DNA LESIONS AS ENDOGENOUS TOPOISOMERASE POISONS

DNA 损伤作为内源性拓扑异构酶毒物

• 批准号: 2910216
• 负责人: NEIL OSHEROFF
• 金额: $ 21.58万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910216
• 关键词: DNA damage; DNA repair; DNA replication; DNA topoisomerases; Drosophilidae; Saccharomyces cerevisiae; active sites; alternatives to animals in research; antineoplastics; cell death; chemical binding; chemical kinetics; cytotoxicity; drug adverse effect; enzyme induction /repression; enzyme mechanism; genetic mapping; isozymes; mutant; pharmacokinetics

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.

拓扑异构酶II是几个最活跃的细胞靶 目前用于治疗人类癌症的抗肿瘤剂。 这些药物通过独特的机制引起其细胞毒性作用。 相当 而不是通过抑制酶，抗癌的催化活性来起作用 药物急剧增加了共价拓扑异构酶II-裂解的水平 正常但短暂的DNA复合物中间 酶的催化循环。 当产生的酶相关时 基因组中存在双链DNA断裂 浓度，它们会产生突变，染色体畸变和 在极端条件下，细胞死亡。 因此，抗癌药毒 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 这些药物代表细胞外源对应物的可能性 诱导DNA重组，诱变或细胞死亡的成分 途径。 该实验室的先前结果表明Abasic 位点，是DNA中最常见的病变，并产生 通过无数的DNA破坏事件，刺激拓扑异构酶II介导 双链DNA裂解。 这种切割刺激的功效 类似于依托泊苷的（最广泛的处方 临床用途中的抗癌剂）。 但是，无聊地点的效力 大约比药物大约2,000倍。 因此， 该提案的最终目标是定义Abasic之间的互动 位点和II型酶，并确定无碱性位点是否 充当内源性拓扑异构酶II毒物。 更具体地说，是 该提案的目的是1）确定abasic的机制 位点增强了拓扑异构酶II介导的裂解，2）描绘 酶识别可碱位点的机制，3）定义 无聊站点的作用机理与 抗癌药物和4）确定无碱性位点是否起作用 拓扑异构酶II毒药在体内。 本研究产生的信息 应该大大提高我们对拓扑异构酶II靶向的理解 代理刺激酶介导的DNA裂解，并最终引起细胞 死亡。 果蝇和酵母将作为此的主要研究模型 学习。 果蝇和酵母酶是最有特征的 II型拓扑异构酶和酵母允许一定程度的遗传操作 任何其他真核系统都无法比拟。 提出的研究 将利用几个最近开发的测定系统。 这 无碱性位点增强酶介导的DNA裂解的机制将 可以通过多种生化，动力学和遗传方法来分析。 拓扑异构酶II对无碱性位点的识别将被表征 通过确定酶如何扫描该病变的DNA并定义 改变酶所需的这种病变的结构特征 活动。 无雅站点与抗癌代理之间的关系将 通过映射这些病变的相互作用域来定义 拓扑异构酶II相对于药物。 最后，生理学 无碱性站点作为拓扑异构酶II毒物的作用将以以下特征 确定这些病变是否诱导拓扑异构酶II介导的细胞 死亡或诱变。