REGULATION OF TRYPANOSOME DNA REPLICATION

动质体鞭毛虫代表了一个独特的机会 研究真核DNA复制机制及其协调 与细胞周期有关。这些寄生原生动物的特征是 存在一个包含大约5，000个 质粒样DNA（微环）的拷贝连接成一个巨大的 网络连同25-50个拷贝的线粒体DNA（大环）。 动质体DNA（kDNA）约占细胞DNA的25%。 与哺乳动物线粒体DNA不同， 核S阶段。 本提案的长期目标是确定 kDNA复制的生化和酶机制 小环在锥虫Crithidia fasciculata和鉴定 微环复制与细胞协调机制的特征 周期 动质体相关蛋白参与启动和可能的 微环复制中的预起始步骤将被纯化并用于 补充粗动基体提取物以形成微环， 依赖的DNA复制系统 参与RNA引发的酶 在小环复制起点处的起始物将被纯化并用于 确定引发的机制和位点。 抗体 纯化的复制蛋白将用于定位单个蛋白质 在动基体内，并确定可能的关联， 具有两个含拓扑异构酶的额外复制蛋白 在以前的研究中，位于kDNA外围的结构 网络 编码动质体相关拓扑异构酶的基因将 的RNA和蛋白质产物的表达。 基因将在细胞周期的不同时间被确定。 蛋白质- 将寻找拓扑异构酶的蛋白质相互作用，如果发现， 用于鉴定和纯化可能的相关蛋白质， 复制装置 该提案解决了现代细胞生物学中的基本问题， 因此，它与正常和异常的理解有关。 细胞和组织的生长和发育。 此外，独特的功能 动质体DNA复制机制的研究可能揭示合适的靶点 用于开发新的化疗药物， 锥虫病和利什曼病。

The kinetoplastid flagellates represent a unique opportunity for investigating eukaryotic DNA replication mechanisms and their coordination with the cell cycle. These parasitic protozoa are distinguished by the presence of a single mitochondrion which contains approximately 5,000 copies of a plasmid-like DNA (minicircles) catenated into an enormous network together with 25-50 copies of the mitochondrial DNA (maxicircles). The kinetoplast DNA (kDNA) accounts for approximately 25% of the cellular DNA and, unlike mammalian mitochndrial DNA, replicates coordinately with the nuclear S phase. The long-term goal of this proposal is to determine the biochemical and enzymatic mechanisms os replication of the kDNA minicircles in the trypanosomatid Crithidia fasciculata and to identify features of the mechanism coordinating minicircle replication with the cell cycle. Kinetoplast-associated proteins involved in initiation and possible pre-initiation steps in minicircle replication will be purified and used to supplement crude kinetoplast extracts in order to develop a minicircle- dependent DNA replication system. The enzymes involved in RNA-primed initiation at minicircle replication origins will be purified and used to determine the mechanism and the sites of initiation. Antibodies against purified replication proteins will be used to localize individual proteins within the kinetoplast and to determine the possible association of additional replication proteins with the two topoisomerase-containing structures located, in previous studies, at the periphery of the kDNA network. The gene encoding the kinetoplast-associated topoisomerase will be cloned and sequenced and the expression of RNA and protein products of the gene will be determined at various times in the cell cycle. Protein- protein interactions of the topoisomerase will be sought and, if found, exploited to identify and purify possible associated proteins of the replication apparatus. This proposal addresses fundamental questions in modern cell biology and therefore is relevant to an understanding of both normal and abnormal growth and development of cells and tissues. In addition, unique features of the kinetoplast DNA replication mechanism may reveal appropriate targets for the development of new chemotherapeutic drugs for the treatment of trypanosomiasis and leishmaniasis.