DNA-SEQUENCE ANALYSIS OF SPONTANEOUS LACI(-D) MUTATIONS IN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE-PROFICIENT AND ALKYLTRANSFERASE-DEFICIENT ESCHERICHIA-COLI

Spontaneous mutagenesis in O-6-alkylguanine-DNA alkyltransferase-profi cient and -deficient (ada ogt mutants) Escherichia coli was studied in two ways: in bacteria growing in nonselective liquid medium and in ba cteria resting on selective agar plates. ATase mutants showed similar spontaneous mutation rates as ATase proficient bacteria during growth phase; an excess of mutants arising in nondividing cells. The resting associated mutagenesis in ada(+) ogt(+) uvr(-) bacteria was biphasic; the high sensitive range being triggered beyond the first 6 days after plating. Contrarily, spontaneous Lac(c) mutants from ada(-) ogt(-) uv r(-) cells steadily increased over the 8 day period of plate incubatio n. These results suggested that, in the absence of nucleotide excision repair, the repair by both the Ada and the Ogt ATases is not saturate d until the cells have been resting for 6 days. The spontaneous lacI(- d) mutation spectrum of ada(+) ogt(+) uvr(-) bacteria growing in non-s elective Liquid medium served as a baseline to determine the mutation events increased in the ATase-deficient derivative upon prolonged incu bation on selective plates. The percentage of G:C-->A:T transitions, p resumably driven by unrepaired O-6-alkylguanine lesions, was increased at the expense of other mutation types. G:C-->A,:T transitions accumu lated with a pronounced 5'-PuG bias, suggesting that the endogenous me tabolite(s) responsible for this mutation class is an S(N)1 type alkyl ating compound(s), Accordingly, the site distribution of G:C-->A:T tra nsitions in nondividing ATase defective bacteria showed similarities w ith the spectra induced by alkylnitrosoureas, particularly with those generating bulky alkylated DNA adducts.