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发生的详细机制酶结构和功能以及mtDNA的水平组织和表达。该提议的具体目标是开始阐明DNA拓扑异构酶在 mtDNA的复制和代谢。特别是 ATP与核和线粒体I型的相互作用拓扑异构酶将进行研究。已经表明与ATP无关的I型拓扑异构酶实际上被抑制 ATP。由于已经观察到这种效果白血病，HeLa和小牛胸腺细胞，我们假设这是 TOPO I活性的ATP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该法规的机制使用体内和体外研究将被阐明。这些酶在线粒体DNA复制中的作用和代谢将通过研究拓扑异构酶I和II与线粒体基因组在体内和体外。这些研究将利用几种抗肿瘤药物已显示出特异性干扰拓扑异构酶 - 催化链破裂和重新加入过程，以便存在蛋白质变性剂，DNA裂解与拓扑异构酶共同附着在DNA的末端分段。特别是，Camptothecin将用于探测 TOPO I-DNA相互作用和4'-（9- acridinylamin） - 甲烷硫-M-苯甲酸硫化物（MAMSA）和附生噬蛋白VP-16 VM-26将用于证明TOPO II-DNA相互作用。这些研究应阐明一些参与的细节 TOPO I和TOPO II在复制和表达中线粒体基因组。拓扑异构酶活性的调节具有一般意义但是，正如提案中所详述的，维护适当染色体和外染色体DNA超固醇是在致癌，诱变和肿瘤发生以及因此，这些研究与癌症病因的问题有关和治疗。