Vaccina Virus DNA Topoisomerase

描述(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家族包括真核细胞的核和线粒体ToplB、痘病毒和拟态病毒拓扑异构酶以及细菌的痘病毒样拓扑异构酶。ToplB酶通过断裂并重新连接DNA双链的一条链来松弛DNA超螺旋。它们通过涉及共价DNA-(3‘-磷酸酪氨酰基)酶中间体的酯交换机制起作用。本实验室以痘苗病毒为模型系统对ToplB进行研究。痘苗病毒编码的ToplB被包装在病毒颗粒中，它通过帮助病毒mRNA的合成在复制适合性中发挥关键作用。痘病毒ToplB的一个显著特征是其在靶序列5‘-(C/T)CCTT处形成共价中间体的特异性。所有的痘病毒TOPO和同源的模拟病毒ToplB酶都能识别这个位点。我们假设DNA靶标识别触发了催化氨基酸侧链的招募，以形成ToplB活性部位。本项目的目的是在单原子分辨率下阐明DNA酯交换的结构基础和靶点特异性，并确定活性中心组装和超螺旋松弛的构象步骤。这将通过一种创新的多学科方法实现，涉及DNA化学、非天然氨基酸的蛋白质修饰和单分子研究，以及“经典”结构导向突变和生物化学。我们还旨在通过评估生化特征的ToplB突变对痘苗病毒复制的影响，从遗传学上剖析痘苗病毒ToplB的哪些特性在体内是重要的。 相关性：了解ToplB的催化机制是一个高度优先的问题，因为：ToplB几乎参与了人类细胞的每一次DNA交易；核ToplB是通过破坏切割-宗教平衡而发挥细胞毒性的抗癌药物的靶标；ToplB酶广泛分布于细菌和病毒病原体中，它们为基于机制的抗感染药物发现提供了未开发的靶点。考虑到天花可能被用作生物恐怖武器，以及如果恢复预防性疫苗接种计划，可能会出现牛痘感染并发症的风险，开发治疗痘病毒感染的新分子靶点是一个紧迫的问题。

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.