Mechanistic Studies of Hexameric Helicases

描述(申请人提供)：我们的长期目标是了解参与DNA复制的酶的机制，以及解旋酶突变如何导致人类疾病。解旋酶是复制机制中不可或缺的组成部分，在复制机制中，解旋酶的运动功能是解开dsDNA和重组DNA分子所必需的。解旋酶功能的缺陷会导致多种人类疾病，包括癌症、早衰和神经肌肉疾病。噬菌体T7编码一个环状解旋酶-启动子酶(T7 GP4)，它与人线粒体DNA解旋酶Twinkle高度同源。T7蛋白是模型蛋白，用于了解与线粒体DNA解旋酶闪烁和线粒体DNA聚合酶伽马突变相关的多种疾病的分子基础。我们在上一个资助期对T7GP4的研究强调了与其相互作用的伙伴研究复制蛋白的重要性。在拟议的研究中，我们将研究T7GP4的解旋酶和启动酶活性与T7DNA聚合酶和T7单链结合蛋白的关系。我们将延长我们对T7 GP4的鉴定，以解决先前结果中出现的关键问题：T7 DNAP如何加速解旋酶的速率？DNAP是否增加了解旋酶的速率和步长(每水解核苷酸一个碱基展开)？启动酶功能如何与解旋酶和聚合酶功能相协调？底漆是什么时候生产的？滞后的链聚合酶不连续地合成DNA，如何跟上领先的链聚合酶？与此同时，我们建议启动对线粒体DNA解旋酶Twinkle的研究，并使用选定的T7GP4突变体作为模式蛋白来了解Twinkle的致病突变。一些未偶联的T7GP4突变体将作为工具来确定机械力化学偶联的原理。这些研究的具体目的如下：(1)研究解旋酶和聚合酶之间的物理和功能相互作用。(2)研究超前和滞后链DNA合成之间的动力学耦合。(3)研究与该病同源的线粒体解旋酶T7GP4突变体，启动T7GP4突变体与线粒体DNA复制的研究。与公共卫生相关：DNA复制是生命的一个基本过程，一旦中断，就会导致基因组不稳定和严重的人类健康问题。人类解旋酶的突变表现在各种疾病中，包括癌症、早衰和线粒体DNA相关疾病，这些疾病是神经系统疾病的主要原因。这项研究将使用T7蛋白作为模型系统，以了解解旋酶Twinkle突变引起的一些线粒体相关疾病的基础。噬菌体T7复制已成为理解DNA复制与人类线粒体复制复合体惊人相似的范例。了解所提出的解旋酶的结构和机制对于寻找复杂的人类疾病和解旋酶缺陷之间的联系以及帮助药物发现过程是重要的。这项基础研究的未来应用还可能是设计基于解旋酶电机的纳米机器，用于运输细胞中的传感器等材料。

DESCRIPTION (provided by applicant): Our long term goal is to understand the mechanisms of enzymes involved in DNA replication and how mutations in helicases lead to human diseases. Helicases are integral components of the replication machinery in which their motor function is required to unwind dsDNA and to recombine DNA molecules. Defects in the functions of helicases cause a variety of human diseases including cancer, premature ageing, and neuromuscular disorders. Phage T7 encodes a ring shaped helicase-primase (T7 gp4), which is highly homologous to the human mitochondrial DNA helicase, Twinkle. The T7 proteins are model proteins to understand the molecular basis for numerous diseases associated with mutations in mtDNA helicase Twinkle and mtDNA polymerase gamma. Our studies of T7 gp4 in the last funding period highlighted the importance of studying replication proteins in association with their interacting partners. In the proposed studies, we will study the helicase and primase activities of T7 gp4 in association with T7 DNA polymerase and T7 single strand binding protein. We will extend our characterization of T7 gp4 from the last funding period to address key questions that arose from prior results: How is helicase rate accelerated by T7 DNAP? Does DNAP increase the rate and the step size of the helicase (bp unwound per nucleotide hydrolyzed)? How is the primase function coordinated with the helicase and polymerase functions? When are primers made? How does the lagging strand polymerase, which synthesizes DNA discontinuously, keep up with the leading strand polymerase? Concomitantly, we propose to initiate studies of the mtDNA helicase, Twinkle, and use selected T7 gp4 mutants as model proteins to understand the disease causing mutants of Twinkle. Some of the uncoupled T7 gp4 mutants will serve as tools to identify the principles of mechanochemical coupling. The studies will be carried out with the following specific aims: (1) to investigate the physical and functional interactions between helicase and polymerase. (2) To investigate the kinetic coupling between leading and lagging strand DNA synthesis. (3) To study T7 gp4 mutants homologous to the disease causing mutants in mitochondrial helicase Twinkle and to initiate studies on Twinkle and mitochondrial DNA replication. PUBLIC HEALTH RELEVANCE: DNA replication is an essential process of life that when disrupted leads to genome instability and serious human health problems. Mutations in human helicases manifest in a variety of diseases including cancer, premature aging, and mtDNA related disorders that are a leading cause of neurological diseases. This study will use T7 proteins as a model system to understand the basis of some of the mitochondrial related diseases caused by mutations in the helicase Twinkle. Phage T7 replication has served as a paradigm to understand DNA replication being remarkably similar to the replication complex of the human mitochondria. Understanding the structure and mechanisms of the proposed helicases is important in finding the link between the complex human diseases and defects in helicases as well as to aid in the drug discovery process. Future applications of this basic research could also be in designing nanomachines based on helicase motor for transporting materials such as sensors in the cell.