VACCINIA VIRUS DNA TOPOISOMERASE

痘苗病毒 DNA 拓扑异构酶

• 批准号: 2444796
• 负责人: Stewart H Shuman
• 金额: $ 20.68万
• 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 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444796
• 关键词: DNA binding protein; DNA topoisomerases; X ray crystallography; antineoplastics; camptothecin; chimeric proteins; crosslink; crystallization; drug interactions; drug resistance; enzyme mechanism; enzyme model; enzyme substrate; genetic recombination; point mutation; protein engineering; protein reconstitution; protein sequence; protein structure function; site directed mutagenesis; vaccinia virus; virus DNA; virus genetics

Type I DNA topoisomerases alter the topology of DNA by transiently breaking and rejoining single DNA strands. The eukaryotic family of type I enzymes, which includes the nuclear enzymes and the topoisomerases encoded by poxviruses, comprises a group of proteins with shared biochemical properties and catalytic mechanism. The involvement of these enzymes in DNA replication, genetic recombination, and transcription, and the fact that topoisomerase I is the target of the camptothecin class of antitumor drugs, mandates a more complete understanding of their mechanism of action. This laboratory is pursuing a combined biochemical and molecular genetic study of eukaryotic topoisomerase I function and pharmacology using vaccinia virus as a model system. Vaccinia is unique among eukaryotic DNA viruses in that it encapsidates a topoisomerase within the infectious virus particle. The viral topoisomerase is similar in its enzymatic properties to the cellular counterpart, but differs from the cellular enzyme in three respects: (i) the 314-amino acid vaccinia topoisomerase is considerably smaller than the cellular type I enzymes (765 to 972-aa); (ii) vaccinia topoisomerase cleaves DNA at a specific recognition element CCCTT; (iii) vaccinia topoisomerase is inherently resistant to camptothecin. In this proposal, we address the specificity of topoisomerase interaction with DNA, and describe novel ways to examine how this specificity might illuminate the role of the topoisomerase I in DNA recombination. A rational mutagenesis strategy is presented that will yield a comprehensive map of vaccinia topoisomerase function and primary protein structure. The hypothesis is made that resistance or sensitivity to camptothecin can be accounted for by defined subdomains of the cellular and viral topoisomerases that influence the interaction of the drug with the covalent enzyme-DNA intermediate. The model will be tested by engineering vaccinia topoisomerase chimeras, by substituting local "patches" of viral sequence with residues unique to the cellular enzymes, in an effort to render the viral enzyme camptothecin sensitive. Mutational analysis of the topoisomerase will be pursued in parallel with efforts to solve the structure of the protein via X-ray crystallography.

I 型 DNA 拓扑异构酶通过瞬时改变 DNA 的拓扑结构 断裂并重新连接单条 DNA 链。真核生物家族类型 I 酶，包括核酶和拓扑异构酶 由痘病毒编码，包含一组具有共享特性的蛋白质 生化性质和催化机制。这些人的参与 DNA复制、基因重组和转录中的酶，以及 事实上，拓扑异构酶 I 是喜树碱类药物的靶标 抗肿瘤药物，需要对其机制有更全面的了解 的行动。 该实验室致力于生物化学和分子遗传学的结合 真核拓扑异构酶 I 功能和药理学研究 牛痘病毒作为模型系统。牛痘在真核 DNA 中是独一无二的 病毒，因为它在传染性内包裹拓扑异构酶 病毒颗粒。病毒拓扑异构酶的酶学性质相似 具有与细胞对应物相同的特性，但与细胞不同 酶在三个方面：(i) 314 个氨基酸的牛痘拓扑异构酶是 比细胞 I 型酶小得多（765 至 972-aa）； (ii) 牛痘拓扑异构酶在特定识别元件处切割 DNA CCCTT； (iii) 牛痘拓扑异构酶具有固有的抗性 喜树碱。在本提案中，我们解决了以下问题的特殊性： 拓扑异构酶与 DNA 的相互作用，并描述了检查拓扑异构酶如何相互作用的新方法 这种特异性可能阐明拓扑异构酶 I 在 DNA 中的作用 重组。提出了一种合理的诱变策略，该策略将 产生痘苗拓扑异构酶功能和初级的综合图谱 蛋白质结构。假设阻力或敏感性 喜树碱可以通过细胞的定义的子域来解释 和影响药物相互作用的病毒拓扑异构酶 共价酶-DNA中间体。该模型将通过以下方式进行测试 工程痘苗拓扑异构酶嵌合体，通过取代局部 病毒序列的“补丁”带有细胞酶特有的残基， 努力使病毒酶喜树碱敏感。突变的 拓扑异构酶的分析将与以下工作同时进行： 通过 X 射线晶体学解析蛋白质的结构。