COMPARATIVE-STUDY OF MUTAGENESIS BY O-6-METHYLGUANINE IN THE HUMAN HA-RAS ONCOGENE IN ESCHERICHIA-COLI AND IN-VITRO

Single residues of O-6-methylguanine (O-6-meG) were introduced into th e first or second position of codon 12 (GGC; positions 12G1 or 12G2, r espectively) or the first position of codon 13 (GGT; position 13G1) of the human Ha-ras oncogene in phage M13-based vectors. After transform ation of E.coli, higher mutant plaque frequencies (MPF) were observed at 12G1 and 13G1 than at 12G2 if O-6-alkylguanine-DNA alkyltransferase (AGT) had been depleted, while similar MPF were observed at all three positions in the presence of active AGT. Taken together, these observ ations suggest reduced AGT repair at 12G2. Kinetic analysis of in Vitr o DNA replication in the same sequences using E.coli DNA polymerase I (Klenow fragment) indicated that variation in polymerase fidelity may contribute to the overall sequence specificity of mutagenesis. By cons tructing vectors which direct methyl-directed mismatch repair to the ( +) or the (-) strand and comparing the MPF values in bacteria proficie nt or deficient in mismatch repair and/or AGT, it was concluded that, while mutS-mediated mismatch repair did not remove O-6-meG from O-6-me G:C pairs, this repair mechanism can affect O-6-meG mutagenesis by rep airing G:T pairs generated through ACT-induced demethylation of O-6-me G:T replication intermediates.