Mutagenesis Induced by a Single 1,N-Ethenodeoxyadenosine Adduct in Human Cells

To study the genotoxic properties of 1,N-ethenodeoxyadenosine (edA) in human cells, a novel site-specific mutagenesis approach was developed, in which a single DNA adduct was uniquely placed in either strand of a shuttle plasmid vector. The analysis of progeny plasmid derived from the modified strand shows that edA, when incorporated into the position of the second A of 5*-CAA (codon 61 of the ras gene), is mutagenic in human cells, inducing A3T, A3G, and A3C mutations. The efficient induction of A3T transversions in experiments using modified doubleand singlestranded DNA substrates supports the hypothesis that A:T3T:A transversions in human and animal tumors induced by vinyl compounds reflect misinsertion of dAMP opposite this adduct. Mutagenic events were similar when the adduct was incorporated into either the leading or the lagging strand. edA was more mutagenic than 8-oxodeoxyguanosine, which induced targeted G3T transversions in HeLa cells. In Escherichia coli, edA did not significantly miscode (<0.27%) even in the presence of induced SOS functions.