Binding and incision activities of UvrABC excinuclease on slipped DNA intermediates that generate frameshift mutations.
Previous in vivo studies involving sequence 5'-CCCG1G2G3-3' (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'-CCCG1G2G3-3'). 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 deltaC/3 and deltaG/3 when modified with the AAF on the third guanine residue in the top strand or deltaC/O and deltaG/O when unmodified. The modified homoduplex was designated SmaI/3. deltaC/O and deltaG/O were weakly recognized by UvrA2B, 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 UvrA2 from the UvrA2B-DNA complex occurred more readily in heteroduplexes than in the homoduplex. SmaI/3 and deltaC/3 were incised with equal efficiency, while deltaG/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.
Delagoutte
E
E
U.P.R., CNRS, Strasborg/Illkirch-Graffenstaden, France.
Bertrand-Burggraf
E
E
Lambert
I B
IB
Fuchs
R P
RP
D015073:2-Acetylaminofluorene / Q000378:metabolism / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D016368:Frameshift Mutation; D006147:Guanine / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; C058093:UvrB protein, E coli; D006147:Guanine; D004247:DNA; D015073:2-Acetylaminofluorene; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases