Regulation of Trypanosome DNA Replication

锥虫 DNA 复制的调控

• 批准号: 8293217
• 负责人: DAN S RAY
• 金额: $ 38.12万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8293217
• 关键词: Address; African; African Trypanosomiasis; Binding Proteins; Biochemical; Cell Respiration; Characteristics; DNA; DNA Ligases; DNA Primase; DNA Sequence; DNA Structure; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Development; Diagnosis; Disease; Dissection; Electron Microscopy; Enzymes; Event; Foundations; Gene Expression; Gene Proteins; Genes; Genetic; Genomics; Goals; Human; In Vitro; Kinetoplast DNA; Ligase; Livestock; MAPK14 gene; Maps; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial RNA; Molecular; Parasites; Parasitic Diseases; Plasmids; Play; Proteins; Proteomics; RNA; RNA Interference; Reaction; Recombinant Proteins; Recombinants; Regulation; Replication Origin; Research; Role; S Phase; Site; Structure; System; Technology; Transcript; Trypanosoma; Trypanosoma brucei brucei; base; drug development; helicase; in vivo; kDNA Minicircles; mitochondrial genome; novel strategies; overexpression; protein function; protein protein interaction; protein purification; public health relevance; rRNA Genes; vector; viral DNA

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.

描述（由申请人提供）：非洲锥虫锥虫Brucei是一种原生动物寄生虫，会引起人类睡眠的疾病和类似的牲畜疾病。这项研究旨在了解调节非洲锥虫线粒体DNA复制的分子机制。长期以来，以一种称为动力学DNA（kDNA）的线粒体DNA的特征形式而闻名。 kDNA由一个拓扑互锁的网络组成，该网络由数千个微圆和20-30个最大环。 Maxicircles编码线粒体核糖体RNA和氧化代谢的基因。通常，DNA复制受到启动水平，而不是伸长的调节。最近发现的Brucei线粒体DNA Primase基因pri 1及其作为重组蛋白的表达将允许开发一种体外系统，以研究小节和Maxicircle DNA复制的启动。 kDNA复制的先前研究取决于RNA干扰等强大但间接的方法。以前不可能使用纯化蛋白质对KDNA复制进行研究，因为DNA聚合酶在体外合成需要底漆，通常是RNA。线粒体DNA起初的可用性将构成使用重组蛋白和含有小圆或最大圆形复制起源的质粒模板的体外系统的基础。基于免疫定位和/或RNA干扰，已经鉴定出许多KDNA复制蛋白。特别是，蛋白质UMSBP和p38与在微小复制起源处的DNA合成起着直接作用有关。两种蛋白质都将作为重组蛋白表示，并作为初始微圆复制系统的组成部分提供。这项研究将确定在微米和最大圆形上启动两个链的DNA合成所需的酶和蛋白质，并将确定DNA链启动的精确位点。将根据其与PRI 1或PRI 2的相互作用（第二次推定的线粒体DNA原始酶）的相互作用来鉴定其他复制蛋白。 PRI 1和PRI 2都被发现对于微小和最大圆的复制都是必不可少的。最后，将基于连接酶的过表达和所得kDNA网络的分析确定基本线粒体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不同人类寄生虫的线粒体基因组复制机制的分子细节，并可能表明可能有新的药物开发目标。 公共卫生相关性：非洲锥虫是原生动物的寄生虫，在人类中引起睡眠和类似的疾病。这项研究旨在确定复制寄生虫线粒体（细胞氧化代谢部位）的遗传信息所涉及的分子机制。这项研究中鉴定出的基因和蛋白质将为寄生疾病的诊断和治疗提供新的方法。