Dominant negative mutator mutations in the mutS gene of Escherichia coli
Department of Pharmacology
Adenosine Triphosphatases; Alleles; Amino Acid Sequence; Bacterial Proteins; Binding Sites; *DNA Repair; DNA, Bacterial; *DNA-Binding Proteins; Drug Resistance, Microbial; Escherichia coli; *Escherichia coli Proteins; *Genes, Bacterial; *Genes, Dominant; Genetic Complementation Test; Hydroxylamine; Hydroxylamines; Molecular Sequence Data; MutS DNA Mismatch-Binding Protein; *Mutagenesis; Nalidixic Acid; Nucleic Acid Heteroduplexes; Plasmids; *Point Mutation; Recombination, Genetic; Rifampin; Sequence Homology, Amino Acid
Life Sciences | Medicine and Health Sciences
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.
J Bacteriol. 1994 Sep;176(17):5393-400.
Journal of bacteriology
Wu, Te-hui and Marinus, Martin G., "Dominant negative mutator mutations in the mutS gene of Escherichia coli" (1994). Open Access Articles. 663.