Structural and Biochemical Analyses of Type II DNA Topoisomerases

摘要 DNA拓扑结构的适当控制对DNA的稳定性和流动性有重大影响。 遗传信息。本申请集中于II型拓扑异构酶， 调节DNA超螺旋和去除染色体的分子机器 通过催化一个DNA双链体的ATP依赖性运输， 另II型拓扑异构酶对于维持基因表达至关重要， 染色体超结构和基因组完整性;它们也作为一线药物 治疗传染病和癌症的靶点。 在上一个项目期间，我们对该机制有了一些新的见解， 调节和II型拓扑异构酶的DNA损伤倾向。这些努力开启 三个新的研究机会，以互补但不相互依赖为中心 II型拓扑异构酶作用的方面，这将促进基础生物学和 知识和治疗干预。目标1试图解释长期未解决的 II型拓扑异构酶如何将ATP依赖性变构反应与 多个DNA片段的选择性接合和释放，并阐明 系统负责启动减数分裂重组增选和修改了 拓扑异构酶支架产生DNA断裂以促进染色体交换。 目标2将在理解真核topo II的调控方面开辟新的天地， 揭示控制酶活性的天然代谢物作为调节 复制、转录和染色体组织过程，这些过程对 DNA拓扑学目的3将确定人类拓扑异构酶IIβ的作用如何驱动异常的 DNA损伤事件以及自然发生的突变如何进一步加强这一点 有害活动。 我们的方法的特点是全面融合了生物化学，结构， 计算的、基于细胞的和化学生物学方法。过去的进展和 未发表的研究结果提供了数据，以确定拟议努力的可行性。我们 研究将影响多个领域，从分子机器的研究和控制， DNA动力学，了解拓扑异构酶活性及其调节 支持特定的生理需求，促进人类生殖和健康。

ABSTRACT The appropriate control of DNA topology has a major impact on the stability and flow of genetic information. The present application focuses on type II topoisomerases, molecular machines that modulate DNA supercoiling and remove chromosome entanglements by catalyzing the ATP-dependent transport of one DNA duplex through another. Type II topoisomerases are critical for maintaining gene expression, chromosome superstructure, and genome integrity; they also serve as frontline drug targets for treating infectious disease and cancer. During the prior project period, we gained several new insights into the mechanism, regulation, and DNA damage propensity of type II topoisomerases. These efforts open up three new research opportunities centered on complementary but non-interdependent aspects of type II topoisomerase action that will advance both fundamental biological knowledge and therapeutic intervention. Aim 1 seeks to explain the long-unresolved question of how type II topoisomerases link ATP-dependent allosteric responses to the selective engagement and release of multiple DNA segments, and to illuminate how a system responsible for initiating meiotic recombination co-opted and modified a topoisomerase scaffold to generate DNA breaks for promoting chromosome exchange. Aim 2 will break new ground in understanding the regulation of eukaryotic topo II, uncovering natural metabolites that control enzyme activity as means to modulate replication, transcription, and chromosome organization processes that are sensitive to DNA topology. Aim 3 will determine how the action of human topo IIβ drives aberrant DNA damaging events and how naturally-occurring mutations may further potentiate this detrimental activity. Our approach is distinguished by a comprehensive blend of biochemical, structural, computational, cell-based, and chemical biology methodologies. Past progress and unpublished findings provide data to establish feasibility for the proposed effort. Our studies will impact multiple fields, from the study of molecular machines and the control of DNA dynamics, to understanding how topoisomerase activity and its regulation support specific physiological needs and promote human reproduction and health.