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DNA TOPOISOMERASES--DNA INTERACTIONS AND AT CONTROL

DNA 拓扑异构酶——DNA 相互作用和控制

基本信息

批准号：
3292118
负责人：
FRANK J CASTORA
金额：
$ 9.29万
依托单位：
EASTERN VIRGINIA MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-09-01 至 1992-02-29
项目状态：
已结题

项目摘要

The long-term objectives of my research are to understand the detailed mechanisms of mitochondrial biogenesis at the level of enzyme structure and function as well as at the level of mtDNA organization and expression. The specific goal of this proposal is to begin to elucidate the roles of DNA topoisomerases in the replication and metabolism of mtDNA. In particular, the interaction of ATP with nuclear and mitochondrial type I topoisomerase will be investigated. It has been shown that the ATP-independent type I topoisomerase is actually inhibited by ATP. Since this effect has been observed with topos from human leukemia, HeLA and calf thymus cells, we hypothesize that this ATP regulation of topo I activity may be a general phenomenon in mammalian cells. The details of the nucleotide-enzyme interaction as well as the mechanism of this regulation using in vivo and in vitro studies will be elucidated. The role of these enzymes in mitochondrial DNA replication and metabolism will be defined by investigating the interactions of topoisomerase I and II with the mitochondrial genome in vivo and in vitro. These studies will utilize several antitumor drugs which have been shown to specifically interfere with the topoisomerase- catalyzed strand breaking and rejoining process such that, in the presence of a protein denaturant, DNA cleavage results with topoisomerase covalently attached to the end of the DNA fragment. In particular, camptothecin will be used to probe for topo I-DNA interactions, and 4'- (9-acridinylamin)-methane sulfon-m-anisidide (mAMSA) and the epipodophyllotoxins VP-16 and VM-26 will be used to prove topo II-DNA interactions. These studies should elucidate some of the details of the involvement of topo I and topo II in replication and expression of the mitochondrial genome. Regulation of topoisomerase activity is of general significance but as detailed in the proposal, the maintenance of proper chromosomal and extrachromosomal DNA superhelicity is important in carcinogenesis, mutagenesis and tumorigenesis and as such these studies are relevant to problems in cancer etiology and therapy.

我研究的长期目标是了解线粒体生物合成的详细机制，酶的结构和功能以及mtDNA水平组织和表达。这项建议的具体目标是开始阐明DNA拓扑异构酶在线粒体DNA的复制和代谢。特别是 ATP与核和线粒体I型相互作用将研究拓扑异构酶。已显示 ATP非依赖性I型拓扑异构酶实际上被 ATP 由于这种效应已被观察到与拓扑斯从人类白血病，HeLa和小牛胸腺细胞，我们假设， ATP对Topo I活性的调节可能是一种普遍现象，哺乳动物细胞核苷酸酶的细节相互作用以及这种调节机制，将阐明体内和体外研究。这些酶在线粒体DNA复制中的作用，代谢将通过研究以下因素的相互作用来定义：拓扑异构酶I和II与线粒体基因组在体内，体外这些研究将利用几种抗肿瘤药物，已经被证明可以特异性地干扰拓扑异构酶催化的链断裂和重新连接过程，蛋白质变性剂的存在下，DNA切割结果，拓扑异构酶共价连接到DNA的末端碎片特别地，喜树碱将用于探测 topo I-DNA相互作用和4 '-（9-吖啶胺）-甲烷磺基间茴香胺（mAMSA）和表鬼臼毒素VP-16 VM-26将用于证明拓扑II-DNA相互作用。这些研究应阐明参与的一些细节， topo I和topo II在复制和表达中的作用线粒体基因组拓扑异构酶活性的调控具有普遍意义但正如提案中详细说明的那样，染色体和染色体外DNA的超螺旋性是在致癌、诱变和肿瘤发生中重要，因此，这些研究与癌症病因学中的问题有关和心理治疗