Mutagenic frequencies of site-specifically located O6-methylguanine in wild-type Escherichia coli and in a strain deficient in ada-methyltransferase.
The adaptive response of Escherichia coli involves protection of the cells against the toxic and mutagenic consequences of exposure to high doses of a methylating agent by prior exposure to low doses of the agent. Ada protein, a major repair activity for O6-methylguanine, is activated to positively control the adaptive response; O6-methylguanine is one of the major mutagenic lesions produced by methylating agents. We investigated the mutation frequency of wild-type Escherichia coli and strains containing the ada-5 mutation in response to site-specifically synthesized O6-methylguanine under conditions in which the adaptive response was not induced. Site-directed mutagenesis and oligonucleotide self-selection techniques were used to isolate the progeny of M13mp18 DNAs constructed to contain O6-methylguanine at any of eight different positions. The progeny were isolated from E. coli strains isogeneic except for deficiency in Ada-methyltransferase repair, UvrABC excision repair, or both. The resulting O6-methylguanine mutation levels at each position were determined by using differential oligonucleotide hybridization. We found that the wild type had up to a 2.6-fold higher mutation frequency than ada-5 mutants. In addition, the mutation frequency varied with the position of the O6-methylguanine in the DNA in the wild type but not in ada-5 mutants; O6-methylguanine lesions at the 5' ends of runs of consecutive guanines gave the highest mutation frequencies. Determination of the mutation frequency of O6-methylguanine in wild-type and mutS cells showed that mismatch repair can affect O6-methylguanine mutation levels.
Rossi
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Department of Pathology, University of North Carolina Medical School, Chapel Hill 27599-7295.
Topal
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D001483:Base Sequence; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019853:O(6)-Methylguanine-DNA Methyltransferase
D006147:Guanine; C008449:O-(6)-methylguanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase