Comparison of Mutagenesis By O-6-Methyl- 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 in Escherichia coli, double-stranded and gapped plasmids were constructed 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 the loss of lacZ' activity on X-gal-containing media resulting in formation of white or sectored (mutant) rather than blue (non-mutant) colonies. Genotoxicity experiments with gapped plasmids containing the modified 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 colonies) than m(6)G (6%) or e(6)G (11%) in repair competent (w.t.) E. coli 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-alkylguanine-DNA alkyltransferases as well as methylation-directed mismatch repair (ada(-)-ogt(-)-mutS(-)). m(6)G in gapped plasmids produced 62% mutant 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 and m(6)G produced very similar distributions of mutant and non-mutant colonies in the ada(-)-ogt(-)-mutS(-) strain. These observations led to 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 compared to m(4)T by alkyltransferase and mismatch repair mechanisms, and did not reflect differences in their respective coding efficiency or their inherent obstructiveness to DNA synthesis as was observed with e(6)G. The combination of alkyltransferase and mismatch repair was concluded to be primarily responsible for the apparent genotoxicity of m(6)G compared to m(4)T in double-stranded plasmids. [References: 25]

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