LOCAL AND GLOBAL CONFORMATIONS OF SUPERCOILED DNA

描述(申请人提供)：DNA超螺旋几乎涉及所有与DNA相关的生化过程，因此，超螺旋DNA的物理、化学和结构特性在过去20年一直是DNA生物物理的重要领域。最近的实验研究和先进的理论分析表明，在基因表达、DNA复制、重组和修复的控制中，DNA动态而不是静态DNA结构起着关键作用。然而，由于缺乏合适的实验技术，在DNA生物物理这一重要领域的实验研究进展非常缓慢。单分子研究的最新进展表明，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，从而可以观察单个分子的结构动力学。最近的研究表明，DNA的局部构象和全局构象处于动态平衡。不同局部结构之间的竞争性转换伴随着全球DNA构象的动态变化，可能在DNA的各种功能中发挥着深远的作用。这一建议利用单分子原子力显微镜和荧光显微镜测试了许多关键假设，并为在这一未开发的分子生物学领域开辟新天地提供了一种现实的手段。(1)十字形和H-DNA的形成可以限制全球DNA的流动性，影响遥远DNA区域的并置，这一假设将得到检验。(2)假设一个局部结构到另一个局部结构的转换是全球DNA结构和动力学的替代分子开关机制。(3)分析涉及远距离DNA区域相互作用的限制性内切酶SfiI的结合机制，并检验局部结构的形成可能调节酶活性的假设。除了假设检验外，还将开发用于DNA动力学研究的新的DNA固定和标记技术。总之，拟议的研究是第一次尝试在不同的水平上观察DNA的动力学，提供关于DNA基本和功能特性的空间和时间信息。样品制备技术的进步和相应仪器的开发将为追求单分子方法的结构生物学家提供新的应用工具，其影响比本提案中概述的目标广泛得多。

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.