Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1.
Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5'-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1' hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences. Prosp. Acad. Lavrent'eva, 8, Novosibirsk, 630090, Russia;
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Miroshnikova
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences. Prosp. Acad. Lavrent'eva, 8, Novosibirsk, 630090, Russia;
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Kuznetsova
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences. Prosp. Acad. Lavrent'eva, 8, Novosibirsk, 630090, Russia; ; Department of Natural Sciences, Novosibirsk State University, Pirogova St., 2, Novosibirsk, 630090 , Russia.
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Kuznetsov
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences. Prosp. Acad. Lavrent'eva, 8, Novosibirsk, 630090, Russia; ; Department of Natural Sciences, Novosibirsk State University, Pirogova St., 2, Novosibirsk, 630090 , Russia.
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Fedorova
apurinic/apyrimidinic site; human AP endonuclease; kinetic mechanism; pre-steady-state kinetics; thermodynamics