Site-specific mutagenesis using a gapped duplex vector: a study of translesion synthesis past 8-oxodeoxyguanosine in E. coli.

We have constructed a gapped plasmid vector in which a single defined lesion is introduced, site-specifically, within a single-strand region. Efficiency of translesional synthesis is determined by the number of colonies recovered following transformation of E. coli. The nucleotide sequence of progeny plasmids in the gapped region of the vector reflects incorporation of bases opposite and near the lesion. The analysis detects non-mutagenic as well as mutagenic events. This system was used to establish the mutagenic potential of 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxodG), a lesion produced by the action of active oxygen species on DNA. The presence of 8-oxodG did not affect the number of transformants recovered. Most transformants (greater than 99%) contained G:C pairs at the site of the lesion; however, a limited number of targeted G----T transversions were observed in the presence and absence of SOS induction. Base substitutions neighboring the lesion, reported for an in vitro system, were not observed. We conclude that the 8-oxodG lesion in DNA is weakly mutagenic in E. coli.