Vaccina Virus DNA Topoisomerase

描述（由申请人提供）：我们的长期目标是了解DNA拓扑异构酶IB（ToplB）的机制和生物学功能。ToplB家族包括真核细胞核和线粒体ToplB、痘病毒和拟病毒拓扑异构酶以及细菌的痘病毒样拓扑异构酶。ToplB酶通过断裂和重新连接DNA双链体的一条链来松弛DNA超螺旋。它们通过涉及共价DNA-（3 '-磷酸-酪氨酰）-酶中间体的酯交换机制起作用。该实验室使用牛痘病毒作为模型系统来研究ToplB。牛痘编码的ToplB被包装在病毒颗粒内，其中它通过帮助病毒mRNA合成在复制适应性中起关键作用。痘病毒ToplB的独特特征是其在靶序列5 '-（C/T）CCTT处形成共价中间体的特异性。所有痘病毒拓扑识别该位点，同源拟病毒ToplB酶也是如此。我们假设DNA靶标识别触发催化氨基酸侧链的募集以形成ToplB活性位点。该项目的目的是阐明在单原子分辨率的DNA酯交换和靶位点特异性的结构基础，并定义活性位点组装和超螺旋松弛的构象步骤。这将通过创新的多学科方法来实现，包括DNA化学，非天然氨基酸的蛋白质修饰，单分子研究，沿着“经典”结构指导诱变和生物化学。我们还旨在通过测定生物化学表征的ToplB突变对牛痘病毒复制的影响，从遗传上剖析牛痘ToplB的哪些特性在体内是重要的。 相关性：理解ToplB的催化机制是高度优先的，因为：ToplB涉及人类细胞中几乎所有的DNA交易;核ToplB是抗癌药物的靶标，其通过破坏切割-再连接平衡来发挥其细胞毒性;并且ToplB酶广泛分布于细菌和病毒病原体中，其中它们为基于机制的抗感染药物发现提供了未开发的靶标。开发新的分子靶点治疗痘病毒感染是一个紧迫的问题，因为人们担心天花可能被用作生物恐怖武器，如果恢复预防性疫苗接种计划，牛痘感染的并发症风险很大。

DESCRIPTION (provided by applicant): Our long-term goals are to understand the mechanism and biological functions of DNA topoisomerase IB (ToplB). The ToplB family includes eukaryotic nuclear and mitochondrial ToplB, poxvirus and mimivirus topoisomerases, and the poxvirus-like topoisomerases of bacteria. ToplB enzymes relax DNA supercoils by breaking and rejoining one strand of the DNA duplex. They act via a transesterification mechanism involving a covalent DNA-(3'-phospho-tyrosyl)-enzyme intermediate. This laboratory uses vaccinia virus as a model system to study ToplB. The vaccinia-encoded ToplB is packaged within the virus particle, where it plays a critical role in replicative fitness by aiding viral mRNA synthesis. A distinctive feature of the poxvirus ToplB is its specificity in forming a covalent intermediate at a target sequence 5'-(C/T)CCTT. All poxvirus topos recognize this site, as does the homologous mimivirus ToplB enzyme. We hypothesize that DNA target recognition triggers the recruitment of catalytic amino acid side chains to form the ToplB active site. An aim of this project is to elucidate at single-atom resolution the structural basis for DNA transesterification and target site specificity and to define the conformational steps for active site assembly and supercoil relaxation. This will be accomplished by an innovative multidisciplinary approach involving DNA chemistry, protein modification with non-natural amino acids, and single-molecule studies, along with "classical" structure-guided mutagenesis and biochemistry. We also aim to dissect genetically which properties of vaccinia ToplB are important in vivo, by gauging the effects of biochemically characterized ToplB mutations on vaccinia virus replication. Relevance: Understanding the catalytic mechanism of ToplB is a high priority because: ToplB is implicated in virtually every DNA transaction in human cells; nuclear ToplB is the target of anticancer drugs that exert their cytotoxicity by perverting the cleavage-religation equilibrium; and ToplB enzymes are distributed widely in bacterial and viral pathogens, where they present untapped targets for mechanism-based anti-infective drug discovery. Exploitation of new molecular targets for treatment of poxvirus infections is a pressing issue, given the concerns that smallpox could be used as a bioterror weapon and the risk of complications of vaccinia infections if a prophylactic vaccination program is resumed.