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Sequence determinants for -2 frameshift mutagenesis at NarI-derived hot spots
1CNRS UPR #9003 Cancérogenèse et Mutagenèse Moléculaire et Structurale ESBS, Strasbourg-Illkirch, France.
Journal of Molecular Biology
|October 6, 1995
Summary
Chemical carcinogen N-2-acetylaminofluorene (AAF) induces -2 frameshift mutations at NarI sites. Flanking nucleotides significantly modulate mutation frequency, with the 3' base (Nb) playing a key role in this process.
Area of Science:
- Molecular Biology
- Genetics
- Chemical Carcinogenesis
Background:
- The NarI restriction enzyme recognition sequence is a known hot spot for -2 frameshift mutations induced by N-2-acetylaminofluorene (AAF).
- Previous studies identified G3 in the template strand as critical for high-level AAF-induced -2 frameshift mutagenesis.
Purpose of the Study:
- To define a consensus sequence for the NarI mutation hot spot.
- To investigate the role of flanking bases (Na and Nb) in the NarI sequence (NaGCGCNb) on AAF-induced mutation frequency.
Main Methods:
- Construction and random modification of 16 plasmids with varying flanking bases (Na, Nb) using AAF.
- Analysis of mutation frequency and type (predominantly CpG deletion) across all 16 sequences.
Main Results:
- All 16 tested sequences were found to be -2 frameshift mutation hot spots, with frequencies 500-5000-fold above background.
- The nucleotide Nb, located 3' to the central GpC repeat, significantly modulated mutation frequency.
- The nucleotide Na, located 5' to the central GpC repeat, had minimal effect on mutation frequency.
Conclusions:
- The 3' flanking nucleotide (Nb) is a critical determinant of mutation frequency at AAF-induced NarI hot spots.
- A model involving template-primer misalignment and a two-nucleotide slipped intermediate explains the modulation of mutagenesis by Nb.
- This finding refines understanding of frameshift mutagenesis mechanisms and sequence context effects.