UNWINDING&TRANSLOCATION OF DNA BY NS3 HELICASE FROM HEPATITIS C

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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.