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Induction of mutations by N-acetoxy-N-acetyl-2-aminofluorene modified M13 viral DNA
P K Gupta1, R G Pandrangi, M S Lee
1Department of Chemical Carcinogenesis, Michigan Cancer Foundation, Detroit 48201.
Abstract:
The specificity of N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (G-8-AAF) adducts in double-stranded DNAs from M13mp8 and M13mp9 bacteriophage was determined following transfection of modified DNA with multiple adducts into competent JM103 cells. Mutant phages were selected by phenotypic screening for colorless or light blue plaques indicating a defective beta-galactosidase marker enzyme. Mutation frequencies of phage DNA with G-8-AAF adducts were increased up to 8-fold in SOS-induced host cells as compared to the uninduced JM103 host cells. DNA sequencing of mutants from SOS-induced host cells indicated approximately 52% frameshifts and 39% base substitutions in M13mp8 DNA and 65% frameshifts and 25% base substitutions in M13mp9 DNA. Mutation spectra exhibited mutations at many sites within the bp 6200-6400 region; one mutational hotspot at position 6343-6347 (5' GGGGG 3') for frameshifts was also observed. The G-8-AAF adduct induced mostly single base deletions at this site. In contrast, a deacetylated adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene (G-8-AF) in our previous experiments induced mostly single base additions at the same position indicating the ability of adduct structure to modulate the specificity of frameshift mutations. A number of other frameshift mutations (11 out of 29) were observed within non-repetitive and non-palindromic sequences. Molecular mechanisms for the induction of these mutations by DNA perturbations produced by the G-8-AAF adducts are discussed.
Insights
The N-acetyl-2-aminofluorene (G-8-AAF) DNA adduct causes mutations, primarily frameshifts, in bacteriophage DNA. Adduct structure influences mutation type, with G-8-AAF inducing deletions and G-8-AF inducing additions.
Area of Science:
- Molecular biology
- Genetics
- Toxicology
Background:
- DNA adducts are chemical modifications to DNA that can lead to mutations.
- The carcinogen 2-aminofluorene (AF) forms DNA adducts, including N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (G-8-AAF).
- Understanding how DNA adducts cause mutations is crucial for assessing their carcinogenic potential.
Purpose of the Study:
- To determine the mutation specificity of G-8-AAF adducts in double-stranded DNA.
- To investigate the role of adduct structure in modulating frameshift mutations.
- To elucidate the molecular mechanisms underlying G-8-AAF-induced mutagenesis.
Main Methods:
- Transfection of modified M13mp8 and M13mp9 bacteriophage DNA containing G-8-AAF adducts into competent JM103 cells.
- Phenotypic screening of mutant phages based on beta-galactosidase activity.
- DNA sequencing of mutants to identify mutation types and hotspots.
- Comparison with previous data on the deacetylated adduct, G-8-AF.
Main Results:
- G-8-AAF adducts increased mutation frequencies up to 8-fold in SOS-induced host cells.
- Mutations included approximately 52% frameshifts and 39% base substitutions in M13mp8 DNA, and 65% frameshifts and 25% base substitutions in M13mp9 DNA.
- A mutational hotspot at 5' GGGGG 3' (positions 6343-6347) primarily induced single base deletions by G-8-AAF, contrasting with single base additions by G-8-AF.
Conclusions:
- The G-8-AAF adduct induces a high frequency of frameshift mutations, predominantly deletions at repetitive sequences.
- The structure of the DNA adduct (acetylated vs. deacetylated) significantly modulates the specificity of frameshift mutations.
- DNA perturbations caused by G-8-AAF adducts are responsible for inducing these mutations through specific molecular mechanisms.