Binding and incision activities of UvrABC excinuclease on slipped DNA intermediates that generate frameshift mutations

Previous in vivo studies involving sequence 5'-CCCG(1)G(2)G(3)-3' (SmaI site) have demonstrated that adducts of N-2-acetylaminofluorene (AAF) to any of the three guanine residues of the SmaI sequence induce, with different efficiencies, two classes of -1 frameshift events, namely -G and -C mutations, referred to as targeted and semitargeted mutations, respectively It has been proposed that both events occur during replication as a consequence of slippage events involving slipped mutagenic intermediates (SMIs). In order to evaluate the potential role of the UvrABC excinuclease in frameshift mutagenesis, we have studied the interaction of this enzyme with DNA molecules mimicking SMIs in vitro. In all of our constructions, when present, the AAF adduct was located on the third guanine residue of the SmaI site (5'-CCCG(1)G(2)G(3)-3'). This strand was referred to as the top strand, the complementary strand being the bottom strand. Double-stranded heteroduplexes mimicking the targeted and semitargeted SMIs contained a deletion of a C and a G within the SmaI sequence in the bottom strand and were designated Delta C/3 and Delta G/3 when modified with the AAF on the third guanine residue in the top strand or Delta C/O and Delta G/O when unmodified. The modified homoduplex was designated SmaI/S. Delta C/O and Delta G/O were weakly recognized by UvrA(2)B, but not incised. All three AAF-modified substrates were recognized with similar efficiency and much more efficiently than unmodified heteroduplexes. With AAF-monomodified substrates, dissociation of UvrA(2) from the UvrA(2)B-DNA complex occurred more readily in heteroduplexes than in the homoduplex. SmaI/3 and Delta C/3 were incised with equal efficiency, while Delta G/3 was less incised. The position of the AAF lesion dictated the position of the incised phosphodiester bonds, suggesting that the presence of a bulge can modulate the yield but not the incision pattern of AAF-modified substrates. The finding that UvrABC excinuclease acts on substrates that mimic SMIs suggests that the nucleotide excision repair pathway may help in fixing frameshift mutations before the following round of replication. (C) 1996 Academic Press Limited