title: Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III.
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 Gln(41) and Leu(81) as DNA lesion sensors.
authors: 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 nikita.kuznetsov@niboch.nsc.ru.
authors: Nikita A
authors: NA
authors: Kuznetsov
authors: 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.
authors: Olga A
authors: OA
authors: Kladova
authors: 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.
authors: Alexandra A
authors: AA
authors: Kuznetsova
authors: the Groupe "Réparation de l'ADN," Université Paris-Sud XI, UMR8200 CNRS, Institute Gustave Roussy, Villejuif Cedex F-94805, France.
authors: Alexander A
authors: AA
authors: Ishchenko
authors: the Groupe "Réparation de l'ADN," Université Paris-Sud XI, UMR8200 CNRS, Institute Gustave Roussy, Villejuif Cedex F-94805, France.
authors: Murat K
authors: MK
authors: Saparbaev
authors: 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.
authors: Dmitry O
authors: DO
authors: Zharkov
authors: 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 fedorova@niboch.nsc.ru.
authors: Olga S
authors: OS
authors: Fedorova
mesh_terms: D001483:Base Sequence; D004260:DNA Repair; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli
keywords: DNA damage; DNA repair; abasic site; dihydrouracil; endonuclease; enzyme kinetics; fluorescence; protein-DNA interaction; stopped-flow enzyme kinetics