PDF(Pubmed)
Abstract：
Apurinic/apyrimidinic endonuclease 1 (APE1) is one of the most important enzymes in base excision repair. Studies on this enzyme have been conducted for a long time, but some aspects of its activity remain poorly understood. One such question concerns the mechanism of damaged-nucleotide recognition by the enzyme, and the answer could shed light on substrate specificity control in all enzymes of this class. In the present study, by pulsed electron-electron double resonance (DEER, also known as PELDOR) spectroscopy and pre-steady-state kinetic analysis along with wild-type (WT) APE1 from Danio rerio (zAPE1) or three mutants (carrying substitution N253G, A254G, or E260A), we aimed to elucidate the molecular events in the process of damage recognition. The data revealed that the zAPE1 mutant E260A has much higher activity toward DNA substrates containing 5,6-dihydro-2\'-deoxyuridine (DHU), 2\'-deoxyuridine (dU), alpha-2\'-deoxyadenosine (αA), or 1,N6-ethenoadenosine (εA). Examination of conformational changes in DNA clearly revealed multistep DNA rearrangements during the formation of the catalytic complex. These structural rearrangements of DNA are directly associated with the capacity of damaged DNA for enzyme-induced bending and unwinding, which are required for eversion of the damaged nucleotide from the DNA duplex and for its placement into the active site of the enzyme. Taken together, the results experimentally prove the factors that control substrate specificity of the AP endonuclease zAPE1.
摘要：
脱嘌呤/脱嘧啶核酸内切酶1（APE1）是碱基切除修复中最重要的酶之一。对这种酶的研究已经进行了很长时间，但其活动的某些方面仍然知之甚少。一个这样的问题涉及酶的受损核苷酸识别的机制，答案可以揭示这类所有酶的底物特异性控制。在本研究中,通过脉冲电子-电子双共振(DEER，也称为PELDOR）光谱学和稳态前动力学分析以及来自Daniorerio（zAPE1）的野生型（WT）APE1或三个突变体（携带取代N253G，A254G,或E260A)，我们旨在阐明损伤识别过程中的分子事件。数据显示，zAPE1突变体E260A对含有5,6-二氢-2'-脱氧尿苷（DHU）的DNA底物具有更高的活性，2'-脱氧尿苷（dU），α-2脱氧腺苷（αA），或1，N6-乙烯腺苷（εA）。对DNA构象变化的检查清楚地揭示了催化复合物形成过程中的多步DNA重排。DNA的这些结构重排与受损DNA的酶诱导的弯曲和展开的能力直接相关。这对于从DNA双链体外翻受损的核苷酸以及将其置于酶的活性位点是必需的。一起来看,实验结果证明了控制AP核酸内切酶zAPE1底物特异性的因素。
