Mutagenesis studies with four stereoisomeric N2-dG benzo[a]pyrene adducts in the identical 5'-CGC sequence used in NMR studies: G->T mutations dominate in each case

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) and a potent mutagen/carcinogen found ubiquitously in the environment. B[a]P is primarily metabolized to diol epoxides, which react principally at N2-dG in DNA. B[a]P-N2-dG adducts have been shown to induce a variety of mutations, notably G-->T, G-->A, G-->C and -1 frameshifts. Four stereoisomers of B[a]P-N2-dG (designated: [+ta]-;, [+ca]-, [-ta] and [-ca]) were studied by NMR in duplex 11mers in a 5'-CGC sequence context, and each adopted a different adduct conformation (Geacintov, et al. (1997) Chem. Res. Toxicol., 10, 111). Herein these four identical B[a]P-containing 11mers are built into duplex plasmid genomes and mutagenesis studied in Escherichia coli following SOS-induction. In nucleotide excision repair (NER) proficient E.coli, no adduct-derived mutants are detected. In NER deficient E.coli, G-->T mutations dominate for all four stereoisomers [+ta]-, [+ca]-, [-ta] and [-ca]-B[a]P-N(2)-dG, and mutation frequency is similar. Thus, the mutagenic pattern for these four B[a]P-N2-dG stereoisomers is the same, in spite of the fact that they adopt dramatically different conformations in ds-oligonucleotides as determined by NMR. These findings suggest that adduct conformation must be fluid enough in the 5'-CGC sequence that the duplex DNA conformation can interconvert to mutagenic and non-mutagenic conformations during lesion-bypass. A comparison of all published studies with these four B[a]P-N2-dG stereoisomers in E.coli reveals that B[a]P-N2-dG adduct stereochemistry tends to have a lesser impact on mutagenic pattern (e.g. G-->T versus G-->A mutations) than does DNA sequence context, which is discussed.