Mechanistic Studies of Hexameric Helicases

描述（由申请人提供）：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中的突变如何导致人类疾病。解旋酶是复制机制的组成部分，其中解旋酶的运动功能是解旋dsDNA和重组DNA分子所必需的。解旋酶功能的缺陷导致多种人类疾病，包括癌症、过早衰老和神经肌肉疾病。噬菌体T7编码一个环状解旋酶引发酶（T7 gp4），它与人线粒体DNA解旋酶Twinkle高度同源。T7蛋白是理解与mtDNA解旋酶Twinkle和mtDNA聚合酶γ突变相关的许多疾病的分子基础的模型蛋白。我们在上一个资助期对T7 gp4的研究突出了研究复制蛋白与其相互作用伙伴的重要性。在本研究中，我们将研究与T7 DNA聚合酶和T7单链结合蛋白相关的T7 gp4的解旋酶和引发酶活性。我们将从上一个资助期扩展我们对T7 gp4的表征，以解决先前结果中出现的关键问题：T7 DNAP如何加速解旋酶速率？DNAP是否增加解旋酶的速率和步长（每水解一个核苷酸解旋的bp）？引发酶的功能是如何与解旋酶和聚合酶的功能相协调的？底漆是什么时候做的？不连续合成DNA的滞后链聚合酶如何跟上前导链聚合酶？同时，我们建议启动研究的mtDNA解旋酶，闪光，并使用选定的T7 gp4突变体作为模型蛋白质，以了解疾病引起的闪光突变体。一些未偶联的T7 gp4突变体将作为工具，以确定机械化学耦合的原则。本论文的主要目的是：（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DNA合成中前导链和滞后链之间的动力学耦合。(3)研究线粒体解旋酶Twinkle突变体T7 gp4的同源突变体，启动Twinkle与线粒体DNA复制的研究。公共卫生相关性：DNA复制是生命的一个重要过程，当它被破坏时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多种疾病中，包括癌症、早衰和作为神经系统疾病的主要原因的mtDNA相关病症。本研究将使用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复制已经作为理解DNA复制与人类线粒体的复制复合物非常相似的范例。了解所提出的解旋酶的结构和机制对于发现复杂的人类疾病与解旋酶缺陷之间的联系以及帮助药物发现过程非常重要。这项基础研究的未来应用也可能是设计基于解旋酶马达的纳米机器，用于在细胞中运输传感器等材料。

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.