Kinetic conformational analysis of human 8-oxoguanine-DNA glycosylase.
7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major DNA lesions formed by reactive oxygen species that can result in transversion mutations following replication if left unrepaired. In human cells, the effects of 8-oxoG are counteracted by OGG1, a DNA glycosylase that catalyzes excision of 8-oxoguanine base followed by a much slower beta-elimination reaction at the 3'-side of the resulting abasic site. Many features of OGG1 mechanism, including its low beta-elimination activity and high specificity for a cytosine base opposite the lesion, remain poorly explained despite the availability of structural information. In this study, we analyzed the substrate specificity and the catalytic mechanism of OGG1 acting on various DNA substrates using stopped-flow kinetics with fluorescence detection. Combining data on intrinsic tryptophan fluorescence to detect conformational transitions in the enzyme molecule and 2-aminopurine reporter fluorescence to follow DNA dynamics, we defined three pre-excision steps and assigned them to the processes of (i) initial encounter with eversion of the damaged base, (ii) insertion of several enzyme residues into DNA, and (iii) enzyme isomerization to the catalytically competent form. The individual rate constants were derived for all reaction stages. Of all conformational changes, we identified the insertion step as mostly responsible for the opposite base specificity of OGG1 toward 8-oxoG:C as compared with 8-oxoG:T, 8-oxoG:G, and 8-oxoG:A. We also investigated the kinetic mechanism of OGG1 stimulation by 8-bromoguanine and showed that this compound affects the rate of beta-elimination rather than pre-excision dynamics of DNA and the enzyme.
Kuznetsov
Nikita A
NA
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Koval
Vladimir V
VV
Nevinsky
Georgy A
GA
Douglas
Kenneth T
KT
Zharkov
Dmitry O
DO
Fedorova
Olga S
OS
D015075:2-Aminopurine / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D003596:Cytosine / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry* / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017382:Reactive Oxygen Species / Q000378:metabolism; D013379:Substrate Specificity
C502319:8-bromoguanine; D008024:Ligands; D009711:Nucleotides; D017382:Reactive Oxygen Species; D006151:Guanosine; C046215:8-hydroxyguanosine; D015075:2-Aminopurine; D006147:Guanine; D003596:Cytosine; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human