Vaccina Virus DNA Topoisomerase

疫苗病毒 DNA 拓扑异构酶

• 批准号: 7989253
• 负责人: Stewart H Shuman
• 金额: $ 14万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-17 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7989253
• 关键词: Active Sites; Alanine; Amino Acids; Anti-Infective Agents; Antineoplastic Agents; Apoenzymes; Bacteria; Binding Sites; Biochemical; Biochemistry; Biological Models; Biological Process; Catalytic Domain; Cells; Chemicals; Chemistry; Chromosome Pairing; Complex; DNA; DNA Binding; DNA Modification Process; DNA Structure; DNA Topoisomerases; Defect; Deinococcus; Enzymes; Equilibrium; Family; Goals; Homologous Gene; Human; In Vitro; Individual; Infection; Laboratories; Left; Major Groove; Messenger RNA; Mitochondria; Modeling; Molecular Target; Mutagenesis; Mutation; Nuclear; Physiology; Play; Post-Translational Protein Processing; Poxviridae; Poxviridae Infections; Property; Proteins; Reaction; Relaxation; Resolution; Risk; Role; Side; Site; Site-Directed Mutagenesis; Smallpox; Specificity; Structure; Superhelical DNA; Testing; Topoisomerase; Transfer RNA; Translations; Type I DNA Topoisomerases; Vaccination; Vaccinia; Vaccinia virus; Viral; Virion; Virus Replication; analog; base; cytotoxicity; drug discovery; fascinate; fitness; functional group; in vivo; innovation; inorganic phosphate; insight; interdisciplinary approach; mutant; nucleobase analog; pathogen; programs; prophylactic; recombinant virus; research study; single molecule; tool; viral DNA; virus core; weapons

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.

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、痘病毒和拟菌病毒拓扑异构酶、以及细菌的类痘病毒拓扑异构酶。 ToplB 酶通过断裂和重新连接 DNA 双链体的一条链来放松 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发挥作用。本实验室使用痘苗病毒作为模型系统来研究ToplB。牛痘编码的 ToplB 包装在病毒颗粒内，通过帮助病毒 mRNA 合成，在复制适应性中发挥关键作用。痘病毒Top1B的显着特征是其在靶序列5'-(C/T)CCTT处形成共价中间体的特异性。所有痘病毒 topos 都识别该位点，同源拟菌病毒 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。该项目的目的是在单原子分辨率下阐明 DNA 酯交换反应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，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”结构引导诱变和生物化学。我们还旨在通过测量生化特征的 Top1B 突变对痘苗病毒复制的影响，从遗传学角度剖析痘苗 Top1B 的哪些特性在体内很重要。 相关性：了解 ToplB 的催化机制是当务之急，因为： ToplB 几乎涉及人类细胞中的每一个 DNA 交易；核ToplB是抗癌药物的靶点，通过破坏裂解-再连接平衡来发挥细胞毒性； ToplB 酶广泛分布于细菌和病毒病原体中，为基于机制的抗感染药物发现提供了尚未开发的靶标。考虑到天花可能被用作生物恐怖武器的担忧以及如果恢复预防性疫苗接种计划则存在牛痘感染并发症的风险，开发新的分子靶标来治疗痘病毒感染是一个紧迫的问题。