title: Separation-of-function mutants unravel the dual-reaction mode of human 8-oxoguanine DNA glycosylase.
abstract: 7,8-Dihydro-8-oxoguanine (8oxoG) is a major mutagenic base lesion formed when reactive oxygen species react with guanine in DNA. The human 8oxoG DNA glycosylase (hOgg1) recognizes and initiates repair of 8oxoG. hOgg1 is acknowledged as a bifunctional DNA glycosylase catalyzing removal of the damaged base followed by cleavage of the backbone of the intermediate abasic DNA (AP lyase/β-elimination). When acting on 8oxoG-containing DNA, these two steps in the hOgg1 catalysis are considered coupled, with Lys249 implicated as a key residue. However, several lines of evidence point to a concurrent and independent monofunctional hydrolysis of the N-glycosylic bond being the in vivo relevant reaction mode of hOgg1. Here, we present biochemical and structural evidence for the monofunctional mode of hOgg1 by design of separation-of-function mutants. Asp268 is identified as the catalytic residue, while Lys249 appears critical for the specific recognition and final alignment of 8oxoG during the hydrolysis reaction.
authors.affiliation: Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet, Oslo University Hospital, N-0027 Oslo, Norway. bjorn.dalhus@rr-research.no
authors.forename: Bjørn
authors.initials: B
authors.lastname: Dalhus
authors.forename: Monika
authors.initials: M
authors.lastname: Forsbring
authors.forename: Ina Høydal
authors.initials: IH
authors.lastname: Helle
authors.forename: Erik Sebastian
authors.initials: ES
authors.lastname: Vik
authors.forename: Rune Johansen
authors.initials: RJ
authors.lastname: Forstrøm
authors.forename: Paul Hoff
authors.initials: PH
authors.lastname: Backe
authors.forename: Ingrun
authors.initials: I
authors.lastname: Alseth
authors.forename: Magnar
authors.initials: M
authors.lastname: Bjørås
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D053837:DNA Cleavage; D045647:DNA Glycosylases / Q000737:chemistry* / Q000235:genetics; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008190:Lyases / Q000737:chemistry; D011119:Polynucleotides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D011119:Polynucleotides; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D000080242:8-Hydroxy-2'-Deoxyguanosine; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; D008190:Lyases; D003849:Deoxyguanosine