Mechanistic Studies of Type II Topoisomerases and Topoisomerase-Targeted Agents

II型拓扑异构酶是一种普遍存在的酶，对正常的染色体结构和 分离并在DNA复制、转录和重组中发挥重要作用。这些酶 放松DNA并通过传递完整的双螺旋(运输)移除遗传物质上的打结和缠结 片段)通过它们在单独的DNA片段(GATE)中产生的瞬时双链断裂 段)。人类编码两种密切相关的II型酶亚型，拓扑异构酶IIα和 拓扑异构酶IIβ。拓扑异构酶IIα对增殖细胞和拓扑异构酶IIβ的生存至关重要 在开发过程中扮演着关键角色。然而，因为这些酶产生必需的双链 DNA在其关键的催化功能中断裂，它们承担着双重角色。虽然对细胞来说是必不可少的 在生存的同时，它们每次行动时也对基因组的完整性构成内在威胁。 除了其重要的生理功能外，拓扑异构酶IIα和IIβ是一些 目前用于治疗人类癌症的最活跃和最广泛的处方药。这些特工 通过稳定共价拓扑异构酶II裂解的DNA复合体(裂解复合体)来杀死细胞 催化DNA链传递反应中的正常但短暂的中间产物。当产生的酶- 相关的DNA断裂在足够的浓度下存在，它们可以触发细胞死亡途径。 以II型酶为靶点的抗癌药物被称为拓扑异构酶II型毒药，因为它们将 这些不可缺少的酶对有效的生理毒素产生作用，从而在处理的细胞中产生DNA损伤。 虽然拓扑异构酶IIα和IIβ是癌症化疗的重要靶点，但它们也具有 有可能引发特定的白血病。例如，一小部分癌症患者或多发性 接受拓扑异构酶II靶向药物米托蒽醌治疗的硬化症患者继续发展为急性 早幼粒细胞白血病(APL)，易位15：17。 尽管II型拓扑异构酶在细胞生长和癌症中的重要性，我们仍然有很多需要了解的东西。 人类酶的功能以及与DNA和抗癌药物的相互作用。因此，这项提案将进一步 确定II型拓扑异构酶的催化机制，并研究酶活性是如何在 手机。它还将定义已建立的和新的拓扑异构酶II靶向药物的机制，环境- 金属化学品和天然产品会增加酶介导的DNA断裂或抑制酶的水平 并确定拓扑异构酶II毒物的细胞后果。的主要研究模型 本研究将用人类拓扑异构酶IIα和IIβ、培养的人类细胞和非洲爪哇的卵子提取液。 来自大肠杆菌和炭疽芽孢杆菌的旋转酶和拓扑异构酶IV将用作对应物 机械实验和结核分枝杆菌旋转酶将被用来评估 针对原核生物和真核生物II型酶的药物作用机制。

Type II topoisomerases are ubiquitous enzymes that are required for proper chromosome structure and segregation and play important roles in DNA replication, transcription, and recombination. These enzymes relax DNA and remove knots and tangles from the genetic material by passing an intact double helix (transport segment) through a transient double-stranded break that they generate in a separate DNA segment (gate segment). Humans encode two closely related isoforms of the type II enzyme, topoisomerase IIα and topoisomerase IIβ. Topoisomerase IIα is essential for the survival of proliferating cells and topoisomerase IIβ plays critical roles during development. However, because these enzymes generate requisite double-stranded DNA breaks during their crucial catalytic functions, they assume a dual persona. Although essential to cell survival, they also pose an intrinsic threat to genomic integrity every time they act. Beyond their critical physiological functions, topoisomerase IIα and IIβ are the primary targets for some of the most active and widely prescribed drugs currently used for the treatment of human cancers. These agents kill cells by stabilizing covalent topoisomerase II-cleaved DNA complexes (cleavage complexes) that are normal, but fleeting, intermediates in the catalytic DNA strand passage reaction. When the resulting enzyme- associated DNA breaks are present in sufficient concentrations, they can trigger cell death pathways. Anticancer drugs that target type II enzymes are referred to as topoisomerase II poisons because they convert these indispensable enzymes to potent physiological toxins that generate DNA damage in treated cells. Although topoisomerase IIα and IIβ are important targets for cancer chemotherapy, they also have the potential to trigger specific leukemias. For example, a small percentage of patients with cancer or multiple sclerosis who are treated with the topoisomerase II-targeted drug mitoxantrone go on to develop acute promyelocytic leukemias (APLs) with 15:17 translocations. Despite the importance of type II topoisomerases in cell growth and cancer, we still have much to learn about how the human enzymes function and interact with DNA and anticancer drugs. Thus, this proposal will further define the catalytic mechanism of type II topoisomerases and examine how enzyme activity is regulated in the cell. It also will define mechanisms by which established and novel topoisomerase II-targeted agents, environ- mental chemicals, and natural products increase levels of enzyme-mediated DNA breaks or inhibit enzyme activity and determine the cellular consequences of topoisomerase II poisons. The primary research models for this study will be human topoisomerase IIα and IIβ, cultured human cells, and Xenopus laevis egg extracts. Gyrase and topoisomerase IV from Escherichia coli and Bacillus anthracis will be used as counterpoints to mechanistic experiments and Mycobacterium tuberculosis gyrase will be used to assess relationships between the mechanisms of action of drugs targeted to prokaryotic and eukaryotic type II enzymes.