Miroshnikova A D, Kuznetsova A A, Kuznetsov N A, Fedorova O S
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences. Prosp. Acad. Lavrent'eva, 8, Novosibirsk, 630090, Russia;
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.
Acta Naturae. 2016 Jan-Mar;8(1):103-10.
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.
脱嘌呤/脱嘧啶（AP）核酸内切酶在DNA修复及AP位点消除的起始过程中发挥着重要作用。DNA修复领域最热门的问题之一是了解涉及人类酶APE1的酶促过程机制，该酶可识别AP位点并有效切割5'-磷酸二酯键。在本研究中，对APE1与含有缺乏C1'羟基的稳定AP位点类似物（F位点）的DNA底物之间的相互作用进行了热力学分析。基于不同温度下的停流动力学数据，对DNA结合、催化及DNA产物释放步骤进行了表征。确定了修复过程中连续特定步骤的标准吉布斯自由能、焓和熵的变化。对数据的热力学分析表明，DNA底物结合的初始步骤包括酶结合表面与DNA之间形成非特异性接触，以及氨基酸残基Arg177和Met270插入双链体，这导致DNA凹槽中的“结晶”水分子被去除。第二个结合步骤涉及F位点翻转过程以及酶活性位点与外翻的5'-磷酸-2'-脱氧核糖残基之间形成特异性接触。结果表明，酶与DNA结合表面之间的非特异性相互作用对DNA产物释放步骤的热力学参数起主要作用。
