Single molecule nucleic acid enzymology

描述（由申请人提供）：DNA解旋酶几乎是DNA代谢的每个方面所必需的，包括复制、修复、重组和转录。这些酶是普遍存在的，已经在病毒、原核生物和真核生物中鉴定。诸如着色性干皮病、科凯恩综合征、布卢姆综合征和沃纳综合征的疾病已经与编码DNA解旋酶的特定基因的缺陷相关联。全面描述这些基本的生化过程需要详细了解解旋酶的机制。对于一些解旋酶，酶活性取决于结合到DNA上的解旋酶分子的数量。这种机制在Dda解旋酶的情况下被称为合作尺蠖模型，或在NS 3 h解旋酶的情况下被称为功能性协同模型。每个单独的解旋酶被认为是通过尺蠖模式的易位发挥作用，但多个分子结合到一个单一的底物表现出更大的活性。协同性可以通过结构或功能相互作用产生。这项R24研究提案的目标是将研究人员聚集在一起，开发出能够在单分子水平上直接可视化解旋酶运动的方法，以测试合作的尺蠖机制，并为研究DNA上的多蛋白复合物奠定基础。解旋酶将用高荧光纳米晶体标记，从而使单个或一组分子能够被直接观察到。将测量在双链DNA解旋（特异性目的1）和单链DNA上的易位（特异性目的2）期间单个和多个解旋酶的直接可视化。将检查具有独特性质的三种解旋酶。Tral是高度进行性解旋酶，而Dda和NS 3 h是不良进行性解旋酶。Dda和NS 3 h被提议协同发挥作用，而Tral被提议作为单体发挥作用。本研究提案是对PA 03-127“研究的综合和协作方法”项目公告的直接回应，该项目旨在促进NIH资助的研究人员之间的协作，这些研究人员使用不同的技术来研究共同的问题。

DESCRIPTION (provided by applicant): DNA helicases are required for virtually every aspect of DNA metabolism, including replication, repair, recombination and transcription. These enzymes are ubiquitous, having been identified in viruses, prokaryotes, and eukaryotes. Diseases such as xeroderma pigmentosum, Cockayne's syndrome, Bloom's syndrome, and Werner's syndrome, have been linked to defects in specific genes coding for DNA helicases. A comprehensive description of these essential biochemical processes requires detailed understanding of helicase mechanisms. For some helicases, enzymatic activity is dependent on the number of helicase molecules that are bound onto the DNA. This mechanism is referred to as the cooperative inchworm model n the case of Dda helicase or the functional cooperativity model in the case of NS3h helicase. Each individual helicase is thought to function via an inchworm mode of translocation, but multiple molecules bound to a single substrate exhibit greater activity. Cooperativity can result through structural or functional interactions. The goal of this R24 research proposal is to bring together investigators to develop methods that will allow direct visualization of helicase movement at the single molecule level in order to test the cooperative inchworm mechanism and lay the groundwork for studying multiprotein complexes on DNA. Helicases will be labeled with highly fluorescent nanocrystals, thereby enabling individual or groups of molecules to be observed directly. Direct visualization of single and multiple helicases during unwinding of double stranded DNA (specific aim 1) and translocation on single stranded DNA (specific aim 2) willl be measured. Three helicases that have unique properties will be examined. Tral is a highly processive helicase whereas Dda and NS3h are poorly processive helicases. Dda and NS3h are proposed to function cooperatively whereas Tral is proposed to function as a monomer. This research proposal is a direct response to the program announcement PA 03-127, "INTEGRATIVE AND COLLABORATIVE APPROACHES TO RESEARCH" which is to facilitate collaboration between NIH-funded investigators who use different technologies to study a common problem.