Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
Conformational dynamics of human 8-oxoguanine-DNA glycosylase
DNA continuously undergoes oxidation damage from both exogenous and endogenous sources, including ionizing radiation, ultraviolet light, and products of metabolism. Replication of damaged DNA sometimes gives rise to mutations, which can contribute to disease and aging. One of the most mutagenic lesions caused by DNA oxidation is 7,8-dihydro-8-oxoguanine (oxoG), which, if not repaired, results in G → T transversions. In human cells, oxoG is repaired through excision by 8-oxoguanine-DNA glycosylase hOGG1. In addition to its glycosylase activity, hOGG1 possesses an AP-lyase activity, which catalyzes elimination of the 3’-phosphate (β-elimination) at the nascent or pre-formed abasic (AP) site. The glycosidic bond breakage is initiated by a nucleophilic attack at C1’ by the Lys-249 residue, resulting in a covalent enzyme–DNA Schiff base intermediate, which then rearranges and undergoes elimination. The three-dimensional structure of hOGG1shows that DNA binding is accompanied with drastic conformational changes, including DNA kinking, eversion of oxoGua from the double helix, and insertion of few amino acid residues into DNA.
Previously (Kuznetsov et al., 2005, 2007), we have studied the stopped-flow kinetics of oxoG and AP site lesions processing by hOGG1. The character of tryptophan and 2-aminopurine fluorescence traces revealed that both the protein and the damaged DNA undergo extensive conformational changes in the course of DNA substrate binding and cleavage.
To understand better the mechanism by which hOGG1 recognizes DNA lesions we have examined the influence of amino acid substitutions on conformational dynamics of hOGG1 and DNA during specific site recognition and conversion. Fluorescence kinetics of enzyme mutant forms F45W, F319W, Y203W, Y203A, H270W, K249Q demonstrated the multi-step character of catalytic process and made clear the role of these amino acid for hOGG1 catalysis.
This research has been supported by The Ministry of Education and Science of Russian Federation, (projects 8092, 8473 and 14.B37.21.0195) and RFBR (projects 12-04-00013 and 12-04-31066).
N. A. Kuznetsov, V. V. Koval, G. A. Nevinsky, K. T. Douglas, D. O. Zharkov & Fedorova, O. S. (2007) Kinetic conformational analysis of human 8-oxoguanine-DNA glycosylase. J. Biol. Chem. 282, 1029-1038.
Nikita A. Kuznetsov
Institute of Chemical Biology and Fundamental Medicine