FUNCTION AND BIOLOGY OF EUKARYOTIC DNA TOPOISOMERASES

描述（改编自申请人的摘要）拓扑异构酶II是一种拓扑异构酶。 一种必需的酶，是正确的染色体结构所必需的， 分离，并在DNA复制和重组中起重要作用。 除了其关键的细胞功能外，它还是一些癌症的主要靶点。 用于治疗人类疾病的最有效和最广泛的处方药 癌的这些药物通过一种机制引起其细胞毒性作用， 与其他药物不同。而不是抑制 拓扑异构酶II的催化活性，靶向该酶的抗癌药物 显著增加共价拓扑异构酶II切割的DNA复合物的水平 它们是正常的，但转瞬即逝的催化中间体。当所得 拓扑异构酶II相关的双链DNA断裂存在于高 浓度，它们产生突变，染色体易位， 触发细胞死亡途径。 虽然拓扑异构酶II是癌症最重要的靶点之一， 化疗，有令人信服的间接证据表明，酶也 有可能引发疾病事实上，继发性白血病 与特定的染色体易位相关， 接受拓扑异构酶II靶向药物治疗的患者。因为拓扑异构酶II 必须在DNA中产生双链断裂才能发挥作用， 对基因组的完整性构成内在的威胁， 遗传物质 尽管拓扑异构酶II对癌症问题至关重要， 酶与DNA和抗癌药物的相互作用还不是很好 表征了因此，本提案的最终目标是进一步界定 拓扑异构酶II进行其基本的细胞 反应和药物改变催化功能的机制， 酵素本研究的研究模型将是人类、草履虫 小球藻病毒-1（PBCV-1）、酵母（酿酒酵母）和果蝇。 该提案的具体目标是：1）进一步界定催化剂 拓扑异构酶II的机制; 2）确定异常的基础 PBCV-1拓扑异构酶II强DNA切割活性; 3）进一步描述 拓扑异构酶II靶向抗癌作用的机制基础 药物;和4）探索拓扑异构酶II和基因组之间的关系 稳定所提出的实验是基于一些新的发现 包括发现PBCV-1 拓扑异构酶II，第一个真核病毒II型酶， 表征了研究将充分利用几种检测方法， 在主要研究者的实验室开发，并将利用 生物化学，物理和遗传方法，以解决既定的目标， 提议

DESCRIPTION (adapted from applicant's abstract) Topoisomerase II is an essential enzyme that is required for proper chromosome structure and segregation and plays important roles in DNA replication and recombination. Beyond its critical cellular functions, it is the primary target for some of the most active and widely prescribed drugs used for the treatment of human cancers. These agents elicit their cytotoxic effects by a mechanism that is markedly different than that of other drugs. Rather than inhibiting the catalytic activity of topoisomerase II, anticancer drugs targeted to the enzyme dramatically increase levels of covalent topoisomerase II-cleaved DNA complexes that are normal, but fleeting, catalytic intermediates. When the resulting topoisomerase II-associated double-stranded DNA breaks are present in high concentrations, they generate mutations, chromosomal translocations, and trigger cell death pathways. Although topoisomerase II is one of the most important targets for cancer chemotherapy, there is compelling circumstantial evidence that the enzyme also has the potential to trigger the disease. Indeed, secondary leukemias associated with specific chromosomal translocations are observed in some patients treated with topoisomerase II-targeted drugs. Because topoisomerase II must create double-stranded breaks in DNA in order to function, the enzyme poses an intrinsic threat to genomic integrity every time it acts on the genetic material. Despite the central importance of topoisomerase II to the cancer problem, interactions of the enzyme with DNA and anticancer drugs have not been well characterized. Thus, the ultimate goal of this proposal is to further delineate the mechanism by which topoisomerase II carries out its fundamental cellular reactions and the mechanism by which drugs alter the catalytic function of the enzyme. Research models for this study will be human, Paramecium bursaria Chlorella virus-1 (PBCV-1), yeast (Saccharomyces cerevisiae), and Drosophila. The specific aims of this proposal are to: 1) further define the catalytic mechanism of topoisomerase II; 2) determine the basis for the exceptionally robust DNA cleavage activity of PBCV-1 topoisomerase II; 3) further delineate the mechanistic basis for the actions of topoisomerase II-targeted anticancer drugs; and 4) explore relationships between topoismerase II and genomic stability. The proposed experiments are based on a number of novel findings made during the previous grant cycle, including the discovery of PBCV-1 topoisomerase II, the first eukaryotic viral type II enzyme to be characterized. Studies will take great advantage of several assays that were developed in the principal investigator's laboratory and will utilize biochemical, physical, and genetic approaches to address the stated aims of the proposal.