Regulation of Trypanosome DNA Replication

描述(申请人提供)：布氏锥虫，非洲锥虫，是一种原虫寄生虫，可引起人类昏睡病和家畜的类似疾病。本研究旨在了解调控非洲锥虫线粒体DNA复制的分子机制。长期以来，锥虫一直被认为是线粒体DNA的一种特征形式，称为动泡体DNA(KDNA)。KDNA由几千个小环和20-30个大环组成的拓扑连锁网络组成。大环编码线粒体核糖体RNA和氧化代谢基因。一般来说，DNA复制是在起始水平上调节的，而不是在伸长水平上调节的。布鲁氏毛滴虫线粒体DNA启动酶基因PrI-1的新发现及其作为重组蛋白的表达，为建立研究小环和上环DNA复制启动的体外系统奠定了基础。以前对kDNA复制的研究依赖于强大的间接方法，如RNA干扰。使用纯化的蛋白质研究kDNA复制以前是不可能的，因为DNA聚合酶的体外合成需要一个引物，通常是RNA。线粒体DNA引物酶的可获得性将构成使用包含小环或上环复制起点的重组蛋白和质粒模板的体外系统的基础。基于基因表达的免疫定位和/或RNA干扰，已经鉴定了许多kDNA复制蛋白。特别是，UMSBP和P38蛋白被认为在小环复制起始处直接启动DNA合成。这两种蛋白都将以重组蛋白的形式表达，并将作为初始微环复制系统的组成部分。这项研究将确定启动DNA合成所需的酶和蛋白质的两条链中的每一条在小环和上环起源，并将确定准确的DNA链起始位置。其他复制蛋白将根据它们与PrI 1或PrI 2的相互作用进行鉴定，PrI 1或PrI 2是另一种假定的线粒体DNA启动酶，也将被研究。已发现PrI 1和PrI 2对小环和上环复制都是必需的。最后，根据连接酶的过度表达和对所产生的kDNA网络的分析，将确定必要的线粒体DNA连接酶在kDNA复制中的作用。总体而言，该项目将揭示调节这种早期分化的人类寄生虫线粒体基因组复制的机制的分子细节，并可能为药物开发指明可能的新靶点。 与公共卫生相关：非洲锥虫是原生动物寄生虫，可引起人类昏睡病和家畜的类似疾病。这项研究旨在确定复制寄生虫线粒体遗传信息的分子机制，线粒体是细胞氧化代谢的部位。这项研究中确定的基因和蛋白质将为寄生虫病的诊断和治疗提供可能的新方法。

DESCRIPTION (provided by applicant): Trypanosoma brucei, the African trypanosome, is a protozoan parasite that causes sleeping sickness in humans and a similar disease in livestock. This research aims to understand the molecular mechanisms involved in regulating replication of the mitochondrial DNA of African trypanosomes. Trypanosomes have long been known for a characteristic form of mitochondrial DNA termed kinetoplast DNA (kDNA). kDNA consists of a topologically interlocked network of several thousand minicircles and 20-30 maxicircles. The maxicircles encode mitochondrial ribosomal RNAs and genes for oxidative metabolism. In general, DNA replication is regulated at the level of initiation, not elongation. The recent discovery of T. brucei mitochondrial DNA primase gene PRI 1 and its expression as a recombinant protein will permit the development of an in vitro system for investigating the initiation of minicircle and maxicircle DNA replication. Prior studies of kDNA replication have relied on powerful, but indirect, methods such as RNA interference. Studies of kDNA replication using purified proteins has not been possible previously since in vitro synthesis by a DNA polymerase requires a primer, which is usually RNA. The availability of a mitochondrial DNA primase will form the basis of an in vitro system using recombinant proteins and plasmid templates containing minicircle or maxicircle replication origins. Many kDNA replication proteins have been identified based on immunolocalization and/or RNA interference of gene expression. In particular, the proteins UMSBP and P38 have been implicated in playing a direct role in initiation of DNA synthesis at the minicircle replication origin. Both proteins will be expressed as recombinant proteins and will be available as components of the initial minicircle replication system. This study will identify the enzymes and proteins required for initiating DNA synthesis of each of the two strands on minicircle and maxicircle origins and will identify precise sites of DNA strand initiation. Additional replication proteins will be identified based on their interaction with PRI 1 or PRI 2, a second putative mitochondrial DNA primase that will also be investigated. Both PRI 1 and PRI 2 have been found to be essential for both minicircle and maxicircle replication. Finally, the role of an essential mitochondrial DNA ligase in kDNA duplication will be determined based on overexpression of the ligase and analysis of resulting kDNA networks. Overall, this project will reveal molecular details of the mechanisms that regulate replication of the mitochondrial genome of this early diverging human parasite and will likely indicate possible new targets for drug development. PUBLIC HEALTH RELEVANCE: African trypanosomes are protozoan parasites that cause sleeping sickness in humans and a similar disease in livestock. This study aims to identify the molecular mechanisms involved in replicating the genetic information of the parasite's mitochondrion, the site of cellular oxidative metabolism. Genes and proteins identified in this study will provide possible new approaches to diagnosis and treatment of parasitic diseases.