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Analysis at the sequence level of mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene
Abstract:
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.
Insights
This study investigates how chemical carcinogen N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) causes DNA mutations. Researchers found that N-AcO-AAF primarily induces frameshift mutations at specific GC base pairs in the tetracycline-resistance gene.
Area of Science:
- Molecular Biology
- Genetics
- Carcinogenesis
Background:
- Chemical carcinogens form DNA adducts, leading to mutations.
- Understanding the conversion of premutational lesions to stable mutations is crucial.
- N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) is a model carcinogen that forms DNA adducts.
Purpose of the Study:
- To analyze the sequence changes of stable mutations induced by N-AcO-AAF.
- To investigate the mechanism by which N-AcO-AAF-induced DNA adducts are converted into mutations.
- To identify mutation hotspots within a specific DNA sequence.
Main Methods:
- In vitro reaction of N-AcO-AAF with DNA to form adducts.
- Directed chemical reaction of the carcinogen to a specific DNA fragment (BamHI-SalI) in the tetracycline-resistance gene of pBR322 plasmid.
- Selection of mutants for ampicillin resistance and tetracycline sensitivity.
- DNA sequencing of the affected gene fragment to analyze mutation types.
Main Results:
- N-AcO-AAF primarily forms a guanine adduct (N-2-(deoxyguanosin-8-yl)-acetylaminofluorene).
- Mutations induced were mainly frameshifts involving GC base pairs.
- Specific base pairs (hotspots) within the targeted DNA fragment were affected at high frequencies.
- The binding of N-AcO-AAF to DNA causes local helix distortion.
Conclusions:
- Frameshift mutations are a major outcome of N-AcO-AAF adducts at GC base pairs.
- Specific DNA sequences act as hotspots for N-AcO-AAF-induced mutations.
- This study provides insight into the molecular mechanisms of chemical mutagenesis.