DNA LESIONS AS ENDOGENOUS TOPOISOMERASE POISONS

拓扑异构酶II是几种最活跃的 目前用于治疗人类癌症的药物。 这些药物通过独特的机制引起其细胞毒性作用。 而 比通过抑制酶的催化活性起作用，抗癌 药物显著增加共价拓扑异构酶II切割的水平， DNA复合物是正常的，但短暂的，中间体， 酶的催化循环。 当产生的酶相关的 双链DNA断裂存在于基因组中， 浓度，它们会产生突变，染色体畸变， 在极端条件下细胞死亡 因此，抗癌药物的毒性 拓扑异构酶II并将其从必需酶转化为 生理毒素 拓扑异构酶II毒物的不寻常的作用机制提高了 这些药物可能代表细胞的外源性对应物， 诱导DNA重组、诱变或细胞死亡的成分 途径。 该实验室先前的结果表明， 位点，这是DNA中最常见的损伤， 通过无数的DNA损伤事件，刺激拓扑异构酶II介导的 双链DNA切割。 这种卵裂刺激的效果 类似于依托泊苷（这是最广泛的规定 在临床上使用的抗癌剂）。 然而，脱碱基位点的效力 是药物的两千倍 因此 该提案的最终目标是定义基础物质之间的相互作用， 位点和II型酶，并确定是否脱碱基位点 作为内源性拓扑异构酶II毒物发挥作用。 更具体而言是 该提案的目的是：1）确定基本 位点增强拓扑异构酶II介导的切割，2）描绘 酶识别脱碱基位点的机制，3）定义 脱碱基位点的作用机制与 抗癌药物，以及4）确定脱碱基位点是否作为 拓扑异构酶II在体内中毒。 本研究产生的信息 这将大大增加我们对拓扑异构酶II靶向 试剂刺激酶介导的DNA切割并最终引起细胞凋亡。 死亡 果蝇和酵母将作为主要的研究模型 study. 果蝇和酵母的酶是最好的特征 II型拓扑异构酶和酵母允许一定程度的遗传操作 这是任何其他真核生物系统都无法比拟的。 拟议的研究 将利用几个最近开发的分析系统。 的 脱碱基位点增强酶介导的DNA切割的机制将 通过各种生化、动力学和遗传学方法进行分析。 拓扑异构酶II的脱碱基位点的识别将被表征 通过确定酶如何扫描DNA寻找损伤， 这种病变的结构特征，需要改变酶 活动 脱碱基位点和抗癌剂之间的关系将 通过将这些病变的相互作用域映射到 相对于药物的拓扑异构酶II。 最后，生理 作为拓扑异构酶II毒物的脱碱基位点的作用的特征在于： 确定这些病变是否诱导拓扑异构酶II介导的细胞凋亡， 死亡或诱变。

Topoisomerase II is the cellular target for several of the most active antineoplastic agents currently used for the treatment of human cancers. These drugs elicit their cytotoxic effects by a unique mechanism. Rather than acting by inhibiting the catalytic activity of the enzyme, anticancer drugs dramatically increase levels of covalent topoisomerase II-cleaved DNA complexes that are normal, but fleeting, intermediates in the catalytic cycle of the enzyme. When the resulting enzyme-associated double-stranded DNA breaks are present in the genome in high concentrations, they generate mutations, chromosomal aberrations, and under extreme conditions, cell death. Thus, anticancer drugs poison topoisomerase II and convert it from an essential enzyme into a physiological toxin. The unusual mechanism of action of topoisomerase II poisons raises the possibility that these drugs represent exogenous counterparts of cellular components that induce DNA recombination, mutagenesis, or cell death pathways. Previous results from this laboratory indicate that abasic sites, which are the most commonly formed lesion in DNA and are generated by a myriad of DNA damaging events, stimulate topoisomerase II-mediated double-stranded DNA cleavage. The efficacy of this cleavage stimulation is similar to that of etoposide (which is the most widely prescribed anticancer agent in clinical use). However, the potency of abasic sites is about 2,000-fold greater than that of the drug. Therefore, the ultimate goals of this proposal are to define interactions between abasic sites and the type II enzyme and to determine whether abasic sites function as endogenous topoisomerase II poisons. More specifically, the aims of this proposal are 1) to determine the mechanism by which abasic sites enhance topoisomerase II-mediated cleavage, 2) to delineate the mechanism by which the enzyme recognizes abasic sites, 3) to define relationships between the mechanism of action of abasic sites and anticancer drugs, and 4) to determine whether abasic sites function as topoisomerase II poisons in vivo. The information generated by this study should greatly increase our understanding of how topoisomerase II-targeted agents stimulate enzyme-mediated DNA cleavage and ultimately cause cell death. Drosophila and yeast will serve as the primary research models for this study. The Drosophila and yeast enzymes are the most well characterized type II topoisomerases and yeast allows a degree of genetic manipulation that is unmatched by any other eukaryotic system. The proposed studies will take advantage of several recently developed assay systems. The mechanism by which abasic sites enhance enzyme-mediated DNA cleavage will be analyzed by a variety of biochemical, kinetic, and genetic approaches. The recognition of abasic sites by topoisomerase II will be characterized by determining how the enzyme scans DNA for this lesion and by defining the structural features of this lesion that are required to alter enzyme activity. Relationships between abasic sites and anticancer agents will be defined by mapping the interaction domain of these lesions on topoisomerase II relative to that of drugs. Finally, the physiological role of abasic sites as topoisomerase II poisons will be characterized by determining whether these lesions induce topoisomerase II-mediated cell death or mutagenesis.