon substitution mutation in Escherichia coli. Mutat Res. 1992 Mar;281(3):221–225. doi: 10.1016/0165-7992(92)90012-7. [DOI] [PubMed] [Google Scholar]
  55. Woodgate R., Ennis D. G. Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage. Mol Gen Genet. 1991 Sep;229(1):10–16. doi: 10.1007/BF00264207. [DOI] [PubMed] [Google Scholar]
  56. Woodgate R., Levine A. S., Koch W. H., Cebula T. A., Eisenstadt E. Induction and cleavage of Salmonella typhimurium UmuD protein. Mol Gen Genet. 1991 Sep;229(1):81–85. doi: 10.1007/BF00264216. [DOI] [PubMed] [Google Scholar]
  57. Woodgate R., Rajagopalan M., Lu C., Echols H. UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD'. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7301–7305. doi: 10.1073/pnas.86.19.7301. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES