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Sequence-dependent modulation of frameshift mutagenesis at NarI-derived mutation hot spots
T H Broschard1, N Koffel-Schwartz, R P Fuchs
1Cancerogenese et Mutagenese Moleculaire et Structurale, CNRS, Blvd Sébastien Brant, Strasbourg, 67400, France.
Journal of Molecular Biology
|May 18, 1999
Summary
The NarI sequence is a mutation hotspot. The flanking nucleotide on the 3' side significantly influences N-2-acetylaminofluorene (AAF)-induced frameshift mutations by stabilizing a slipped mutagenic intermediate.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The NarI sequence is recognized as a potent mutation hotspot for induced frameshift mutagenesis.
- A single N-2-acetylaminofluorene (AAF) adduct can trigger -2 frameshift mutations at a rate exceeding 10^7-fold over background levels in Escherichia coli.
- The mechanism involves a two-nucleotide primer-template misalignment during replication of the adduct-containing DNA.
Purpose of the Study:
- To investigate the origin of the NarI sequence's extreme susceptibility to frameshift mutagenesis.
- To analyze AAF-induced mutagenesis at sequences containing the GCGC repeat flanked by variable nucleotides (Na and Nb).
- To determine how flanking nucleotides influence the frequency and mechanism of frameshift mutations.
Main Methods:
- Mutagenesis assays using modified DNA sequences with N-2-acetylaminofluorene (AAF) adducts.
- Analysis of AAF-induced mutagenesis at 5 omino-NaGCGAAFCNb-3 omino sequences.
- Investigation of SOS-inducibility and umuDC-dependence of mutations.
- Replication event analysis using strand markers to assess DNA slippage propensity.
Main Results:
- The nucleotide at the 3 omino-side (Nb) strongly modulated -2 frameshift mutagenesis frequency (up to 30-50 fold), while the 5 omino-side nucleotide (Na) had minimal effect.
- Mutagenesis was SOS-inducible but umuDC-independent, consistent with the NarI sequence.
- Sequences like NarI (GGCGCC) and TGCGCA were highly mutable, whereas AGCGCT showed significantly lower induced mutagenesis (30-50 fold reduction).
- Differences in mutagenesis frequency correlated with intrinsic differences in sequence slippage propensity during replication.
Conclusions:
- The flanking nucleotide on the 3 omino-side of an adduct-containing sequence plays a critical role in stabilizing the slipped mutagenic intermediate (SMI).
- This stabilization directly impacts the frequency of frameshift mutagenesis.
- Understanding these sequence-context effects is crucial for deciphering mutation mechanisms and DNA repair pathways.