Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
The mechanism of the specific protein-DNA interactions during DNA lesion search and damaged base recognition by Endonuclease VIII
Endonuclease VIII (Nei) is a DNA repair enzyme that removes a wide range of damaged pyrimidine bases from DNA such as 5,6-dihydrouracil (DHU), thymine glycol, 5,6-dihydrothymine, 5-hydroxypyrimidines, etc. Accordingto X-ray data the recognition of DNA lesions by Nei involves several conformational changes in both protein and DNA, such as DNA kinking, damaged base flipping out from DNA helix, insertion into the enzyme's active site and the intrusion of the enzyme loop (a triad: Gln-69, Leu-70, and Tyr-71) into the void created in DNA after eversion of the damaged base. The excision of the damaged base by Nei proceeds through several chemical steps: N-glycosidic bond cleavage, β-elimination and δ-elimination of the phosphates flanking the damaged nucleoside. Despite detailed knowledge on the chemical mechanisms of the main steps of the repair processes and the atomic structures of the complexes of enzyme with damaged and non-damaged DNA it is still unclear how the search for the lesion proceeds and what the initial steps of the enzyme/DNA interactions are that lead to discrimination damaged and non-damaged bases.
The interrogation of the lesions in DNA by Nei may consist of multiple steps, including the encounter complex formation, sliding/rotation the enzyme along the DNA chain into a specific position, lesion recognition, catalytic steps and the release of product from the enzyme complex. It is obvious that mutual structural adjustment of Nei and the DNA is responsible for the high specific affinity of the enzyme and that there are some crucial conformational changes, which determine whether the following steps of the enzyme/substrate interaction will proceed.
In the present work, we aim to gain a better understanding of the recognition process of specific DHU-substrate and non-damaged DNA by Nei. The Nei mutants, which contain substitutions of functionally important amino acids, have been constructed. Real-time conformational rearrangements of the enzyme in the course of interaction with DNA were visualized through intrinsic tryptophan (Trp) fluorescence changes (Kuznetsov N. A. et al. 2012; Koval V. V. et al. 2010) Using pre-steady state stopped-flow measurements the reaction's progress from the substrate binding to the product release and the catalytic turnover has been monitored, as reported (Kuznetsova A. A. et al. 2014ab). In most cases, specific amino acids were substituted with fluorescent Trp, which permits monitoring the conformational transitions in the vicinity of these residues. These studies allowed: (i) to observe conformational changes of the enzyme in real time during lesion search, damaged base recognition and catalysis; (ii) to specify the role of certain catalytically important amino acids during enzymatic pathway; and (iii) to describe step-by-step their participation in the sequential mechanism of DNA lesion recognition and processing by Nei. Based on crystal structures of the enzyme, free and in complex with the target DNA, biochemical means and stopped flow studies of the protein dynamics, a mechanism was proposed to explain how Nei process on the DNA substrate.
Funding. This work was supported by the Program of the Russian Academy of Sciences "Molecular & Cell Biology" [6.11]; O.S.F. and A.A.K. are supported by Russian Foundation for Basic Research (13-04-00013 and 14-04-31174, respectively); N.A.K. and O.A.K. are supported by the Grant from Russian Scientific Foundation [14-14-00063].
2. Koval V. V. et al. (2010) Mutation Research 685, 3-10.
3. Kuznetsova A. A. et al. (2014a) Biochim. Biophys. Acta 1840, 387-395.
4. Kuznetsova A. A. et al. (2014b), PLoS ONE 9, e100007.
N. A. Kuznetsov*
Institute of Chemical Biology and Fundamental Medicine