Th. Broschard et al., Sequence-dependent modulation of frameshift mutagenesis at NarI-derived mutation hot spots, J MOL BIOL, 288(1), 1999, pp. 191-199
The NarI sequence is known to be the strongest mutation hot spot for induce
d frameshift mutagenesis. indeed, a single N-2-acetylaminofluorene (AAF) ad
duct 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-te
mplate misalignment event during replication of the adduct-containing seque
nce. The slipped mutagenic intermediate (SMI) that is thus formed is strong
ly stabilised by the AAF residue, in order to understand the origin of the
extreme susceptibility of this sequence to frameshift mutagenesis, we analy
sed AAF-induced mutagenesis at sequences 5'-N(a)GCG(AAF)CN(b)-3' containing
the core dinucleotide GCGC repeat present in the NarI 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 t
o 30 to 50-fold), while little if any effect could be attributed to nucleot
ide N-a. The induction of -2 frameshifts, regardless of nucleotides N-a and
N-b, was found to be SOS-inducible but umu/DC-independent as previously fo
und for the authentic NarI sequence. The NarI sequence (GGCGCC) and sequenc
e TGCGCA (N-a = T, N-b = A) were found to be equally "hot" for -2 frameshif
t mutation induction compared to the sequence AGCGCT where induced mutagene
sis 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 na
ture of the nucleotide flanking the adduct on its 3'-side (N-b) differentia
lly stabilises the SMI will be discussed in the light of recent structural
data and theoretical models. (C) 1999 Academic Press.