Molecular mechanisms of mutagenesis by aromatic amines and amides.
The mutagenic properties of 2-acetylaminofluorene-derived DNA adducts, including N-(deoxyguanosin-8-yl)-2-acetylaminofluorene, N-(deoxyguanosin-8-yl)-2-aminofluorene, N-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, and several minor oxidation products have been explored, using site-specific techniques. Oligodeoxynucleotides containing a single AAF-derived DNA adduct were prepared by postsynthetic modification and used as templates in primer extension reactions catalyzed by bacterial and mammalian DNA polymerases. Base substitutions and deletions occurring during DNA synthesis were quantified. dG-C8-AAF promoted one- and two-base deletions and small amounts of incorporation of dCMP, dAMP, and/or dTMP opposite the lesion in reactions catalyzed by the 3'-->5' exonuclease-free Klenow fragment of DNA polymerase 1 (exo-) and polymerase alpha. dG-C8-AF did not miscode in reactions catalyzed by exo-; however, base misincorporation and deletions were observed in reactions with pol alpha. dG-N2-AAF promoted small amounts of dAMP incorporation in reactions catalyzed by exo-. The miscoding potential of minor oxidation products of dG-C8-AF was much higher than that of other adducts. Steady-state kinetics were used to measure frequencies of nucleotide insertion opposite the lesion and chain extension from the 3' terminus. Kinetic data were consistent with the results of primer extension studies. A mutation 'hot spot' was constructed and the influence of sequence context on the frequency of deletions generated by dG-C8-AAF was explored systematically in reactions catalyzed by exo-. Based on our results with aminofluorene DNA adducts, we propose a general mechanism for frameshift deletion mutagenesis. Site-specific methods also were used to establish the mutagenic potential of AAF-derived DNA adducts in mammalian cells. dG-C8-AAF and dG-C8-AF exhibited similar mutagenic specificities, predicting the occurrence of G-->T transversions and G-->A transitions in mammalian cells.
Shibutani
S
S
Department of Pharmacological Sciences, State University of New York at Stony Brook 11794-8651, USA.
Grollman
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AP
D015073:2-Acetylaminofluorene / Q000633:toxicity*; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D018736:DNA Adducts; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D016296:Mutagenesis; D009152:Mutagenicity Tests; D017384:Sequence Deletion
D018736:DNA Adducts; D015073:2-Acetylaminofluorene; D004259:DNA-Directed DNA Polymerase