title: Pre-steady-state kinetic study of substrate specificity of Escherichia coli formamidopyrimidine--DNA glycosylase.
abstract: Formamidopyrimidine-DNA glycosylase (Fpg) is responsible for removal of 8-oxoguanine (8-oxoG) and other oxidized purine lesions from DNA and can also excise some oxidatively modified pyrimidines [such as dihydrouracil (DHU)]. Fpg is also specific for a base opposite the lesion, efficiently excising 8-oxoG paired with C but not with A. We have applied stopped-flow kinetics using intrinsic tryptophan fluorescence of the enzyme and fluorescence of 2-aminopurine-labeled DNA to analyze the conformational dynamics of Escherichia coli Fpg during processing of good substrates (8-oxoG.C), poor substrates (8-oxoG.A), and substrates of unclear specificity (such as DHU and 8-oxoG opposite T or G). The analysis of fluorescence traces allows us to conclude that when the enzyme encounters its true substrate, 8-oxoG.C, the complex enters the productive catalytic reaction after approximately 50 ms, partitioning the substrate away from the competing dissociation process, while poor substrates linger in the initial encounter complex for longer. Several intermediate ES complexes were attributed to different structures that exist along the reaction pathway. A likely sequence of events is that the damaged base is first destabilized by the enzyme binding and then everted from DNA, followed by insertion of several amino acid residues into DNA and isomerization of the enzyme into a pre-excision complex. We conclude that rejection of the incorrect substrates occurs mostly at the early stage of formation of the pre-eversion recognition complex, supporting the role of indirect readout in damage recognition.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Novosibirsk State University, 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: Yuri N
authors.initials: YN
authors.lastname: Vorobjev
authors.forename: Georgy A
authors.initials: GA
authors.lastname: Nevinsky
authors.forename: Kenneth T
authors.initials: KT
authors.lastname: Douglas
authors.forename: Olga S
authors.initials: OS
authors.lastname: Fedorova
mesh_terms: D001483:Base Sequence; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
chemical_list: D009838:Oligodeoxyribonucleotides; C024829:8-hydroxyguanine; D006147:Guanine; D014364:Tryptophan; D043382:DNA-Formamidopyrimidine Glycosylase