Mutagenic properties of 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, a persistent acetylaminofluorene-derived DNA adduct in mammalian cells.

The carcinogen 2-acetylaminofluorene is metabolically activated in cells and reacts with DNA to form N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N 2 -yl)-2-acetylaminofluorene (dG-N 2 -AAF) DNA adducts. The dG-N 2 -AAF adduct is the least abundant of the three isomers, but it persists in the tissues of animals treated with this carcinogen. The miscoding and mutagenic properties of dG-C8-AAF and dG-C8-AF have been established; these adducts are readily excised by DNA repair enzymes engaged in nucleotide excision repair. In the present study, oligodeoxynucleotides modified site-specifically with dG-N2-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol a were strongly blocked at a position one base before dG-N 2 -AAF and also opposite this lesion. In contrast, during translesion synthesis catalyzed by pol η or pol K nucleotides were incorporated opposite the lesion. Both pol η and pol K incorporated dCMP, the correct base, opposite dG-N 2 -AAF. In reactions catalyzed by pol η, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol K, significant dTMP misincorporation was observed opposite the lesion. Steady-state kinetic analysis confirmed the results obtained from primer extension studies. Single-stranded shuttle vectors containing 5 ' TCCTCCTCXCCTCTC (X = dG-N 2 -AAF, dG-C8-AAF, or dG) were used to establish the frequency and specificity of dG-N2-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G → T transversions overall, with dG-N2-AAF being less mutagenic than dG-C8-AAF (3.4% vs 12.5%). We conclude from this study that dG-N 2 -AAF, by virtue of its persistence in tissues, contributes significantly to the mutational spectra observed in AAF-induced mutagenesis and that pol η, but not pol K, may play a role in this process.