TARGETED A-]T AND G-]T MUTATIONS INDUCED BY SITE-SPECIFIC DEOXYADENOSINE AND DEOXYGUANOSINE ADDUCTS, RESPECTIVELY, FROM THE (-ANTI-DIOL EPOXIDE OF DIBENZ[AJ]ANTHRACENE IN M13MP7L2())

The studies described in this report directly examined the mutagenicit y in Escherichia coli of both a deoxyadenosine (dAdo) and a deoxyguano sine (dGuo) adduct derived from (+)-anti-dibenz[a,j]-anthracene-3,4-di ol 1,2-epoxide [(+)anti-DB[a,j]A-DE] that were site-specifically place d in a single-stranded M13mp7l2 replication vector. An 11-base oligonu cleotide (5'-CTC (A) under bar C (G) under bar CTT CT-3') containing e ither a single (+)anti-DB[a,j]A-DE-trans-N-2-dGuo or (+)anti-DB[a,j]A- DE-trans-N-6-dAdo adduct was successfully incorporated into single-str anded M13mp7L2 plasmid via ligation. In vitro studies using E. coli DN A polymerase I (Klenow fragment) indicated that both adducts were effe ctive blocks for polymerase action. E. coli strains JM103 and JM103 uv rA6 were subsequently transformed with control (unadducted) and adduct -containing M13mp7L2 constructs followed by analysis of progen DNA. In both JM103 and JM103 uvrA6 cells, plaque yields were markedly reduced with adduct containing vectors compared to control vectors. Activatio n of the inducible bacterial DNA repair system (SOS) by UV light only slightly increased the number of plaques recovered from either bacteri al strain transformed with adduct-containing vectors. Targeted mutatio ns were obtained with both adduct-containing vectors in both bacterial strains, whereas no mutations were detected in plaques recovered from control M13mp7L2 vectors. In JM103 cells, (+)anti-DB[a,j]A-DE-N-6-dAd o induced exclusively A --> T transversions and (+)anti-DB[a,j]A-DE-N- 2-dGuo induced exclusively G --> T transversions. In JM103 uvrA6 cells , similar targeted transversion mutations were also obtained except th at a few C deletions (i.e., similar to 10% of the mutations) were dete cted immediately 3' to the dAdo adduct. While mutagenesis was SOS depe ndent in JM103 cells [<0.15% (-SOS) vs similar to 1.3% (+SOS)], it app eared to be SOS independent in JM103 uvrA6 cells (similar to 1-2% in t he presence or absence of SOS induction). It is argued that adduct-ind uced G --> T mutations can be rationalized by either misinformational or noninformational mechanisms. In contrast, A --> T mutations are unl ikely to arise via a misinformational pathway, which provides the stro ngest support to date that bulky DNA adducts can induce mutations via a noninformational pathway.