TOPOISOMERASE I STRUCTURE AND REGULATION

这项建议旨在加强目前对 DNA拓扑异构酶I与抑制性抗肿瘤的相互作用 毒品。对两国之间相互作用的因素的了解 酶和抑制剂，如喜树碱，将有益于 这些药物的临床应用。 在Aim L中提出的研究是对先前工作的扩展，其中 发现了喜树碱耐药的新机制。 涉及拓扑异构酶I第361位的点突变。 初步研究表明，这种突变对 喜树碱对酶的影响，以及额外的突变 这一区域损害了Topo 1催化循环的DNA切割步骤。 这些发现将通过使用 进行寡核苷酸裂解分析和凝胶位移分析 细菌表达的突变蛋白。更多的研究将评估 这些突变对喜树碱结合的影响以及对 非喜树碱类拓扑异构酶T毒物的抑制活性 Hoechst染料33342和放线菌素D。 由于细胞对拓扑异构酶I抑制的反应可能是 受与酶相关的蛋白质的影响，特异性目标2是 用来识别这类蛋白质。利用亲和力进行的初步工作 用固定的拓扑异构酶I融合蛋白进行层析 鉴定了几个可能的结合蛋白，显微测序已经 鉴定出其中一种蛋白质是核仁蛋白。这一目标的研究是 旨在证实这一发现并定位结合到 拓扑异构酶I通过使用缺失突变体。更多的研究将 尝试鉴定其他结合蛋白并研究其影响 这些蛋白质对拓扑异构酶I功能和相互作用的影响 拓扑异构酶I和喜树碱等抑制剂之间的关系。

This proposal is designed to enhance the current understanding of the interaction between DNA topoisomerase I and inhibitory antineoplastic drugs. Knowledge of factors involved in the interaction between the enzyme and inhibitors such as camptothecin would be of benefit in the clinical application of these agents. The studies proposed in Aim l are an extension of prior work in which a novel mechanism of cellular resistance to camptothecin was identified, involving a point mutation at position 361 in topoisomerase I. Preliminary studies suggest that this mutation confers resistance to the effects of camptothecin on the enzyme, and that additional mutations in this region impair the DNA cleavage step of the topo 1 catalytic cycle. These findings will be confirmed and extended by the use of oligonucleotide cleavage assays and gelshift assays performed with bacterially expressed mutant proteins. Additional studies will evaluate the effects of these mutations on camptothecin binding and on the inhibitory activity of noncamptothecin topoisomerase T poisons such as Hoechst dye 33342 and actinomycin D. Since the cellular response to topoisomerase I inhibition may be influenced by proteins associating with the enzyme, Specific Aim 2 is designed to identify such proteins. Preliminary work using affinity chromatography with an immobilized topoisomerase I fusion protein has identified several putative binding proteins, and microsequencing has identified one of these proteins as nucleolin. Studies in this Aim are designed to confirm this finding and to localize the site of binding to topoisomerase I by the use of deletion mutants. Additional studies will attempt to identify other binding proteins and to investigate the effects of these proteins on topoisomerase I function and on the interaction between topoisomerase I and inhibitors such as camptothecin.