Vaccina Virus DNA Topoisomerase

疫苗病毒 DNA 拓扑异构酶

• 批准号: 8098235
• 负责人: Stewart H Shuman
• 金额: $ 58.27万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8098235
• 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; Vaccinium; 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家族包括真核核和线粒体TOPLB，poxvirus和mimivirus topoisomerases，以及细菌的孢子病毒样拓扑异构酶。 TOPLB酶通过打破DNA双链体的一条链来放松DNA超驾驶仪。它们通过涉及共价DNA-（3'-磷酸苯基） - 酶中间体的式式机制起作用。该实验室使用离甲酸病毒作为研究TOPLB的模型系统。疫苗编码的TOPLB包装在病毒颗粒中，在该病毒颗粒中，它通过帮助病毒mRNA合成在复制适应性中起关键作用。 POXVIRUS TOPLB的一个独特特征是它在目标序列5' - （C/T）CCTT形成共价中间体方面的特异性。所有的痘病毒拓扑都可以识别此部位，同源的Mimivirus 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上阐明DNA持续测定和目标位点特异性的结构基础，并定义主动位点组装和超级侧侧放松的构象步骤。这将通过涉及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”结构引导的诱变和生物化学的创新多学科方法来实现。我们还旨在通过鉴定生物化学表征的TOPLB突变对疫苗病毒复制的影响，从而在遗传上剖析遗传上哪些特性在体内很重要。 相关性：理解TOPLB的催化机制是一个很高的优先级，因为：TOPLB几乎与人类细胞中的所有DNA交易有关；核TOPLB是抗癌药物的靶标，通过扭曲裂解齐平衡来发挥其细胞毒性。 TOPLB酶在细菌和病毒病原体中广泛分布，在那里它们为基于机制的抗感染药物发现提供了未开发的靶标。考虑到可以将天花用作生物疗法武器的担忧，并且如果恢复预防性疫苗，疫苗感染并发症的风险。