LOCAL AND GLOBAL CONFORMATIONS OF SUPERCOILED DNA

描述（由申请人提供）：DNA超卷绕涉及几乎所有DNA相关的生化过程，因此，在过去的二十年中，超卷绕DNA的物理、化学和结构特性一直是DNA生物物理学的重要领域。最近的实验研究和先进的理论分析表明，DNA动态而不是静态DNA结构在基因表达、DNA复制、重组和修复的控制中起着关键作用。然而，由于缺乏适当的实验技术，在DNA生物物理学这一重要领域的实验研究进展非常低。单分子研究的最新进展，允许观察单个分子的结构动力学，表明单分子方法可以提供关于生物分子结构和动力学的独特信息。最近的研究表明，局部和全局DNA构象处于动态平衡状态。不同局部结构之间的竞争转变伴随着整体DNA构象的动态变化，可能在各种DNA功能中起着深远的作用。该建议利用单分子AFM和荧光显微镜测试了一些关键的假设，并为在分子生物学这一未开发的领域开辟新的领域提供了一种现实的手段。(1)十字形和H-DNA的形成可以限制DNA的整体流动性，从而影响远距离分离的DNA区域并置的假设将得到验证。(2)假设一个局部结构转化为另一个局部结构是DNA整体结构和动力学的另一种分子开关机制。(3)将分析限制性内切酶SfiI的结合机制，涉及远端DNA区域的相互作用，并测试局部结构的形成可能调节酶活性的假设。除了假设测试，新的DNA固定和标记技术的DNA动力学研究将被开发。总的来说，提出的研究是第一次尝试在不同的水平上观察DNA的动态，提供关于DNA的基本和功能特性的空间和时间信息。样品制备技术的进步与相应仪器的开发相结合，将为结构生物学家提供追求单分子方法的新应用工具，其影响远远超出本提案中概述的目标。

DESCRIPTION (provided by applicant): DNA supercoiling is involved with virtually all DNA-related biochemical processes, therefore physical chemical and structural properties of supercoiled DNA have been important areas of DNA biophysics over last two decades. Recent experimental studies and advanced theoretical analyses suggest that DNA dynamics rather than static DNA structure plays a critical role in the control of gene expression, DNA replication, recombination, and repair. However, progress in experimental studies in this important area of DNA biophysics has been very low due to the lack of appropriate experimental techniques. Recent advances in single molecule studies, allowing the observation of structural dynamics of individual molecules, showed that the single molecule approaches could provide unique information on the structure and dynamics of biological molecules. Recent studies showed that local and global DNA conformations are in a dynamic equilibrium. Competing transitions between different local structures accompanied by dynamic changes of global DNA conformations may have a profound role in various DNA functions. This proposal utilizing single molecule AFM and fluorescence microscopy tests a number of critical hypotheses and provides a realistic means for breaking new ground in this undeveloped area of molecular biology. (1) The hypothesis that the formation of cruciforms and H-DNA can limit global DNA mobility affecting the juxtaposition of distantly separated DNA regions will be tested. (2) The hypothesis that conversion of one local structure into another is an alternative molecular switch mechanism for the global DNA structure and dynamics (3) The mechanism of binding of the restriction enzyme SfiI involving the interaction of distant DNA regions will be analyzed and the hypothesis that the formation of local structures may regulate the enzyme activity will be tested. In addition to the hypotheses testing, novel DNA immobilization and labeling techniques for DNA dynamics studies will be developed. Altogether, the proposed studies are the first attempt to look at the dynamics of DNA at various levels providing spatial and temporal information about the fundamental and functional properties of DNA. Advances in sample preparation techniques combined with corresponding instrument development will provide structural biologists pursuing single molecule approaches with novel tools for applications, ramifications considerably wider than the objectives outlined in this proposal.