title: Conformational dynamics of the interaction of Escherichia coli endonuclease VIII with DNA substrates.
abstract: Endonuclease VIII (Nei) from Escherichia coli is a DNA repair enzyme that removes a wide range of oxidized pyrimidine bases from DNA. As inferred from the crystal structures and biochemical studies, recognition of DNA lesions by Nei involves several conformational changes in both protein and DNA, such as DNA kinking, damaged base eversion into the enzyme's active site, and insertion of a loop of the enzyme into the void formed by the eversion. Excision of the damaged base by Nei also proceeds through several chemical steps: N-glycosidic bond breakage, β-elimination and δ-elimination of the phosphates flanking the lesion. We have used stopped-flow kinetics with fluorescence detection to follow conformational changes in the Nei molecule when the enzyme binds normal DNA, damaged but uncleavable DNA, or several cleavable damaged DNA substrates. Binding normal or damaged uncleavable DNA proceeded in two fluorescently discernible reversible stages, while processing of cleavable substrates involved three reversible stages followed by and irreversible stage and equilibrium with the reaction product. Individual rate constants were calculated for each reaction step. Based on the stopped-flow data, crystal structure, and a comparison with the stopped-flow kinetics of E. coli formamidopyrimidine-DNA glycosylase, a homolog of Nei, we propose the nature of some of the steps that may be involved into the recognition and excision of damaged bases by Nei.
authors.affiliation: SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., Novosibirsk 630090, Russia.
authors.forename: Nikita A
authors.initials: NA
authors.lastname: Kuznetsov
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Koval
authors.forename: Dmitry O
authors.initials: DO
authors.lastname: Zharkov
authors.forename: Olga S
authors.initials: OS
authors.lastname: Fedorova
mesh_terms: D000595:Amino Acid Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D043224:Deoxyribonuclease (Pyrimidine Dimer); C468346:Nei protein, E coli