Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.
Biochemistry. 2012 Feb 14;51(6):1306-21. doi: 10.1021/bi201444m. Epub 2012 Jan 30.
Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from exposure to UV light, ionizing radiation, alkylating agents, and oxygen radicals. In human cells, AP endonuclease 1 (APE1) recognizes this mutagenic lesion and initiates its repair via a specific incision of the phosphodiester backbone 5' to the AP site. We have investigated a detailed mechanism of APE1 functioning using fluorescently labeled DNA substrates. A fluorescent adenine analogue, 2-aminopurine, was introduced into DNA substrates adjacent to the abasic site to serve as an on-site reporter of conformational transitions in DNA during the catalytic cycle. Application of a pre-steady-state stopped-flow technique allows us to observe changes in the fluorescence intensity corresponding to different stages of the process in real time. We also detected an intrinsic Trp fluorescence of the enzyme during interactions with 2-aPu-containing substrates. Our data have revealed a conformational flexibility of the abasic DNA being processed by APE1. Quantitative analysis of fluorescent traces has yielded a minimal kinetic scheme and appropriate rate constants consisting of four steps. The results obtained from stopped-flow data have shown a substantial influence of the 2-aPu base location on completion of certain reaction steps. Using detailed molecular dynamics simulations of the DNA substrates, we have attributed structural distortions of AP-DNA to realization of specific binding, effective locking, and incision of the damaged DNA. The findings allowed us to accurately discern the step that corresponds to insertion of specific APE1 amino acid residues into the abasic DNA void in the course of stabilization of the precatalytic complex.
无碱基（AP）位点是大量的 DNA 损伤，由暴露于紫外光、电离辐射、烷化剂和氧自由基引起。在人类细胞中，AP 内切核酸酶 1（APE1）识别这种诱变损伤，并通过在 AP 位点 5' 磷酸二酯骨架的特异性切割起始其修复。我们使用荧光标记的 DNA 底物研究了 APE1 功能的详细机制。将荧光腺嘌呤类似物 2-氨基嘌呤引入到紧邻无碱基位点的 DNA 底物中，作为催化循环中 DNA 构象转变的现场报告器。应用预稳态停流技术使我们能够实时观察与过程不同阶段相对应的荧光强度变化。我们还在与含有 2-aPu 的底物相互作用期间检测到酶的固有色氨酸荧光。我们的数据揭示了 APE1 处理的无碱基 DNA 的构象灵活性。荧光痕迹的定量分析得出了一个包含四个步骤的最小动力学方案和适当的速率常数。停流数据的结果表明，2-aPu 碱基位置对某些反应步骤的完成有很大影响。通过对 DNA 底物的详细分子动力学模拟，我们将 AP-DNA 的结构扭曲归因于特定结合、有效锁定和损伤 DNA 的切割的实现。这些发现使我们能够准确辨别在稳定前催化复合物过程中，对应于特定 APE1 氨基酸残基插入无碱基 DNA 空隙的步骤。
