Regulation of Trypanosome DNA Replication

描述（由申请方提供）：布氏锥虫，非洲锥虫，是一种原生动物寄生虫，可引起人类昏睡病和牲畜类似疾病。本研究旨在了解参与调节非洲锥虫线粒体DNA复制的分子机制。锥虫长期以来一直被认为是线粒体DNA的特征形式，称为动基体DNA（kDNA）。kDNA由数千个微环和20-30个大环的拓扑互锁网络组成。大环编码线粒体核糖体RNA和氧化代谢基因。一般来说，DNA复制是在起始水平而不是延伸水平受到调节。T.布氏杆菌线粒体DNA引物酶基因PRI 1及其重组蛋白的表达，将有助于建立一个研究小环和大环DNA复制起始的体外系统。先前对kDNA复制的研究依赖于强大但间接的方法，如RNA干扰。使用纯化的蛋白质研究kDNA复制以前是不可能的，因为通过DNA聚合酶的体外合成需要引物，其通常是RNA。线粒体DNA引发酶的可用性将形成使用含有小环或大环复制起点的重组蛋白和质粒模板的体外系统的基础。许多kDNA复制蛋白已被确定基于免疫定位和/或RNA干扰的基因表达。特别是，蛋白质UMSBP和P38已被牵连在小环复制起点的DNA合成起始中发挥直接作用。这两种蛋白质将表达为重组蛋白，并将作为初始小环复制系统的组分。本研究将确定启动小环和大环起源的两条链中的每一条的DNA合成所需的酶和蛋白质，并将确定DNA链起始的精确位点。其他复制蛋白将根据它们与PRI 1或PRI 2的相互作用来鉴定，PRI 1或PRI 2是第二种推定的线粒体DNA引发酶，也将进行研究。已经发现PRI 1和PRI 2对于小环和大环复制都是必不可少的。最后，一个必不可少的线粒体DNA连接酶在kDNA复制的作用将被确定的基础上过表达的连接酶和分析所产生的kDNA网络。总的来说，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机制的分子细节，并可能为药物开发提供新的靶点。 公共卫生相关性：非洲锥虫是一种原生动物寄生虫，可引起人类昏睡病和牲畜类似疾病。本研究的目的是确定参与复制的寄生虫的寄生虫，细胞氧化代谢的网站的遗传信息的分子机制。本研究所鉴定的基因和蛋白质将为寄生虫病的诊断和治疗提供新的途径。

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.