Analysis at the sequence level of mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene.
The covalent binding of an ultimate carcinogen to the DNA bases or phosphate groups creates a premutational lesion that in vivo is processed by the repair, replication and recombination enzymes, and eventually may be converted into a mutation. Being interested in the way that an initial premutational event is converted into a stable heritable mutation, we have sequenced stable mutations in a gene that has formed covalent adducts in vitro with N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF, a model for the ultimate metabolite of the rat liver carcinogen 2-acetylaminofluorene, AAF). In vivo studies have shown the mutagenicity of AAF and its derivatives in both bacterial and eukaryotic systems. N-AcO-AAF reacts in vitro with DNA leading mainly to the formation of a guanine adduct, N-2-(deoxyguanosin-8-yl)-acetylaminofluorene (80%) and to at least three minor adducts. Studies by our group showed that binding of N-AcO-AAF to DNA resulted in a local distortion of the DNA helix around the C-8 adduct (the insertion-denaturation model). We describe here the analysis of forward mutations induced in the tetracycline-resistance gene of pBR322 by directing the chemical reaction of the carcinogen to a small restriction fragment (BamHI-SalI) inside the antibiotic-resistance gene. Mutants are selected for ampicillin (Ap) resistance and tetracycline (Tc) sensitivity. The plasmid DNA of such mutants was analyzed for sequence changes in the fragment where the AAF binding had been directed. We show here that the mutations are mainly frameshifts involving GC base pairs and that certain base pairs (hotspots) are affected at high frequencies.
D015073:2-Acetylaminofluorene / Q000031:analogs & derivatives*; D000099:Acetoxyacetylaminofluorene / Q000494:pharmacology*; D000667:Ampicillin / Q000494:pharmacology; D001483:Base Sequence / Q000187:drug effects; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000187:drug effects*; D009152:Mutagenicity Tests; D009153:Mutagens; D010403:Penicillin Resistance; D013754:Tetracyclines / Q000494:pharmacology
D004269:DNA, Bacterial; D009153:Mutagens; D013754:Tetracyclines; D000099:Acetoxyacetylaminofluorene; D000667:Ampicillin; D015073:2-Acetylaminofluorene