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. 1990 Nov;172(11):6223–6231. doi: 10.1128/jb.172.11.6223-6231.1990

LexA-independent expression of a mutant mucAB operon.

K P McNally 1, N E Freitag 1, G C Walker 1
PMCID: PMC526803  PMID: 2228957

Abstract

pKM101 is a naturally occurring plasmid that carries mucAB, an analog of the umuDC operon, the gene products of which are required for the SOS-dependent processing of damaged DNA necessary for most mutagenesis. Genetic studies have indicated that mucAB expression is controlled by the SOS regulatory circuit, with LexA acting as a direct repressor. pGW16 is a pKM101 derivative obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis that was originally identified on the basis of its ability to cause a modest increase in spontaneous mutation rate. In this report, we show that pGW16 differs from pKM101 in being able to enhance methyl methanesulfonate mutagenesis and to confer substantial resistance to UV killing in a lexA3 host. The mutation carried by pGW16 is dominant and was localized to a 2.4-kb region of pGW16 that includes the mucAB coding region and approximately 0.6 kb of the 5'-flanking region. We determined the sequence of a 119-bp fragment containing the region upstream of mucAB and identified a single-base-pair change in that region, a G.C-to-A.T transition that alters a sequence homologous to known LexA-binding sites. DNA gel shift experiments indicate that LexA protein binds poorly to a 125-bp fragment containing this mutation, whereas a fragment containing the wild-type sequence is efficiently bound by LexA. This mutation also alters an overlapping sequence that is homologous to the -10 region of Escherichia coli promoters, moving it closer to the consensus sequence. The observation that the synthesis of pGW16-encoded mucAB proteins in maxicells is increased relative to that of pKM101-encoded mucAB proteins even in the absence of a lexA+ plasmid suggests that this mutation also increases the activity of the mucAB promoter.

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Selected References

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  1. 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]
  2. Brouwer J., Adhin M. R., van de Putte P. Effect of pKM101 on cell killing and specificity of mutation induction by cis-diaminedichloroplatinum(II) in Escherichia coli K-12. J Bacteriol. 1983 Dec;156(3):1275–1281. doi: 10.1128/jb.156.3.1275-1281.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Elledge S. J., Walker G. C. Proteins required for ultraviolet light and chemical mutagenesis. Identification of the products of the umuC locus of Escherichia coli. J Mol Biol. 1983 Feb 25;164(2):175–192. doi: 10.1016/0022-2836(83)90074-8. [DOI] [PubMed] [Google Scholar]
  6. Elledge S. J., Walker G. C. The muc genes of pKM101 are induced by DNA damage. J Bacteriol. 1983 Sep;155(3):1306–1315. doi: 10.1128/jb.155.3.1306-1315.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Langer P. J., Shanabruch W. G., Walker G. C. Functional organization of plasmid pKM101. J Bacteriol. 1981 Mar;145(3):1310–1316. doi: 10.1128/jb.145.3.1310-1316.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Langer P. J., Walker G. C. Restriction endonuclease cleavage map of pKM101: relationship to parental plasmid R46. Mol Gen Genet. 1981;182(2):268–272. doi: 10.1007/BF00269669. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Marsh L., Walker G. C. Cold sensitivity induced by overproduction of UmuDC in Escherichia coli. J Bacteriol. 1985 Apr;162(1):155–161. doi: 10.1128/jb.162.1.155-161.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Marsh L., Walker G. C. New phenotypes associated with mucAB: alteration of a MucA sequence homologous to the LexA cleavage site. J Bacteriol. 1987 May;169(5):1818–1823. doi: 10.1128/jb.169.5.1818-1823.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  19. Mortelmans K. E., Stocker B. A. Segregation of the mutator property of plasmid R46 from its ultraviolet-protecting property. Mol Gen Genet. 1979 Jan 2;167(3):317–327. doi: 10.1007/BF00267425. [DOI] [PubMed] [Google Scholar]
  20. Mount D. W., Low K. B., Edmiston S. J. Dominant mutations (lex) in Escherichia coli K-12 which affect radiation sensitivity and frequency of ultraviolet lght-induced mutations. J Bacteriol. 1972 Nov;112(2):886–893. doi: 10.1128/jb.112.2.886-893.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Oliver P., Stacey K. A. The effect of a drug-resistance factor on recombination and repair of DNA in Escherichia coli K12. J Gen Microbiol. 1977 Jul;101(1):93–98. doi: 10.1099/00221287-101-1-93. [DOI] [PubMed] [Google Scholar]
  23. Pang P. P., Walker G. C. The Salmonella typhimurium LT2 uvrD gene is regulated by the lexA gene product. J Bacteriol. 1983 Jun;154(3):1502–1504. doi: 10.1128/jb.154.3.1502-1504.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Peterson K. R., Ossanna N., Thliveris A. T., Ennis D. G., Mount D. W. Derepression of specific genes promotes DNA repair and mutagenesis in Escherichia coli. J Bacteriol. 1988 Jan;170(1):1–4. doi: 10.1128/jb.170.1.1-4.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rambach A., Hogness D. S. Translation of Drosophila melanogaster sequences in Escherichia coli. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5041–5045. doi: 10.1073/pnas.74.11.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  28. Sancar A., Wharton R. P., Seltzer S., Kacinski B. M., Clarke N. D., Rupp W. D. Identification of the uvrA gene product. J Mol Biol. 1981 May 5;148(1):45–62. doi: 10.1016/0022-2836(81)90234-5. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Shanabruch W. G., Walker G. C. Localization of the plasmid (pKM101) gene(s) involved in recA+lexA+-dependent mutagenesis. Mol Gen Genet. 1980;179(2):289–297. doi: 10.1007/BF00425456. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Shinagawa H., Kato T., Ise T., Makino K., Nakata A. Cloning and characterization of the umu operon responsible for inducible mutagenesis in Escherichia coli. Gene. 1983 Aug;23(2):167–174. doi: 10.1016/0378-1119(83)90048-3. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Taylor D. P., Cohen S. N. Structural and functional analysis of cloned DNA segments containing the replication and incompatibility regions of a miniplasmid derived from a copy number mutant of NR1. J Bacteriol. 1979 Jan;137(1):92–104. doi: 10.1128/jb.137.1.92-104.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Walker G. C., Dobson P. P. Mutagenesis and repair deficiencies of Escherichia coli umuC mutants are suppressed by the plasmid pKM101. Mol Gen Genet. 1979 Apr 17;172(1):17–24. doi: 10.1007/BF00276210. [DOI] [PubMed] [Google Scholar]
  37. Walker G. C. Inducible DNA repair systems. Annu Rev Biochem. 1985;54:425–457. doi: 10.1146/annurev.bi.54.070185.002233. [DOI] [PubMed] [Google Scholar]
  38. Walker G. C. Isolation and characterization of mutants of the plasmid pKM101 deficient in their ability to enhance mutagenesis and repair. J Bacteriol. 1978 Mar;133(3):1203–1211. doi: 10.1128/jb.133.3.1203-1211.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Walker G. C. Plasmid (pKM101)-mediated enhancement of repair and mutagenesis: dependence on chromosomal genes in Escherichia coli K-12. Mol Gen Genet. 1977 Mar 28;152(1):93–103. doi: 10.1007/BF00264945. [DOI] [PubMed] [Google Scholar]
  41. Winans S. C., Elledge S. J., Krueger J. H., Walker G. C. Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J Bacteriol. 1985 Mar;161(3):1219–1221. doi: 10.1128/jb.161.3.1219-1221.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Winans S. C., Walker G. C. Entry exclusion determinant(s) of IncN plasmid pKM101. J Bacteriol. 1985 Jan;161(1):411–416. doi: 10.1128/jb.161.1.411-416.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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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