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，其响应于细胞触发因子而经历序列特异性结构变化，所述细胞触发因子对于基本过程如复制、转录、重组和DNA修复是必不可少的。DNA经历功能优化的构象变化的机制仍然知之甚少。有越来越多的证据表明，内在序列特异性的灵活性引导DNA结构转变沿着功能途径，然而，这一假设的直接测试一直受到阻碍，缺乏技术，可用于可视化DNA变形在原子尺度。固有的灵活性也指导DNA对细胞超螺旋和弯曲力的动态响应。尽管越来越多的证据表明，这种力量可以显着影响DNA的结构和功能，目前的DNA结构-功能范式几乎完全是基于DNA的研究，在实验上更容易获得放松双链体的形式。该提案的目标是开发NMR方法，辅之以分子动力学模拟和生物化学测定，在存在和不存在超螺旋的情况下，在原子尺度上可视化序列和损伤特异性DNA的灵活性。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力学系综中“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-tracts，二核苷酸CpA步骤，它们的组合，并将探索序列特异性的适应性识别的灵活性的生物学意义。具体目标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的修改灵活性并从动态系综中“捕获”瞬态，而不是通过“诱导拟合”诱导新的瞬态。这些研究将集中在碱基对切除途径酶人烷基腺嘌呤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NMR系统，用于在超螺旋存在下以原子分辨率实验表征DNA结构动力学。我们将测试这一假设，即超螺旋显着影响DNA的基本结构和动力学特性，导致增加残基之间的运动相关性，促进B到Z的转换，并提高A-束和受损DNA的构象变形性，从而提供了一种机制，为远程信号和通信。 公共卫生相关性：越来越多的证据表明，序列特异性DNA灵活性在关键的遗传交易中起着重要作用，如复制，转录，重组和DNA修复，当功能不正常时会导致病理。了解序列特异性DNA的灵活性对于合理设计与DNA特异性结合的小分子也至关重要，因此可以作为研究各种生物学问题的治疗或化学工具。

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.