DNA LESIONS AS ENDOGENOUS TOPOISOMERASE POISONS

描述(改编自《调查者摘要》)：拓扑异构酶II 一种必需的酶，它是正确的染色体结构和 分离，并在DNA复制和重组中发挥重要作用。 除了其关键的细胞功能外，拓扑异构酶II是主要的靶标 用于治疗的一些最活跃和最广泛的处方药 人类癌症。这些药物通过一种机制诱导它们的细胞毒作用。 这与其他药物有明显的不同。而不是抑制 靶向抗癌药物拓扑异构酶II的催化活性 显著增加共价拓扑异构酶II裂解的DNA复合体水平 这些是正常的，但转瞬即逝的催化中间体。当所产生的 拓扑异构酶II相关的双链DNA断裂在高 浓度，它们会产生突变，染色体易位，以及 触发细胞死亡途径。因为以拓扑异构酶II为靶点的抗癌药物 将这种可有可无的酶转化为一种有效的生理毒素，它们 称为拓扑异构酶II毒物。尽管拓扑异构酶II是 癌症化疗的最重要的目标，有令人信服的 间接证据表明，这种酶也有可能触发 疾病。以及拓扑异构酶II独特的作用机制 毒物，这表明拓扑异构酶II靶向药物可能代表着 诱导DNA重组的外源细胞成分的对应物， 突变，或细胞死亡途径。本实验室以前的研究结果 表明基本部位，DNA中最常见的损伤，刺激 拓扑异构酶II介导的双链DNA切割 比依托泊苷高1000倍以上，是应用最广泛的 临床上使用的抗癌处方药。因此，的最终目标是 该提案旨在进一步定义拓扑异构酶II和 DNA损伤，并确定DNA损伤在体内是否起内源性作用 拓扑异构酶II毒物。这项提议的具体目的是：1)进一步 确定改变II型催化功能的DNA损伤的光谱 拓扑异构酶；2)确定DNA损伤增强的机制 拓扑异构酶II介导的DNA切割；3)确定碱性 碱基切除修复产生的中间产物可以触发形成 永久性拓扑异构酶II介导的双链DNA断裂；和4) 确定DNA损伤是否在细胞中起到拓扑异构酶II毒物的作用。这个 这项研究的主要酶学模型将是人类拓扑异构酶IIα 还有贝塔。生理学研究将使用人类细胞系和酵母 (酿酒酵母)。细胞DNA损伤将由一种 化学方法和遗传方法的结合。

DESCRIPTION (As Adapted From the Investigator'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, topoisomerase II 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. Because topoisomerase II-targeted anticancer drugs convert this dispensable enzyme into a potent physiological toxin, they are referred to as topoisomerase II poisons. 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. Together with the unique mechanism of action of topoisomerase II poisons, this suggests that topoisomerase II-targeted drugs may represent exogenous counterparts of cellular components that induce DNA recombination, mutagenesis, or cell death pathways. Previous results form this laboratory indicated that abasic sites, the most commonly formed lesions in DNA, stimulate topoisomerase II-mediated double-stranded DNA cleavage with a potency that is greater than 1000-fold higher than that of etoposide, one of the most widely prescribed anticancer drugs in clinical use. Therefore, the ultimate goals of the proposal are to further define interactions between topoisomerase II and DNA damage and to determine whether DNA lesions function in vivo as endogenous topoisomerase II poisons. The specific aims of this proposal are to: 1) further define the spectrum of DNA damage that alters the catalytic function of type II topoisomerases; 2) define the mechanism by which DNA lesions enhance topoisomerase II-mediated DNA cleavage; 3) determine whether abasic intermediates generated by base excision repair can trigger the formation of permanent topoisomerase II-mediated double-stranded DNA breaks; and 4) determine whether DNA lesions act as topoisomerase II poisons in the cell. The primary enzymological model for this study will be human topoisomerase II alpha and beta. Physiological studies will employ human cell lines and yeast (Saccharomyces cerevisiae). Cellular DNA damage will be induced by a combination of chemical and genetic approaches.