ROLE OF TOPOISOMERASE I IN DNA REPAIR AND CHEMOTHERAPY

拓扑异构酶 I 在 DNA 修复和化疗中的作用

• 批准号: 3195203
• 负责人: ARTHUR B PARDEE
• 金额: $ 19.22万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-03-01 至 1994-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3195203
• 关键词: DNA repair; DNA topoisomerases; antineoplastics; camptothecin; enzyme inhibitors; enzyme mechanism; enzyme substrate; mutant; naphthoquinones; protein purification; transfection

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.

我们已经报道，β-拉帕醌，一种植物生物碱， 增加了多种DNA损伤剂对哺乳动物的致死率 戴尔斯 该药物被证明可以增加DNA的双链断裂， 损伤的细胞，并降低单个细胞的消失率， 链断裂。 它也增加了纯化的蛋白质的解旋活性。 拓扑异构酶T我们从这些结果推测拓扑异构酶I 参与修复受损的DNA 与此相一致，我们发现 喜树碱，一种拓扑异构酶I的特异性抑制剂， 细胞中DNA损伤的致命性。 我们的三个具体目标是：（1） 为了验证拓扑异构酶I参与切除的假设， 修复受损的DNA (2)为了确定喜树碱或 β-拉帕酮通过以下方式增强了DNA损伤剂的致死性 改变拓扑异构酶I活性。(3)与上述致死率相关 随着单链DNA损伤向双链DNA损伤的转化增加， 搁浅断裂。 更详细描述的实验方法 下面，取决于建立系统的三个主要变化， 研究拓扑异构酶I，即（A）使用带缺口的，随机的 与完整的DNA相比，受损的DNA作为酶的底物。 (B)为了确定这两种药物对受损细胞和 纯化系统中的拓扑异构酶1活性。(C)使…突变 拓扑异构酶I，并与它们进行比较， 野生型酶，无论是在体内还是在纯化的制剂中。 这项研究提出-在此赠款是为了填补几个 我们知识的差距。 第一个是关于拓扑异构酶I的作用， DNA修复DNA修复过程是如何依赖于 初始病变？拓扑异构酶I是否参与了各种 DNA损伤？从实践的角度来看， 拓扑异构酶I抑制剂与DNA损伤剂可能导致增强的 抗肿瘤活性，同时降低宿主毒性，从而改善 已建立的抗肿瘤药物的治疗指数。