Binding and Incision Activities of UvrABC Excinuclease on Slipped DNA Intermediates that Generate Frameshift Mutations |
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| Institution: | 1. U.P.R. 9003 CNRS, “Cancérogenèse et Mutagenèse Moléculaire et Structurale” Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, 67400 Strasborg/Illkirch-Graffenstaden, France;1. MOE Key Laboratory of Bioinformatics and Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China;2. Tsinghua-Peking Center for Life Sciences, Beijing 100084, China;3. Institute of Genetics and Biotechnology, Hungarian University of Agriculture and Life Sciences, Szent-Györgyi Albert u. 4, Gödöll? 2100, Hungary;4. Department of Cell and Systems Biology, University of Toronto, 25 Willcocks Street, Toronto, ON M5S 3B2, Canada;5. Department of Plant and Microbial Biology, North Carolina State University, Raleigh NC 27695, USA;6. Research Centre for Plant RNA Signaling, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China;7. School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK;8. School of Science and the Environment, University of Worcester, Worcester WR2 6AJ, UK |
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| Abstract: | Previousin vivostudies involving sequence 5′-CCCG1G2G3-3′ (SmaI site) have demonstrated that adducts ofN-2-acetylaminofluorene (AAF) to any of the three guanine residues of theSmaI 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 SMIsin vitro.In all of our constructions, when present, the AAF adduct was located on the third guanine residue of theSmaI 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 theSmaI sequence in the bottom strand and were designated ΔC/3 and ΔG/3 when modified with the AAF on the third guanine residue in the top strand or ΔC/O and ΔG/O when unmodified. The modified homoduplex was designatedSmaI/3.ΔC/O and ΔG/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 UvrA2from the UvrA2B- DNA complex occurred more readily in heteroduplexes than in the homoduplex.SmaI/3 and ΔC/3 were incised with equal efficiency, while ΔG/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. |
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