DNA LESIONS AS ENDOGENOUS TOPOISOMERASE POISONS

拓扑异构酶II是几个最活跃的 目前用于治疗人类癌症的抗肿瘤药物。 这些药物通过一种独特的机制产生细胞毒作用。宁可 而不是通过抑制酶的催化活性来起作用，抗癌 药物显著增加共价拓扑异构酶II-裂解水平 DNA复合体是正常的，但转瞬即逝的，在 酶的催化循环。当产生的酶与 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，以及 在极端条件下，细胞死亡。因此，抗癌药物有毒 拓扑异构酶II，并将其从一种必需的酶转化为 生理毒素。 拓扑异构酶II毒物的不寻常作用机制提高了 这些药物可能代表细胞的外源性对应物 诱导DNA重组、突变或细胞死亡的成分 小路。这个实验室以前的研究结果表明， 部位，这是DNA中最常见的损伤，并产生 通过无数的DNA损伤事件，刺激拓扑异构酶II介导的 双链DNA裂解。这种卵裂刺激的效果 类似于依托泊苷(这是最广泛的处方 临床使用的抗癌剂)。然而，碱性位点的效力 大约是这种药物的2000倍。因此， 这项提议的最终目标是定义基础知识之间的互动 和II型酶，并确定是否有碱性位点 起到内源性拓扑异构酶II毒物的作用。更具体地说， 这项建议的目的是1)确定基本的 位点增强拓扑异构酶II介导的切割，2)描绘 酶识别基本位点的机制，3)定义 碱性位点的作用机制与它们的关系 抗癌药物，以及4)确定基本部位是否起作用 拓扑异构酶II在体内中毒。这项研究产生的信息 应该会大大增加我们对拓扑异构酶II靶向的理解 药物刺激酶介导的DNA切割，最终导致细胞 死亡。 果蝇和酵母将作为这方面的主要研究模型 学习。果蝇和酵母酶是最有特点的 II型拓扑异构酶和酵母允许一定程度的基因操作 这是任何其他真核系统都无法比拟的。建议进行的研究 将利用最近开发的几个检测系统。这个 碱性位点促进酶介导的DNA裂解的机制 通过各种生化、动力学和遗传学方法进行分析。 将表征拓扑异构酶II对碱性位点的识别 通过确定酶如何扫描DNA以发现这种损伤，并通过定义 改变酶所需的病变的结构特征 活动。碱性位点与抗癌药物之间的关系将 通过将这些病变的相互作用域映射到 拓扑异构酶II相对于药物而言。最后，生理学的 基本位置作为拓扑异构酶II毒物的作用将由 确定这些病变是否诱导拓扑异构酶II介导的细胞 死亡或突变。

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.