ROLE OF TOPOISOMERASE I IN DNA REPAIR AND CHEMOTHERAPY

我们已经报道了β-拉帕酮，一种植物生物碱，极大地 增加各种DNA损伤剂对哺乳动物的杀伤力 戴尔斯。这种药物被证明增加了DNA的双链断裂 损伤的细胞，降低单个细胞的消失率 链子断了。它还增加了纯化的解离活性。 拓扑异构酶T。我们从这些结果推测，拓扑异构酶I 参与了受损DNA的修复。与此一致的是，我们展示了 喜树碱，一种拓扑异构酶I的特异性抑制剂，也增强了 细胞中DNA损伤的致命性。我们的三个具体目标是：(一) 为了验证拓扑异构酶I参与切除的假设 修复受损的DNA。(2)确定喜树碱或 β-拉帕酮通过以下途径提高抗肿瘤DNA损伤剂的杀伤力 改变拓扑异构酶I的活性。(3)将上述致命性关联起来 随着单链DNA损伤转化率增加到双倍 搁浅的休息。更详细地描述了实验方法 以下，取决于已建立的系统的三个主要变体 研究拓扑异构酶I，即(A)利用缺口缺口，并随机 与完整的DNA相比，破坏了作为酶底物的DNA。 (B)确定这两种药物对受损细胞和 纯化体系中拓扑异构酶1的活性。(C)进行突变 拓扑异构酶I及其与上述现象的对比研究 对于野生型酶，在体内和纯化制剂中都是如此。 这项拨款中提出的研究旨在填补几个 我们知识上的鸿沟。第一个涉及拓扑异构酶I在细胞内的作用。 DNA修复。DNA修复过程如何依赖于 最初的损伤？拓扑异构酶I是否参与了各种神经细胞的修复 DNA损伤？从实践的角度来看， 拓扑异构酶I抑制剂与DNA损伤剂联合使用可能导致增强 抗肿瘤活性，减少宿主毒性，从而改善 已有抗肿瘤药物的治疗指标。

We have reported that beta-lapachone, a plant alkaloid, greatly increases the lethality of a variety of DNA damaging agents to mammalian dells. The drug was shown to increase the double strand breaks in DNA of the damaged cells, and to decrease the rate of disappearance of single strand breaks. It also-increased the unwinding activity of purified topoisomerase T. We hypothesized from these results that topoisomerase I is involved in repair of damaged DNA. Consistent with this, we showed that camptothecin, a specific inhibitor of topoisomerase I, also enhanced the lethality of DNA damage in cells. Our three specific aims are: (1) To test the hypothesis that topoisomerase I is involved in excision repair of damaged DNA. (2) To determine whether camptothecin or beta-lapachone enhance lethality of antineoplastic DNA damaging agents by altering topoisomerase I activity. (3) To correlate the above lethality with the increased conversion of single stranded DNA damage to double stranded breaks. The experimental approach, described in more detail below, depends upon three principle variations of established systems for studying topoisomerase I, namely (A) To use nicked gapped, and randomly damaged DNA's as substrates for the enzyme in comparison with intact DNA. (B) To determine the effects of the two drugs on both damaged cells and topoisomerase 1 activity in purified systems. (C) To make mutations of topoisomerase I and investigate the above phenomena with them as compared to the wild type enzyme, both in vivo and in purified preparations. The research proposed- in-this grant is intended to fill several gaps in our knowledge. The first concerns the role of topoisomerase I in DNA repair. How are DNA repair processes dependent upon the type of initial lesion? Is topoisomerase I involved in the repair of all kinds of DNA lesions? From a practical point of view, the combination of topoisomerase I inhibitors with DNA damaging agents may lead to enhanced antitumor activity with diminished host toxicity, and thereby improve the therapeutic indices of established antineoplastic agents.