title: DNA damage processing by human 8-oxoguanine-DNA glycosylase mutants with the occluded active site.
abstract: 8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation.
authors: From the Siberian Branch of the Russian Academy of Sciences Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, Novosibirsk 630090 and.
authors: Maria V
authors: MV
authors: Lukina
authors: Alexander V
authors: AV
authors: Popov
authors: Vladimir V
authors: VV
authors: Koval
authors: Yuri N
authors: YN
authors: Vorobjev
authors: Olga S
authors: OS
authors: Fedorova
authors: Dmitry O
authors: DO
authors: Zharkov
mesh_terms: D001483:Base Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D004249:DNA Damage; D045647:DNA Glycosylases / Q000378:metabolism*; D005453:Fluorescence; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010649:Phenylalanine / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: C502319:8-bromoguanine; D050505:Mutant Proteins; D010649:Phenylalanine; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human
keywords: 8-Oxoguanine; Abasic Site; DNA Damage; DNA Repair; Enzyme Kinetics; Enzyme Mutation; Molecular Modeling; OGG1; Presteady-state Kinetics; Substrate Recognition
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