Single molecule nucleic acid enzymology

描述(由申请人提供)：DNA解旋酶几乎是DNA新陈代谢的各个方面所必需的，包括复制、修复、重组和转录。这些酶普遍存在于病毒、原核生物和真核生物中。诸如色素性干皮病、Cockayne综合征、Bloom‘s综合征和Werner’s综合征等疾病都与编码DNA解旋酶的特定基因缺陷有关。对这些基本生化过程的全面描述需要对解旋酶机制有详细的了解。对于某些解旋酶，酶的活性取决于与DNA结合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协同尺寸虫模型，在NS3h解旋酶的情况下被称为功能协同性模型。每个单独的解旋酶被认为是通过尺寸虫易位模式发挥作用，但与单一底物结合的多个分子表现出更大的活性。协作性可以通过结构或功能的相互作用而产生。这项R24研究计划的目标是将研究人员聚集在一起，开发能够在单分子水平上直接可视化解旋酶运动的方法，以测试合作尺虫机制，并为研究DNA上的多蛋白复合体奠定基础。解旋酶将被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。在双链DNA解离(特定目标1)和单链DNA易位(特定目标2)过程中，将测量单链和多个螺旋酶的直接可视化。三种具有独特性质的解旋酶将被研究。Tral是一种高度进行性的解旋酶，而Dda和NS3h是一种弱进行性的解旋酶。DDA和NS3h被认为是协同作用，而Tral被认为是作为单体发挥作用。这项研究提案是对PA 03-127项目公告的直接回应，该项目公告的目的是促进NIH资助的研究人员之间的合作，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.