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Nucleic Acids Research 2005 33(12):3919-3931; doi:10.1093/nar/gki694
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Published online 15 July 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase

Nikita A. Kuznetsov1,2, Vladimir V. Koval1,2, Dmitry O. Zharkov1,2, Georgy A. Nevinsky1,2, Kenneth T. Douglas3 and Olga S. Fedorova1,2,*

1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences Novosibirsk 630090, Russia 2Novosibirsk State University Novosibirsk 630090, Russia 3School of Pharmacy and Pharmaceutical Sciences, University of Manchester Manchester M13 9PL, UK

*To whom correspondence should be addressed. Tel: +7 383 330 92 74; Fax: +7 383 333 36 77; Email: fedorova{at}nibosch.nsc.ru

Received February 3, 2005. Revised May 4, 2005. Accepted June 16, 2005.

Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and ß-elimination of its 3'-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to ß-elimination since it is the very step facilitated by 8-BrG.


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