Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III |
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Authors: | Nikita A Kuznetsov Olga A Kladova Alexandra A Kuznetsova Alexander A Ishchenko Murat K Saparbaev Dmitry O Zharkov Olga S Fedorova |
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Institution: | From the ‡Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentyev Ave., Novosibirsk 630090, Russia.;the §Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia, and ;the ¶Groupe “Réparation de l''ADN,” Université Paris-Sud XI, UMR8200 CNRS, Institute Gustave Roussy, Villejuif Cedex F-94805, France |
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Abstract: | Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3′-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln41 and Leu81 as DNA lesion sensors. |
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Keywords: | DNA damage DNA repair endonuclease enzyme kinetics fluorescence protein-DNA interaction abasic site dihydrouracil stopped-flow enzyme kinetics |
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