Sequence-dependent modulation of frameshift mutagenesis at NarI-derived mutation hot spots

Abstract The Nar I sequence is known to be the strongest mutation hot spot for induced frameshift mutagenesis. Indeed, a single N -2-acetylaminofluorene (AAF) adduct induces −2 frameshift mutations (5′-GGCG AAF CC → 5′-GGCC) more than 10 7 -fold over background mutagenesis in Escherichia coli . The mechanism of induction of the frameshift mutation involves a two nucleotide primer-template misalignment event during replication of the adduct-containing sequence. The slipped mutagenic intermediate (SMI) that is thus formed is strongly stabilised by the AAF residue. In order to understand the origin of the extreme susceptibility of this sequence to frameshift mutagenesis, we analysed AAF-induced mutagenesis at sequences 5′- N a GCG AAF C N b -3′ containing the core dinucleotide GCGC repeat present in the Nar I sequence flanked by variable nucleotides N a and N b . The nature of nucleotide N b was found to strongly modulate the frequency of induced −2 frameshift mutagenesis (up to 30 to 50-fold), while little if any effect could be attributed to nucleotide N a . The induction of −2 frameshifts, regardless of nucleotides N a and N b , was found to be SOS-inducible but umuDC -independent as previously found for the authentic Nar I sequence. The Nar I sequence (GGCGCC) and sequence TGCGCA ( N a = T, N b = A) were found to be equally “hot” for −2 frameshift mutation induction compared to the sequence AGCGCT where induced mutagenesis was 30 to 50-fold lower. The analysis of replication events using constructions containing a strand marker across from the adduct site allowed us to demonstrate that the large difference in −2 frameshift mutagenesis is due to an intrinsic difference in the propensity of these sequences to slip during replication. How the nature of the nucleotide flanking the adduct on its 3′-side ( N b ) differentially stabilises the SMI will be discussed in the light of recent structural data and theoretical models.