TOPOISOMERASES

拓扑异构酶

• 批准号: 7953773
• 负责人: LYNN ZECHIEDRICH
• 金额: $ 0.87万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7953773
• 关键词: ATPase Domain; Antineoplastic Agents; Biochemical; Catenated DNA; Complex; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; DNA; DNA Maintenance; DNA topoisomerase II alpha; Data; Dimensions; Electron Microscopy; Enzymes; Funding; Grant; Human; Individual; Institution; Kinetics; Metabolism; Neurofibrillary Tangles; Organism; Pharmacologic Substance; Proteins; Reaction; Research; Research Personnel; Resources; Source; Structure; Superhelical DNA; Topoisomerase; United States National Institutes of Health; Yeasts; enzyme structure; macromolecule; particle; reconstruction; three dimensional structure

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Topoisomerases are essential enzymes that impact nearly every aspect of DNA metabolism through the maintenance of DNA supercoiling, untangling DNA knots, and resolving DNA catenanes. These enzymes are highly conserved in all organisms and are major pharmaceutical targets. Our current challenges focus on how human topoisomerase II alpha, a type-2 topoisomerase, untangles rather than tangles its DNA substrates. Although there are extensive biochemical data available for the enzyme, there exists no atomic structure information on any complete type-2 topoisomerase. Atomic structures are available of individual domains of eukaryotic type-2 enzymes, such as the ATPase domain for both the yeast and human enzymes and the cleavage-religation region of the yeast enzyme; however, there is no atomic structure information available for 22% of the C-terminus of the human protein. Electron cryomicroscopy and single particle reconstruction will be used to determine the three-dimensional structure of the complete enzyme alone or complexed with its DNA substrate in the presence and absence of anticancer drugs. In addition, reconstructions of isolated reaction intermediates will be used to determine the conformational changes of the enzyme. Human topoisomerase II alpha is a homodimer of 340 kDa and our initial multiple refinement convergence reconstructions of the enzyme alone show the dimensions of the particles to be roughly 65 A x 70 A x 75 A. With the structures of the enzyme alone, complexed with DNA, with anticancer drugs, and each trapped kinetic intermediate, we will be able to interpret how the machine moves and progresses through its catalytic cycle.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 拓扑异构酶是重要的酶，通过维持 DNA 超螺旋、解开 DNA 结和解析 DNA 链，影响 DNA 代谢的几乎各个方面。 这些酶在所有生物体中高度保守，是主要的药物靶点。 我们当前的挑战集中在人类拓扑异构酶 II α（一种 2 型拓扑异构酶）如何解开而不是缠结其 DNA 底物。 尽管该酶有大量的生化数据，但没有任何完整 2 型拓扑异构酶的原子结构信息。 真核 2 型酶的各个结构域均可获得原子结构，例如酵母和人类酶的 ATP 酶结构域以及酵母酶的裂解-再连接区域；然而，人类蛋白质 22% 的 C 末端没有可用的原子结构信息。 电子冷冻显微镜和单粒子重建将用于确定在存在或不存在抗癌药物的情况下单独的完整酶或与其 DNA 底物复合的完整酶的三维结构。 此外，分离的反应中间体的重建将用于确定酶的构象变化。 人类拓扑异构酶 II α 是 340 kDa 的同二聚体，我们对单独酶的初始多重细化收敛重建显示，颗粒的尺寸约为 65 A x 70 A x 75 A。通过单独酶的结构、与 DNA、抗癌药物以及每个捕获的动力学中间体的复合物，我们将能够解释机器如何移动和 通过其催化循环进展。