Dynamics of Normal and Damaged DNA Under Relaxed and Supercoiled Conditions

描述（由申请人提供）：DNA是一种高度动态的生物聚合物，在细胞触发因子的作用下发生序列特异性结构变化，这些因素对复制、转录、重组和DNA修复等基本过程至关重要。DNA经历功能优化构象变化的机制仍然知之甚少。越来越多的证据表明，固有的序列特异性灵活性引导DNA结构沿着功能途径转变；然而，由于缺乏可以在原子尺度上可视化DNA可变形性的技术，对这一假设的直接测试受到了阻碍。固有的灵活性也指导DNA对细胞超卷曲和弯曲力的动态响应。尽管越来越多的证据表明这种力可以显著地影响DNA的结构和功能，但目前的DNA结构-功能范式几乎完全基于对更易实验获得的松弛双工形式的DNA的研究。本提案的目标是发展核磁共振方法，辅以分子动力学模拟和生化分析，在存在和不存在超卷曲的情况下，在原子尺度上可视化序列和损伤特异性DNA灵活性。具体目标1将测试DNA经历序列特异性和空间非随机热诱导波动的假设，以及触发因素，如蛋白质，利用这种灵活性并通过从预先存在的动态集合中“捕获”不同的构象来诱导DNA结构的特定变化。这些研究将集中在可变长度的a链、二核苷酸CpA步骤及其组合上，并将探索序列特异性灵活性在适应性识别中的生物学意义。Specific Aim 2将测试DNA损伤诱导与序列特异性灵活性相关的假设，以及修复酶利用受损DNA的修饰灵活性并从动态集合中“捕获”瞬时状态，而不是通过“诱导匹配”诱导新的状态。这些研究将集中在碱基对切除途径酶人类烷基腺嘌呤DNA糖基化酶的受损DNA底物上。具体目标3将开发微环作为模型核磁共振系统实验表征DNA结构动力学在原子分辨率存在超卷曲。我们将验证一个假设，即超卷曲会显著影响DNA的基本结构和动力学特性，导致残基之间的运动相关性增加，促进b到z的转变，增强a束和受损DNA的构象变形能力，从而为远程信号传导和通信提供了一种机制。

DESCRIPTION (provided by applicant): DNA is a highly dynamic biopolymer that undergoes sequence-specific structural changes in response to cellular trigger factors that are essential for fundamental processes such as replication, transcription, recombination, and DNA repair. The mechanism by which DNA undergoes functionally optimized conformational changes remains poorly understood. There is growing evidence that intrinsic sequence-specific flexibility guides DNA structural transitions along functional pathways; however, a direct test of this hypothesis has been hindered by lack of techniques that can be used to visualize DNA deformability at the atomic scale. Intrinsic flexibility also guides the DNA dynamic response to cellular supercoiling and bending forces. Despite growing evidence that such forces can dramatically affect DNA structure and function, the current DNA structure-function paradigm is based almost exclusively on studies of DNA in the more experimentally accessible relaxed duplex form. The goal of this proposal is to develop NMR methods, complemented by molecular dynamics simulations and biochemical assays, to visualize sequence and damage-specific DNA flexibility at the atomic scale in the presence and absence of supercoiling. Specific Aim 1 will test the hypothesis that DNA undergoes sequence-specific and spatially non-random thermally-induced fluctuations and that trigger factors, such as proteins, take advantage of this flexibility and induce specific changes in DNA structure by "capturing" distinct conformations from a pre-existing dynamical ensemble. These studies will focus on variable length A-tracts, dinucleotide CpA steps, their combination, and will explore the biological significance of sequence-specific flexibility in adaptive recognition. Specific Aim 2 will test the hypothesis that DNA damage induction is correlated to sequence-specific flexibility and that repair enzymes exploit the modified flexibility of damaged DNA and "capture" transient states from a dynamical ensemble rather than induce new ones by "induced fit". These studies will focus on damaged DNA substrates of the base pair excision pathway enzyme human alkyladenine DNA glycosylase. Specific Aim 3 will develop minicircles as a model NMR system for experimentally characterizing DNA structural dynamics at atomic resolution in the presence of supercoiling. We will test the hypothesis that supercoiling dramatically affects the basic structural and dynamical properties of DNA, causing an increase in motional correlations between residues, promoting B-to-Z transitions, and enhancing the conformational deformability of A-tracts and damaged DNA, thus providing a mechanism for long-range signaling and communication. PUBLIC HEALTH RELEVANCE: There is growing evidence that sequence-specific DNA flexibility plays a fundamental role in key genetic transactions such as replication, transcription, recombination, and DNA repair that lead to pathology when improperly functioning. Understanding sequence-specific DNA flexibility is also of key importance for rationally designing small molecules that specifically bind to DNA and thus act as therapeutics or chemical tools for investigating diverse biological questions.