title: Mutagenic properties of 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, a persistent acetylaminofluorene-derived DNA adduct in mammalian cells.
abstract: 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-N(2)-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol alpha 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 eta or pol kappa nucleotides were incorporated opposite the lesion. Both pol eta and pol kappa incorporated dCMP, the correct base, opposite dG-N(2)-AAF. In reactions catalyzed by pol eta, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol kappa, 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-N(2)-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G --> T transversions overall, with dG-N(2)-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 eta, but not pol kappa, may play a role in this process.
authors.affiliation: Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, USA.
authors.forename: Manabu
authors.initials: M
authors.lastname: Yasui
authors.forename: Huan
authors.initials: H
authors.lastname: Dong
authors.forename: Radha R
authors.initials: RR
authors.lastname: Bonala
authors.forename: Naomi
authors.initials: N
authors.lastname: Suzuki
authors.forename: Haruo
authors.initials: H
authors.lastname: Ohmori
authors.forename: Fumio
authors.initials: F
authors.lastname: Hanaoka
authors.forename: Francis
authors.initials: F
authors.lastname: Johnson
authors.forename: Arthur P
authors.initials: AP
authors.lastname: Grollman
authors.forename: Shinya
authors.initials: S
authors.lastname: Shibutani
mesh_terms: D015073:2-Acetylaminofluorene / Q000031:analogs & derivatives* / Q000737:chemistry / Q000633:toxicity*; D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D018736:DNA Adducts / Q000737:chemistry / Q000633:toxicity*; D004249:DNA Damage / Q000235:genetics; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000633:toxicity; D003854:Deoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D005822:Genetic Vectors; D007700:Kinetics; D008968:Molecular Conformation; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000737:chemistry / Q000633:toxicity*; D013698:Templates, Genetic
chemical_list: D018736:DNA Adducts; D003854:Deoxyribonucleotides; D009153:Mutagens; D015073:2-Acetylaminofluorene; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine