Dominant negative mutator mutations in the mutL gene of Escherichia coli.
The mutL gene product is part of the dam-directed mismatch repair system of Escherichia coli but has no known enzymatic function. It forms a complex on heteroduplex DNA with the mismatch recognition MutS protein and with MutH, which has latent endonuclease activity. An N-terminal hexahistidine-tagged MutL was constructed which was active in vivo. As a first stop to determine the functional domains of MutL, we have isolated 72 hydroxylamine-induced plasmid-borne mutations which impart a dominant-negative phenotype to the wild-type strain for increased spontaneous mutagenesis. None of the mutations complement a mutL deletion mutant, indicating that the mutant proteins by themselves are inactive. All the dominant mutations but one could be complemented by the wild-type mutL at about the same gene dosage. DNA sequencing indicated that the mutations affected 22 amino acid residues located between positions 16 and 549 of the 615 amino acid protein. In the N-terminal half of the protein, 12 out of 15 amino acid replacements occur at positions conserved in various eukaryotic MutL homologs. All but one of the sequence changes affecting the C-terminal end of the protein are nonsense mutations.
Aronshtam
A
A
Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Marinus
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MG
D000251:Adenosine Triphosphatases; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D018628:Gene Dosage; D005798:Genes, Bacterial / Q000235:genetics*; D005816:Genetic Complementation Test; D000070956:MutL Proteins; D016296:Mutagenesis / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins