Investigation of energetics of sharp DNA bending

DNA急剧弯曲的能量学研究

• 批准号: 10437942
• 负责人: Harold D Kim
• 金额: $ 29.74万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-06 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10437942
• 关键词: 3-Dimensional; Affect; Affinity; Base Pair Mismatch; Base Pairing; Base Sequence; Binding; Biological Assay; Biological Models; Biology; Biophysics; Cell physiology; Cells; Chromatin Loop; Clustered Regularly Interspaced Short Palindromic Repeats; Conflict (Psychology); Confusion; Coupling; Cyclization; DNA; DNA Binding; DNA Sequence; DNA Structure; Defect; Dependence; Disease; Entropy; Enzyme Kinetics; Enzymes; Face; Fluorescence Resonance Energy Transfer; Freezing; Funding; Genes; Genetic Carriers; Genetic Code; Genetic Transcription; Genetic Variation; Genome; Genomics; Geometry; Goals; Grain; Individual; Investigation; Kinetics; Laws; Lead; Length; Ligation; Light; Link; Major Groove; Measurement; Measures; Mechanics; Methods; Minor Groove; Mismatch Repair; Modeling; Molecular Conformation; Monitor; Mutation; Paper; Physics; Polymers; Predisposition; Probability; Process; Property; Proteins; Proxy; RNA; Reaction; Regulation; Research Proposals; Rotation; Shapes; Signal Transduction; Site; Statistical Mechanics; Stress; Structure; Surface; System; Testing; Thermodynamics; Time; base; design; genetic information; human disease; innovation; insight; mechanical behavior; mechanical properties; molecular dynamics; novel; novel strategies; nuclease; repaired; single molecule; single-molecule FRET; tool; transcription factor

Genetic information is carried by DNA, a polymeric molecule governed by the laws of physics. Strongly bent and twisted DNA is associated with many genomic processes including packaging, transcription, repair, and editing, which suggests that these processes are aided by the intrinsic deformability of DNA. Hence, altered deformability of DNA due to damage or mutation can perturb the regulatory state of the genome, thus increasing the susceptibility to disease. Understanding how deformability of DNA changes with base sequence can thus provide a missing link between genetic variation and cell physiology. DNA is a double helical ladder of base pair steps with the major and minor grooves. This groove asymmetry confers DNA with asymmetric bendability and bend- twist coupling, properties truly unique to DNA, but these properties have not been thoroughly investigated by experimental means. Extreme bending or twisting of a single base pair step can lead to large changes in the three-dimensional DNA conformation, but the thermodynamics and sequence dependence of extreme bendability and twistability remain largely unknown due to the lack of experimental methods. Deformed base pair steps will likely affect how enzymes and transcription factors interact with DNA, but testing this idea requires fine control of base-pair step deformation. The PI has investigated the thermodynamics of strong DNA bending by the combined use of short DNA with sticky ends and surface-based single-molecule assays. During the first funding period of this R01, looping and unlooping rates were measured from DNA molecules of different lengths and base sequences including mismatched bases. The results from these studies elucidated the kinetics of loop formation and helped us to design new approaches to quantifying asymmetric bendability and bend-twist coupling of DNA. Furthermore, they revealed DNA loop geometries that enable measurement of the bending and twist stiffness of individual base pair steps. Building upon these key results and insights from the first R01, this proposal will measure extreme deformability of DNA and investigate its consequence on the kinetics of a DNA targeting protein. The experimental approach is to combine singe-molecule FRET with small DNA loops of different geometries. Four specific aims are proposed: Aim 1, quantifying the bending asymmetry and bend-twist coupling of DNA; Aim 2, quantifying bending stiffness of mismatched base pairs at different bending angles; Aim 3, measuring coaxial stacking and unstacking rates of individual base pair steps; and Aim 4, measuring the reaction kinetics of Cas12, an RNA-guided DNA targeting protein of the CRISPR system, on curved and twisted DNA substrates. These studies will shed light on the generic mechanics-function relationship of DNA.

遗传信息是由由物理定律控制的聚合物分子DNA携带的。强烈弯曲和 扭曲的DNA与许多基因组过程有关，包括包装，转录，修复和编辑， 这表明这些过程得到了DNA的固有变形性的帮助。因此，变形性改变 由于损伤或突变而引起的DNA可能会扰动基因组的调节状态，从而增加 对疾病的敏感性。了解DNA的可变形性如何通过碱基序列变化可以提供 遗传变异与细胞生理学之间缺失的联系。 DNA是基本台阶的双螺旋梯 带有主要和小凹槽。这种凹槽不对称赋予了DNA，具有不对称的弯曲性和弯曲 扭曲耦合，DNA真正独特的属性，但是这些特性尚未通过 实验手段。单个基对台阶的极端弯曲或扭曲可能会导致大幅变化 三维DNA构象，但是极端的热力学和序列依赖性 由于缺乏实验方法，弯曲性和可扭曲性在很大程度上仍然未知。变形的基对 步骤可能会影响酶和转录因子与DNA相互作用的方式，但是测试此想法需要罚款 控制碱基对阶跃变形。 PI通过合并使用短DNA与 粘性末端和基于表面的单分子测定法。在此R01的第一个资金期间，循环和 从不同长度和基本序列的DNA分子（包括 不匹配的基地。这些研究的结果阐明了循环形成的动力学，并帮助我们 设计新方法来量化DNA的不对称弯曲性和弯曲扭转​​耦合。此外， 他们揭示了DNA环的几何形状，可以测量个体的弯曲和扭曲刚度 基本对步骤。 在第一个R01的这些关键结果和见解的基础上，该提案将衡量极端可变形性 DNA并研究其对DNA靶向蛋白动力学的后果。实验方法 是将单分子物与不同几何形状的小型DNA环相结合。四个具体目标是 提出：目标1，量化DNA的弯曲不对称和弯曲扭曲耦合；目标2，量化弯曲 不同弯曲角的底座对刚度的刚度； AIM 3，测量同轴堆叠和拆卸 单个基对步骤的速率； AIM 4，测量CAS12的反应动力学，RNA引导的DNA 靶向CRISPR系统的蛋白质，在弯曲和扭曲的DNA底物上。这些研究将阐明 DNA的通用力学 - 功能关系。