Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Energetics of damaged bases recognition by DNA glycosylases
DNA N-glycosylases are a key enzymes responsible for initiating the base excision repair of red/ox damaged bases in DNA. In our studies a thermodynamic analysis of the interaction of DNA glycosylases (human OGG1, E.coli Fpg and Nei) with specific and non-specific DNA-substrates was performed using stopped-flow kinetic data (Kuznetsov et al. 2012, 2014). The standard Gibbs energies, enthalpies and entropies of specific stages of the repair processes were determined via kinetic measurements over a temperature range based on the vant Hoff approach. The steps which are accompanied with changes in the enzyme and DNA conformations were detected via tryptophan (Kuznetsov et al. 2009, 2014) and 2-aminopurine (Kuznetsov et al. 2014ab), pyrrolocytosine (Kuznetsov et al. 2012), 1,3-diaza-2-oxophenoxazine fluorescence (Kuznetsov et al. 2014b), respectively, in the processes of binding and recognition of damaged bases. The thermodynamic data demonstrate that the initial steps of the DNA substrates binding are mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which result in negative enthalpy change. Discrimination of non-specific base versus specific modified base is occurring in few steps where DNA is kinked, damaged base is flipped out from DNA helix and inserted into the enzyme's active site, the enzyme amino acid are intruded into the void created in DNA after eversion of the damaged base. These steps are accompanied with the gain of compactness of enzyme-DNA complexes and require energy consumptions which are compensated by the positive entropy contributions. The last recognition step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy and originated from the dehydration of the surface between DNA grooves and enzyme. The data suggest that not only enthalpy-driven exothermic lesion recognition but also the desolvation accompanied entropy-driven enzyme-substrate complex adjustment into the catalytically active state play equally important roles in the overall process.
Funding.This research has been supported by the Program of the Russian Academy of Sciences "Molecular & Cell Biology" #6.11; the Grants from Russian Foundation for Basic Research #13-04-00013 (O. S. F.) and Russian Scientific Foundation #14-14-00063 (N. A. K.).
Kuznetsov N. A., Vorobjev Y. N., Krasnoperov L. N., and Fedorova O. S. (2012) Thermodynamics of the Multi-Stage DNA Lesion Recognition and Repair by Formamidopyrimidine-DNA Glycosylase Using Pyrrolocytosine Fluorescence - Stopped-Flow Pre-Steady-State Kinetics. Nucleic Acids Res. 40, 7384-7392.
Kuznetsov N. A., Kuznetsova A. A., Vorobjev Y. N., Krasnoperov L. N., and Fedorova O. S. (2014a) Thermodynamics of the DNA Damage Repair Steps of Human 8-Oxoguanine DNA Glycosylase. PLoS ONE, 9(6): e98495.
Kuznetsov N. A., Zharkov D. O., Koval V. V., Buckle M., and Fedorova O. S. (2009) Reversible DNA cleavage and rate-limiting enzyme regeneration in the reaction catalyzed by formamidopyrimidine-DNA-glycosylase. Biochemistry 48, 11335-11343.
Kuznetsova A. A., Kuznetsov N. A., Vorobjev Y. N., Barthes N. P. F., Michel B. Y., Burger A., and Fedorova O. S. (2014b) New environment-sensitive multichannel DNA fluorescent label for investigation of the protein-DNA interactions. PLoS ONE 9(6), e100007.
Olga S. Fedorova*
Institute of Chemical Biology and Fundamental Medicine