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J. Biol. Chem., Vol. 282, Issue 2, 1029-1038, January 12, 2007

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Kinetic Conformational Analysis of Human 8-Oxoguanine-DNA Glycosylase^*

Nikita A. Kuznetsov¹, Vladimir V. Koval¹, Georgy A. Nevinsky, Kenneth T. Douglas^¶, Dmitry O. Zharkov, and Olga S. Fedorova²

From the Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia, Novosibirsk State University, Novosibirsk 630090, Russia, and ^¶School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, United Kingdom

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 -elimination reaction at the 3'-side of the resulting abasic site. Many features of OGG1 mechanism, including its low -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 -elimination rather than pre-excision dynamics of DNA and the enzyme.

Received for publication, June 16, 2006 , and in revised form, August 10, 2006.

^* This research was made possible in part by grants from the Wellcome Trust (United Kingdom) (070244/Z/03/Z); the Presidium of the Russian Academy of Sciences (MCB Program, 10.5 and 10.6); the Russian Foundation for Basic Research (RFBR 04-04-48171, 04-04-48254, and 05-04-48619); the Russian Ministry of Education and Science (NS-1419.2003.4 and ZN-359-05); and the U. S. Civilian Research & Development Foundation (CRDF Y1-B-08-16 and Y2-B-08-08). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Young Scientist Fellowships 05-109-4159 and 04-83-3849, respectively, from INTAS (International Association for the Promotion of Cooperation with Scientists from the New Independent States of the Former Soviet Union).

² To whom correspondence should be addressed: Inst. of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia. Tel.: 7-383-335-6274; Fax: 7-383-333-3677; E-mail: fedorova{at}niboch.nsc.ru.

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