UNWINDING&TRANSLOCATION OF DNA BY NS3 HELICASE FROM HEPATITIS C

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 丙型肝炎病毒(丙型肝炎病毒)感染全球1.7亿人(1-3)。它是美国肝脏疾病的主要原因，也是大多数肝脏移植的原因。目前对这种传染病的治疗是不够的，因此必须开发新的治疗方法(4)。为了发现干扰病毒复制的新靶点，有必要了解病毒复制的机制。该提案的重点是由丙型肝炎病毒编码的非结构蛋白3(NS3)。NS3蛋白具有ATPase、解旋酶和蛋白水解酶活性。 NS3蛋白属于解旋酶超家族-2。NS3对于病毒复制是必不可少的。鉴于这一观察结果，NS3是一个重要的治疗靶点(5)。NS3的特性(包括阐明其核酸转位/解离机制)对于未来疫苗和/或治疗的发展非常重要。 使用首席研究员(马特洛克博士)建立和发表的方法，将研究NS3(NS3h)解旋酶结构域与核酸底物的相互作用。这项建议的具体目的是阐明NS3结合、解离和沿核酸底物移位的机制。攻击计划是使用一种成熟的、灵敏的、实时的荧光方法，这种方法已经被证明可以准确地报告双链DNA的分离(6)。 去年夏天(2006年)，在Kevin Raney博士的指导下，我们的团队开发了一种灵敏的实时荧光方法，以全长NS3和NS3的解旋酶结构域(NS3h)作为模型系统来研究核酸易位。在不同的实验条件下，研究了NS3和NS3h在核酸底物上的移位。 这种已建立的实时方法的使用和本文提出的使用来自丙型肝炎病毒的NS3的研究将提供对NS3的机制的洞察，到目前为止，这些机制很难用传统方法来检验。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Hepatitis C Virus (HCV) infects over 170 million persons worldwide (1-3). It is the leading cause of liver disease in the U.S. and is responsible for most liver transplants. Current treatments for this infectious disease are inadequate; therefore new therapies must be developed (4). It is necessary to understand the mechanism of viral replication in order to discover new targets for disruption of viral replication. The focus of this proposal is non-structural protein 3 (NS3) that is encoded by HCV. The NS3 protein exhibits ATPase, helicase, and protease activities. The NS3 protein is within helicase superfamily-2. NS3 is essential for viral replication. Given this observation, NS3 is an important therapeutic target (5). Characterization of NS3 (to include the elucidation of its nucleic acid translocation/unwinding mechanism) is important for the future development of a vaccine and/or cure. Using established and published methods by the principal investigator (Dr. Matlock), the interaction of the helicase domain of NS3 (NS3h) with a nucleic acid substrate will be studied. The specific aim of this proposal is to elucidate the mechanism of NS3 binding, unwinding, and translocation along a nucleic acid substrate. The plan of attack is to employ an established, sensitive, real-time fluorescence method that has been shown to accurately report the separation of double-stranded DNA (6). Last summer (2006) under the direction of Dr. Kevin Raney, our group developed a sensitive, real-time fluorescence method to study nucleic acid translocation using full-length NS3 and the helicase domain of NS3 (NS3h) as a model system. The translocation of NS3 and NS3h along a nucleic acid substrate was examined under a variety of experimental conditions. The use of this established real-time method and the proposed studies herein using NS3 from HCV will provide insight into the mechanism of NS3 that to date have been difficult to examine using conventional methods.