Mechanistic Studies of Type II Topoisomerases and Topoisomerase-Targeted Agents

II型拓扑异构酶是普遍存在的酶，其是正确的染色体结构所需的， 分离，并在DNA复制，转录和重组中发挥重要作用。这些酶 通过传递完整的双螺旋（运输）来放松DNA并去除遗传物质中的结和缠结 片段）通过它们在单独的DNA片段（门）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II α和 拓扑异构酶II β。拓扑异构酶II α对增殖细胞的存活至关重要，拓扑异构酶II β 在发育过程中起着关键作用。然而，由于这些酶产生必需的双链 DNA在其关键的催化功能期间断裂，它们承担双重角色。虽然对细胞至关重要 为了生存，它们每次行动都对基因组完整性构成内在威胁。 除了它们的重要生理功能之外，拓扑异构酶II α和II β是一些肿瘤的主要靶点。 是目前用于治疗人类癌症的最有效和最广泛的处方药。这些试剂 通过稳定共价拓扑异构酶II-切割的DNA复合物（切割复合物）杀死细胞， 正常的，但短暂的，在催化DNA链通道反应的中间体。当生成的酶- 如果相关的DNA断裂以足够的浓度存在，它们可以触发细胞死亡途径。 靶向II型酶的抗癌药物被称为拓扑异构酶II毒药，因为它们将 这些不可或缺的酶，以强大的生理毒素，产生DNA损伤处理细胞。 虽然拓扑异构酶II α和II β是癌症化疗的重要靶点，但它们也具有 可能引发特定的白血病。例如，一小部分患有癌症或多种癌症的患者， 用拓扑异构酶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），具有15：17易位。 尽管II型拓扑异构酶在细胞生长和癌症中的重要性，我们仍然有很多东西要了解 人类酶如何发挥作用以及如何与DNA和抗癌药物相互作用。因此，该提案将进一步 定义II型拓扑异构酶的催化机制，并研究酶活性如何在 cell.它还将定义机制，建立和新的拓扑异构酶II靶向药物，环境， 金属化学品和天然产物增加酶介导的DNA断裂水平或抑制酶 活性和确定拓扑异构酶II毒物的细胞后果。主要研究模型 本研究将人类拓扑异构酶II α和II β，培养的人类细胞，以及非洲爪蟾卵提取物。 来自大肠杆菌和炭疽杆菌的促旋酶和拓扑异构酶IV将被用作对应物， 机制实验和结核分枝杆菌促旋酶将用于评估 靶向原核和真核II型酶的药物的作用机制。

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.