COMPARISON OF MUTAGENESIS BY O-6-METHYLGUANINE AND O-6-ETHYLGUANINE AND O-4-METHYLTHYMINE IN ESCHERICHIA-COLI USING DOUBLE-STRANDED AND GAPPED PLASMIDS

To compare mutagenesis by O-6-methylguanine (m(6)G), O-4-methylthymine (m(4)T) and O-6-ethylguanine (e(6)G), and assess their genotoxicity i n Escherichia coli, double-stranded and gapped plasmids were construct ed containing a single m(6)G, e(6)G or m(4)T in the initiation codon ( ATG) of a lacZ' gene. Modified base induced mutations were scored by t he loss of lacZ' activity on X-gal-containing media resulting in forma tion of white or sectored (mutant) rather than blue (non-mutant) colon ies. Genotoxicity experiments with gapped plasmids containing the modi fied bases indicated that m(4)T produced a greater number of bacterial colonies than m(6)G or e(6)G, m(4)T was more mutagenic (45% mutant co lonies) than m(6)G (6%) or e(6)G (11%) in repair competent (w.t.) E. c oli when incorporated in double-stranded plasmids. In gapped plasmids, m(4)T produced 99% mutant colonies (as was observed previously for e( 6)G) in both w.t. E. coli or E. coli deficient in both O-6-alkylguanin e-DNA alkyltransferases as well as methylation-directed mismatch repai r (ada(-)-ogt(-)-mutS(-)). m(6)G in gapped plasmids produced 62% mutan t colonies in wt. E. coli, but this percentage increased to 94% in the ada(-)-ogt(-)-mutS(-)strain. In double-stranded plasmids both m(4)T a nd m(6)G produced very similar distributions of mutant and non-mutant colonies in the ada(-)-ogt(-)-mutS(-) strain. These observations led t o the conclusion that differences in the mutagenicity of m(6)G and m(4 )T in w.t. E. coli were a result of preferential repair of m(6)G compa red to m(4)T by alkyltransferase and mismatch repair mechanisms, and d id not reflect differences in their respective coding efficiency or th eir inherent obstructiveness to DNA synthesis as was observed with e(6 )G. The combination of alkyltransferase and mismatch repair was conclu ded to be primarily responsible for the apparent genotoxicity of m(6)G compared to m(4)T in double-stranded plasmids.