EUKARYOTIC TOPOISOMERASE I

拟议研究的长期目标是详细的分子 理解人的结构与 拓扑异构酶I及其在细胞DNA旋转中的作用 复制、RNA合成、染色质组装和重组。 基于 关于即将到来的晶体结构和使用定点 诱变，我们将研究化学缺口关闭 反应，结构决定因素的核苷酸偏见，在 切口位点上游的四个碱基对，接头的作用 区域的拓扑异构化，和有效的行动方式 拓扑异构酶I毒药喜树碱 生物化学和 结构分析将用于区分股- 通道和自由旋转模型的弛豫反应。 的 人磷酸二酯酶，被认为是重要的 拓扑异构酶I介导的损伤的修复将被纯化和表征。 编码磷酸二酯酶的cDNA将被克隆和测序， 这种酶可能与人类癌症有关， 评价对喜树碱的敏感性。 由于细胞毒性 在细胞培养物中已经显示与喜树碱诱导的 给药后的损害，将开发一种测定方法， 为了定量拓扑异构酶I-DNA共价加合物的水平， 细胞 这样的分析方法可能是有用的测试新的喜树碱 衍生物和评估个体患者肿瘤是否会 对药物治疗有反应。 最后，我们最近发现， 亲脂性胺增强喜树碱的细胞毒性， 追求了解效果的基础，并确定是否 联合用药方案可能具有治疗潜力， 抗癌治疗

The long range goal of the proposed research is a detailed molecular understanding of the relationship between the structure of human topoisomerase I and its role in the cell of providing swivels for DNA replication, RNA synthesis, chromatin assembly and recombination. Based on the impending crystal structure and the use of site-directed mutagenesis, we will investigate the chemistry of the nicking-closing reaction, the structural determinants for the nucleotide bias in the four base pairs upstream of the nicking site, the role of the linker region in topoisomerization, and the mode of action of the potent topoisomerase I poison, camptothecin. A combination of biochemistry and structural analyses will be used to distinguish between the strand- passage and the free-rotation models for the relaxation reaction. The human phosphodiesterase that is believed to be the important for the repair of topo I-mediated damage will be purified and characterized. The cDNA encoding the phosphodiesterase will be cloned and sequenced and the possible involvement of the enzyme in human cancers and susceptibility to camptothecin will be evaluated. Since cytotoxicity in cell culture has been shown to correlate with camptothecin-induced damage following administration of the drug, an assay will be developed to quantitate the level of topoisomerase I-DNA covalent adducts in the cells. Such an assay could be useful in testing new camptothecin derivatives and in evaluating whether individual patient tumors will respond to drug treatment. Finally, our recent discovery that lipophilic amines enhance the cytotoxicity of camptothecin will be pursued to understand the basis of the effect and to determine whether a combination drug regimen might have therapeutic potential in anticancer treatment.