Effect of benzo-ring hydroxyl groups on site-specific mutagenesis by tetrahydrobenzo[a]pyrene adducts at N-6 of deoxyadenosine

We have previously investigated the mutations induced on replication in Esc herichia coli of the M13mp7L2 genome containing each of the eight possible adducts derived from the four optically active 7,8-diol 9,10-epoxide metabo lites of benzo[a]pyrene (B[a]P) by alkylation of a specific deoxyadenosine (dAdo) residue at N-6. Observed mutational frequencies depended in part on the relative spatial orientations of the three hydroxyl groups in these add ucts. To determine how the presence or absence of these hydroxyl groups aff ects mutational response, we have synthesized 16-mer oligonucleotides with the same sequence as one of those previously studied with the diol epoxide adducts, but containing B[a]P-dAdo adducts in which two or all three of the adduct hydroxyl groups were replaced by hydrogen. Transfection of the addu cted M13 constructs into SOS-induced Escherichia coli consistently gave few er infective centers than the control construct, with viabilities ranging f rom 8.4 to 44.9% relative to control. In general, decreasing the number of adduct hydroxyls decreased the total frequency of substitution mutations in duced. For all but one of the present adducts, the total mutational frequen cy was lower than that for any of the previously reported diol epoxide addu cts in the same sequence. Remarkably, this (9S,10R)-adduct with cis orienta tion of the dAdo residue and the 9-OH group gave the highest mutational fre quency of all the B[a]P adducts studied in this sequence, including the dio l epoxide adducts. With the present adducts, A --> T transversions predomin ated, with smaller numbers of A --> G transitions and even fewer A --> C tr ansversions.