title: Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition.
abstract: Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination.
authors.affiliation: SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., Novosibirsk 630090, Russia Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia.
authors.forename: Nikita A
authors.initials: NA
authors.lastname: Kuznetsov
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Bergonzo
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Campbell
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Haoquan
authors.initials: H
authors.lastname: Li
authors.affiliation: SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., Novosibirsk 630090, Russia.
authors.forename: Grigory V
authors.initials: GV
authors.lastname: Mechetin
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Carlos
authors.initials: C
authors.lastname: de los Santos
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Arthur P
authors.initials: AP
authors.lastname: Grollman
authors.affiliation: SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., Novosibirsk 630090, Russia Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia fedorova@niboch.nsc.ru.
authors.forename: Olga S
authors.initials: OS
authors.lastname: Fedorova
authors.affiliation: SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave., Novosibirsk 630090, Russia Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia dzharkov@niboch.nsc.ru.
authors.forename: Dmitry O
authors.initials: DO
authors.lastname: Zharkov
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, USA Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY 11794, USA carlos.simmerling@stonybrook.edu.
authors.forename: Carlos
authors.initials: C
authors.lastname: Simmerling
mesh_terms: D020029:Base Pairing; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation
chemical_list: C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D043382:DNA-Formamidopyrimidine Glycosylase; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase