Dynamics of Normal and Damaged DNA Under Relaxed and Supercoiled Conditions

描述(申请人提供)：DNA是一种高度动态的生物聚合物，在复制、转录、重组和DNA修复等基本过程中，细胞触发因子是必不可少的，它会经历特定序列的结构变化。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结构沿功能途径发生转变；然而，由于缺乏可用于在原子尺度上可视化DNA变形能力的技术，这一假说的直接测试一直受到阻碍。固有的灵活性也引导DNA对细胞超螺旋和弯曲力的动态反应。尽管越来越多的证据表明，这种作用力可以显著影响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容易在实验中获得的松弛双链形式的DNA的研究。这项建议的目标是开发核磁共振方法，辅之以分子动力学模拟和生化分析，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。特定目标1将测试这样的假设，即DNA经历序列特定的和空间上非随机的热诱导波动，以及触发因素，如蛋白质，利用这种灵活性，通过从预先存在的动态集合中“捕捉”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。特定目标2将测试这样的假设，即DNA损伤诱导与序列特定的灵活性相关，修复酶利用受损DNA的修改后的灵活性并从动态集合中“捕捉”瞬时状态，而不是通过“诱导匹配”来诱导新的瞬时状态。这些研究将集中在碱基对切割途径酶人烷基腺嘌呤DNA糖基酶的受损DNA底物上。特殊目标3将开发微环作为一个模型核磁共振系统，用于在存在超螺旋的情况下以原子分辨率实验表征DNA结构动力学。我们将检验这一假说，即超螺旋显著影响DNA的基本结构和动力学性质，导致残基之间运动相关性的增加，促进B-Z转变，并增强A-链和受损DNA的构象变形能力，从而为远程信号和通信提供机制。 与公共卫生相关：越来越多的证据表明，序列特异性DNA灵活性在复制、转录、重组和DNA修复等关键遗传交易中发挥着基础性作用，这些交易在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特异结合的小分子，从而作为研究各种生物学问题的治疗或化学工具也是至关重要的。

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.