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. 1994 Sep;176(17):5393–5400. doi: 10.1128/jb.176.17.5393-5400.1994

Dominant negative mutator mutations in the mutS gene of Escherichia coli.

T H Wu 1, M G Marinus 1
PMCID: PMC196726  PMID: 8071216

Abstract

The MutS protein of Escherichia coli is part of the dam-directed MutHLS mismatch repair pathway which rectifies replication errors and which prevents recombination between related sequences. In order to more fully understand the role of MutS in these processes, dominant negative mutS mutations on a multicopy plasmid were isolated by screening transformed wild-type cells for a mutator phenotype, using a Lac+ papillation assay. Thirty-eight hydroxylamine- and 22 N-methyl-N'-nitro-N-nitrosoguanidine-induced dominant mutations were isolated. Nine of these mutations altered the P-loop motif of the ATP-binding site, resulting in four amino acid substitutions. With one exception, the remaining sequenced mutations all caused substitution of amino acids conserved during evolution. The dominant mutations in the P-loop consensus caused severely reduced repair of heteroduplex DNA in vivo in a mutS mutant host strain. In a wild-type strain, the level of repair was decreased by the dominant mutations to between 12 to 90% of the control value, which is consistent with interference of wild-type MutS function by the mutant proteins. Increasing the wild-type mutS gene dosage resulted in a reversal of the mutator phenotype in about 60% of the mutant strains, indicating that the mutant and wild-type proteins compete. In addition, 20 mutant isolates showed phenotypic reversal by increasing the gene copies of either mutL or mutH. There was a direct correlation between the levels of recombination and mutagenesis in the mutant strains, suggesting that these phenotypes are due to the same function of MutS.

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Selected References

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

  1. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  2. 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]
  3. Connolly D. M., Winkler M. E. Genetic and physiological relationships among the miaA gene, 2-methylthio-N6-(delta 2-isopentenyl)-adenosine tRNA modification, and spontaneous mutagenesis in Escherichia coli K-12. J Bacteriol. 1989 Jun;171(6):3233–3246. doi: 10.1128/jb.171.6.3233-3246.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cupples C. G., Miller J. H. A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5345–5349. doi: 10.1073/pnas.86.14.5345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feinstein S. I., Low K. B. Hyper-recombining recipient strains in bacterial conjugation. Genetics. 1986 May;113(1):13–33. doi: 10.1093/genetics/113.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fishel R., Lescoe M. K., Rao M. R., Copeland N. G., Jenkins N. A., Garber J., Kane M., Kolodner R. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell. 1993 Dec 3;75(5):1027–1038. doi: 10.1016/0092-8674(93)90546-3. [DOI] [PubMed] [Google Scholar]
  8. Fujii H., Shimada T. Isolation and characterization of cDNA clones derived from the divergently transcribed gene in the region upstream from the human dihydrofolate reductase gene. J Biol Chem. 1989 Jun 15;264(17):10057–10064. [PubMed] [Google Scholar]
  9. Glickman B. W., Radman M. Escherichia coli mutator mutants deficient in methylation-instructed DNA mismatch correction. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1063–1067. doi: 10.1073/pnas.77.2.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grafstrom R. H., Hoess R. H. Nucleotide sequence of the Escherichia coli mutH gene. Nucleic Acids Res. 1987 Apr 10;15(7):3073–3084. doi: 10.1093/nar/15.7.3073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Haber L. T., Pang P. P., Sobell D. I., Mankovich J. A., Walker G. C. Nucleotide sequence of the Salmonella typhimurium mutS gene required for mismatch repair: homology of MutS and HexA of Streptococcus pneumoniae. J Bacteriol. 1988 Jan;170(1):197–202. doi: 10.1128/jb.170.1.197-202.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haber L. T., Walker G. C. Altering the conserved nucleotide binding motif in the Salmonella typhimurium MutS mismatch repair protein affects both its ATPase and mismatch binding activities. EMBO J. 1991 Sep;10(9):2707–2715. doi: 10.1002/j.1460-2075.1991.tb07815.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Karran P., Marinus M. G. Mismatch correction at O6-methylguanine residues in E. coli DNA. Nature. 1982 Apr 29;296(5860):868–869. doi: 10.1038/296868a0. [DOI] [PubMed] [Google Scholar]
  14. Kleckner N., Bender J., Gottesman S. Uses of transposons with emphasis on Tn10. Methods Enzymol. 1991;204:139–180. doi: 10.1016/0076-6879(91)04009-d. [DOI] [PubMed] [Google Scholar]
  15. Le O., Shen B., Iismaa S. E., Burgess B. K. Azotobacter vinelandii mutS: nucleotide sequence and mutant analysis. J Bacteriol. 1993 Dec;175(23):7707–7710. doi: 10.1128/jb.175.23.7707-7710.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Linton J. P., Yen J. Y., Selby E., Chen Z., Chinsky J. M., Liu K., Kellems R. E., Crouse G. F. Dual bidirectional promoters at the mouse dhfr locus: cloning and characterization of two mRNA classes of the divergently transcribed Rep-1 gene. Mol Cell Biol. 1989 Jul;9(7):3058–3072. doi: 10.1128/mcb.9.7.3058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lundblad V., Kleckner N. Mismatch repair mutations of Escherichia coli K12 enhance transposon excision. Genetics. 1985 Jan;109(1):3–19. doi: 10.1093/genetics/109.1.3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marinus M. G., Morris N. R. Pleiotropic effects of a DNA adenine methylation mutation (dam-3) in Escherichia coli K12. Mutat Res. 1975 Apr;28(1):15–26. doi: 10.1016/0027-5107(75)90309-7. [DOI] [PubMed] [Google Scholar]
  19. McGraw B. R., Marinus M. G. Isolation and characterization of Dam+ revertants and suppressor mutations that modify secondary phenotypes of dam-3 strains of Escherichia coli K-12. Mol Gen Genet. 1980;178(2):309–315. doi: 10.1007/BF00270477. [DOI] [PubMed] [Google Scholar]
  20. Modrich P. Mechanisms and biological effects of mismatch repair. Annu Rev Genet. 1991;25:229–253. doi: 10.1146/annurev.ge.25.120191.001305. [DOI] [PubMed] [Google Scholar]
  21. New L., Liu K., Crouse G. F. The yeast gene MSH3 defines a new class of eukaryotic MutS homologues. Mol Gen Genet. 1993 May;239(1-2):97–108. doi: 10.1007/BF00281607. [DOI] [PubMed] [Google Scholar]
  22. Norkin L. C. Marker-specific effects in genetic recombination. J Mol Biol. 1970 Aug;51(3):633–655. doi: 10.1016/0022-2836(70)90013-6. [DOI] [PubMed] [Google Scholar]
  23. Parker B. O., Marinus M. G. Repair of DNA heteroduplexes containing small heterologous sequences in Escherichia coli. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1730–1734. doi: 10.1073/pnas.89.5.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Peterson K. R., Mount D. W. Analysis of the genetic requirements for viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants. J Bacteriol. 1993 Nov;175(22):7505–7508. doi: 10.1128/jb.175.22.7505-7508.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Priebe S. D., Hadi S. M., Greenberg B., Lacks S. A. Nucleotide sequence of the hexA gene for DNA mismatch repair in Streptococcus pneumoniae and homology of hexA to mutS of Escherichia coli and Salmonella typhimurium. J Bacteriol. 1988 Jan;170(1):190–196. doi: 10.1128/jb.170.1.190-196.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Prudhomme M., Méjean V., Martin B., Claverys J. P. Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation. J Bacteriol. 1991 Nov;173(22):7196–7203. doi: 10.1128/jb.173.22.7196-7203.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pukkila P. J., Peterson J., Herman G., Modrich P., Meselson M. Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli. Genetics. 1983 Aug;104(4):571–582. doi: 10.1093/genetics/104.4.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rayssiguier C., Thaler D. S., Radman M. The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature. 1989 Nov 23;342(6248):396–401. doi: 10.1038/342396a0. [DOI] [PubMed] [Google Scholar]
  29. Reenan R. A., Kolodner R. D. Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mitochondrial and nuclear functions. Genetics. 1992 Dec;132(4):975–985. doi: 10.1093/genetics/132.4.975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reenan R. A., Kolodner R. D. Isolation and characterization of two Saccharomyces cerevisiae genes encoding homologs of the bacterial HexA and MutS mismatch repair proteins. Genetics. 1992 Dec;132(4):963–973. doi: 10.1093/genetics/132.4.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rewinski C., Marinus M. G. Mutation spectrum in Escherichia coli DNA mismatch repair deficient (mutH) strain. Nucleic Acids Res. 1987 Oct 26;15(20):8205–8215. doi: 10.1093/nar/15.20.8205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rydberg B. Bromouracil mutagenesis and mismatch repair in mutator strains of Escherichia coli. Mutat Res. 1978 Oct;52(1):11–24. doi: 10.1016/0027-5107(78)90091-x. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Schlensog V., Böck A. The Escherichia coli fdv gene probably encodes mutS and is located at minute 58.8 adjacent to the hyc-hyp gene cluster. J Bacteriol. 1991 Dec;173(23):7414–7415. doi: 10.1128/jb.173.23.7414-7415.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shanabruch W. G., Rein R. P., Behlau I., Walker G. C. Mutagenesis, by methylating and ethylating agents, in mutH, mutL, mutS, and uvrD mutants of Salmonella typhimurium LT2. J Bacteriol. 1983 Jan;153(1):33–44. doi: 10.1128/jb.153.1.33-44.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Siegel E. C., Bryson V. Mutator gene of Escherichia coli B. J Bacteriol. 1967 Jul;94(1):38–47. doi: 10.1128/jb.94.1.38-47.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Siegel E. C., Wain S. L., Meltzer S. F., Binion M. L., Steinberg J. L. Mutator mutations in Escherichia coli induced by the insertion of phage mu and the transposable resistance elements Tn5 and Tn10. Mutat Res. 1982 Mar;93(1):25–33. doi: 10.1016/0027-5107(82)90122-1. [DOI] [PubMed] [Google Scholar]
  38. Skopek T. R., Hutchinson F. Frameshift mutagenesis of lambda prophage by 9-aminoacridine, proflavin and ICR-191. Mol Gen Genet. 1984;195(3):418–423. doi: 10.1007/BF00341442. [DOI] [PubMed] [Google Scholar]
  39. Su S. S., Modrich P. Escherichia coli mutS-encoded protein binds to mismatched DNA base pairs. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5057–5061. doi: 10.1073/pnas.83.14.5057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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