BINDING AND INCISION ACTIVITIES OF UVRABC EXCINUCLEASE ON SLIPPED DNAINTERMEDIATES THAT GENERATE FRAMESHIFT MUTATIONS

Previous in vivo studies involving sequence 5'-CCCG(1)G(2)G(3)-3' (Sma I site) have demonstrated that adducts of N-2-acetylaminofluorene (AAF ) to any of the three guanine residues of the SmaI sequence induce, wi th different efficiencies, two classes of -1 frameshift events, namely -G and -C mutations, referred to as targeted and semitargeted mutatio ns, respectively It has been proposed that both events occur during re plication as a consequence of slippage events involving slipped mutage nic intermediates (SMIs). In order to evaluate the potential role of t he 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 str and 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 Del ta C/3 and Delta G/3 when modified with the AAF on the third guanine r esidue in the top strand or Delta C/O and Delta G/O when unmodified. T he 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-mod ified substrates were recognized with similar efficiency and much more efficiently than unmodified heteroduplexes. With AAF-monomodified sub strates, dissociation of UvrA(2) from the UvrA(2)B-DNA complex occurre d more readily in heteroduplexes than in the homoduplex. SmaI/3 and De lta C/3 were incised with equal efficiency, while Delta G/3 was less i ncised. The position of the AAF lesion dictated the position of the in cised phosphodiester bonds, suggesting that the presence of a bulge ca n modulate the yield but not the incision pattern of AAF-modified subs trates. The finding that UvrABC excinuclease acts on substrates that m imic SMIs suggests that the nucleotide excision repair pathway may hel p in fixing frameshift mutations before the following round of replica tion. (C) 1996 Academic Press Limited