Function and Biology of Eukaryotic DNA Topoisomerases

真核 DNA 拓扑异构酶的功能和生物学

• 批准号: 8112443
• 负责人: NEIL OSHEROFF
• 金额: $ 33.85万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 2012-07-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8112443
• 关键词: 11q23; Address; Antineoplastic Agents; Benzene; Biology; Catalytic DNA; Cell Death; Cell Survival; Cells; Chemicals; Chemotherapy-Oncologic Procedure; Chlorella; Chromosome Band; Chromosome Segregation; Chromosome Structures; Cleaved cell; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Topoisomerases; DNA biosynthesis; DNA strand break; Development; Disease; Drug Delivery Systems; Drug Prescriptions; Enzymes; Epigallocatechin Gallate; Exposure to; Generations; Genetic Materials; Genetic Recombination; Genetic Transcription; Genomics; Goals; Health; Human; Infant; Ions; Kinetics; MLL gene; Malignant Neoplasms; Maternal Exposure; Mediating; Neurofibrillary Tangles; Nucleic Acids; Pathway interactions; Patients; Pharmaceutical Preparations; Physiological; Play; Poison; Pregnancy; Proliferating; Protein Isoforms; Reaction; Regimen; Research; Role; Saccharomyces cerevisiae; Structure; Study models; Superhelical DNA; Time; Topoisomerase; Topoisomerase II; Toxin; Type I DNA Topoisomerases; Virus; Yeasts; alpha-Thalassemia; cytotoxic; cytotoxicity; divalent metal; genotoxicity; leukemia; novel; polyphenol; research study; response; single molecule

DESCRIPTION (provided by applicant): Type II topoisomerases are ubiquitous enzymes that are required for proper chromosome structure and segregation. In addition, these enzymes play important roles in DNA replication, transcription, and recombination. Type II topoisomerases relax DNA and remove knots and tangles from the genetic material by passing an intact double helix through a transient double-stranded break that they generate in a separate DNA segment. Humans encode two closely related isoforms of the type II enzyme, topoisomerase II1 and topoisomerase II2. Topoisomerase II1 is essential for the survival of proliferating cells and topoisomerase II2 plays critical roles during development. However, since these enzymes generate requisite double-stranded DNA breaks in order to carry out their crucial catalytic functions, they assume a dual persona. While essential to cell survival, they also pose an intrinsic threat to genomic integrity every time they act. Beyond their important physiological functions, topoisomerase II1 and 2 are the primary targets for some of the most active and widely prescribed drugs currently used for the treatment of human cancers. These agents elicit their cytotoxic effects by dramatically increasing levels of covalent topoisomerase II-cleaved DNA 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 trigger cell death. 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 II1 and 2 are important targets for cancer chemotherapy, evidence suggests that they also have the potential to trigger the disease. Leukemias that carry genomic translocations involving the MLL gene at chromosome band 11q23 are associated with exposure to topoisomerase II poisons. Despite the central importance of type II topoisomerases to the cancer problem, interactions between topoisomerase II1/2, DNA, and anticancer drugs (or other enzyme poisons) have not been well characterized. Thus, the specific aims of this proposal are to further delineate the mechanism by which type II topoisomerases carry out their essential catalytic reactions, to define the mechanism by which drugs and other topoisomerase II-active chemicals alter the catalytic function of these enzymes, and to characterize the response of cells to enzyme-generated DNA strand breaks. Research models for this study will be primarily human topoisomerase II1 and 2, but yeast (Saccharomyces cerevisiae) and Chlorella virus topoisomerase II may also be employed for specific experiments. A variety of approaches, including ensemble and single molecule kinetic studies, novel nucleic acid substrates and topoisomerase II-targeted drugs, and cellular studies will be used to address the stated aims of the proposal. PUBLIC HEALTH RELEVANCE Type II topoisomerases are ubiquitous enzymes that remove knots and tangles from the genetic material by passing an intact double helix through a transient double-stranded break that they generate in a separate DNA segment. These enzymes are targets for important anticancer drugs, but are also believed to trigger to specific types of leukemias. The aims of this proposal are to determine how type II topoisomerases function, how drugs and other chemicals alter their activities, and how cells respond to the actions of these enzymes.

描述（由申请人提供）：II型拓扑异构酶是适当的染色体结构和隔离所需的无处不在的酶。另外，这些酶在DNA复制，转录和重组中起重要作用。 II型拓扑异构酶通过将完整的双螺旋穿过瞬态双链断裂，从而从遗传物质中释放DNA，并从遗传物质中取出结和缠结，它们在单独的DNA段中产生。人类编码II型酶，拓扑异构酶II1和拓扑异构酶II2的两个密切相关的同工型。拓扑异构酶II1对于增殖细胞的存活和拓扑异构酶II2在发育过程中起关键作用至关重要。但是，由于这些酶会产生必要的双链DNA断裂以执行其关键的催化功能，因此它们假定双重角色。尽管对细胞的生存至关重要，但它们每次行动都会对基因组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II1和2是目前用于治疗人类癌症的一些最活跃和处方药的主要目标。这些药物通过大大增加了正常但转瞬即逝的中间体的共价拓扑异构酶II裂解的DNA复合物的共价拓扑异构酶II裂解的DNA复合物，从而引起了细胞毒性作用。当产生的酶相关的DNA断裂以足够的浓度存在时，它们会触发细胞死亡。靶向II型酶的抗癌药被称为拓扑异构酶II毒物，因为它们将这些必不可少的酶转化为有效的生理毒素，从而在处理过的细胞中产生DNA损伤。尽管拓扑异构酶II1和2是癌症化学疗法的重要靶标，但证据表明它们也有可能触发疾病。携带涉及染色体带11q23 MLL基因的基因组易位的白血病与暴露于拓扑异构酶II毒物有关。尽管II型拓扑异构酶对癌症问题的重要性，但拓扑异构酶II1/2，DNA和抗癌药物（或其他酶毒物）之间的相互作用尚未得到很好的特征。 Thus, the specific aims of this proposal are to further delineate the mechanism by which type II topoisomerases carry out their essential catalytic reactions, to define the mechanism by which drugs and other topoisomerase II-active chemicals alter the catalytic function of these enzymes, and to characterize the response of cells to enzyme-generated DNA strand breaks.这项研究的研究模型将主要是人拓扑异构酶II1和2，但是酵母（酿酒酵母）和小球藻病毒拓扑异构酶II也可能用于特定实验。各种方法，包括集合和单分子动力学研究，新型核酸底物和拓扑异构酶II靶向药物，以及细胞研究将用于解决该提案的明确目的。公共卫生相关性II型拓扑异构酶是普遍存在的酶，通过通过瞬态双螺旋中的完整双螺旋通过它们在单独的DNA段中产生的瞬态双链断裂，从遗传材料中消除结和缠结。这些酶是重要的抗癌药物的靶标，但也被认为会触发特定类型的白血病。该提案的目的是确定II型拓扑异构酶如何起作用，药物和其他化学物质如何改变其活性以及细胞如何应对这些酶的作用。