Mutagenesis induced by a single 1,N-6-ethenodeoxyadenosine adduct in humancells

To study the genotoxic properties of 1,N-6-ethenodeoxyadenosine (epsilon dA ) 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 shu ttle plasmid vector. The analysis of progeny plasmid derived from the modif ied strand shows that epsilon dA, when incorporated into the position of th e second A of 5'-CAA (codon 61 of the ras gene), is mutagenic in human cell s, inducing A-->T, A-->G, and A-->C mutations. The efficient induction of A -->T transversions in experiments using modified double- and single-strande d DNA substrates supports the hypothesis that A:T-->T:A transversions in hu man and animal tumors induced by vinyl compounds reflect misinsertion of dA MP opposite this adduct. Mutagenic events were similar when the adduct was incorporated into either the leading or the lagging strand. epsilon dA was more mutagenic than 8-oxodeoxyguanosine, which induced targeted G-->T trans versions in HeLa cells. In Escherichia coli, epsilon dA did not significant ly miscode (<0.27%) even in the presence of induced SOS functions.