EUKARYOTIC TOPOISOMERASE I

这项拟议研究的长期目标是一种详细的分子 对人的结构关系的认识 拓扑异构酶I及其在DNA旋转提供细胞中的作用 复制、RNA合成、染色质组装和重组。基座 论临界性晶体结构及其定位法的使用 突变，我们将研究切开-关闭的化学 反应中，核苷酸偏向的结构决定因素 断裂位点上游的四个碱基对，即连接子的作用 在拓扑异构化中的区域和有效的作用方式 拓扑异构酶I中毒，喜树碱。生物化学和生物化学的结合 将使用结构分析来区分链- 松弛反应的通道模型和自由旋转模型。这个 人类磷酸二酯酶被认为是 Topo I介导的损伤的修复将被提纯和鉴定。 编码磷酸二酯酶的cDNA将被克隆和测序 该酶可能参与人类癌症和 将评估对喜树碱的敏感性。因为细胞毒性 已经证明在细胞培养中与喜树碱诱导的 给药后的损害，将建立一种分析方法 目的：定量测定大鼠脑内拓扑异构酶I-DNA共价加合物的水平。 细胞。这种检测方法可用于检测新的喜树碱。 在评估个别患者的肿瘤是否会 对药物治疗有反应。最后，我们最近的发现是 亲脂胺增强喜树碱的细胞毒作用 以了解影响的基础并确定是否 联合用药方案可能有治疗潜力 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.