Trypanosomatid Mitochondrial DNA Polymerases

锥虫线粒体 DNA 聚合酶

• 批准号: 6957756
• 负责人: Michele M Klingbeil
• 金额: $ 15.65万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6957756
• 关键词: DNA directed DNA polymerase; DNA replication; RNA interference; SDS polyacrylamide gel electrophoresis; Trypanosoma brucei; cell line; enzyme activity; fluorescent in situ hybridization; gene induction /repression; immunoprecipitation; intracellular parasitism; mass spectrometry; mitochondrial DNA; polymerase chain reaction; protein biosynthesis; protein localization; protein protein interaction; yeast two hybrid system

DESCRIPTION (provided by the applicant): Trypanosoma brucei is a protist parasite that causes fatal African sleeping sickness in humans, and a related disease in cattle called nagana. Current drug treatments are inadequate, often toxic, and no vaccine is available. T. brucei and other trypanosomatids are distinguished from all other eukaryotes by an unusual mitochondrial genome that is a catenated network of maxicircles (tens) and minicircles (thousands) called kinetoplast DNA (kDNA). No replicative mitochondrial DNA polymerase (pol) has been identified from trypanosomatids. However, we have identified four T. brucei mitochondrial proteins (Tbpol IA, IB, IC and ID) related to bacterial pol I. This is unique because yeast and vertebrates utilize just a single mitochondrial replicative enzyme, pol gamma. Our long term goal is to understand the particular roles these multiple mitochondrial pols play in maintaining kDNA which is essential for parasite viability. The specific hypothesis is that multiple pol I-like proteins are the kDNA replicative enzymes. We base our hypothesis on the following observations: 1) pol IB and IC localize near the kDNA where minicircle replication initiates, 2) single gene RNA interference (RNAi) experiements indicate that both are required for normal growth of procyclic parasites and 3) silencing of IB or IC results in kDNA network shrinkage and an accumulation of minicircle replication intermediates, but not a complete block in minicircle replication. We will use a multifaceted approach including genetics, molecular biology, and biochemistry to identify replicative properties of the mitochondrial pol I-like proteins. Our three specific aims are focused on pol IB, IC and ID Aim 1. Clarify the cellular role(s) of the pol I-like proteins by using various RNAi cell lines including single, and multiple knockdown constructs. Aim 2. Characterize the enzymatic properties of recombinant pol I-like proteins, including processivity, fidelity, and inhibitor studies. Aim 3. Identify associated proteins that contribute to the specific pol cellular roles. Completion of these specific aims will provide important new information about kDNA replication, a process conserved in trypanosomatids, and possibly the opportunity to exploit differences between trypanosomatid and mammalian mitochondrial DNA pols to develop new strategies for drug treatment. The T. brucei pol I-like protein family also offers the unique opportunity to uncover insights into the evolutionary strategies used to produce a replicative polymerase.

描述（由申请人提供）：布鲁氏锥虫是一种原生寄生虫，可引起人类致命的非洲昏睡病，以及牛的一种相关疾病，称为纳迦纳病。目前的药物治疗是不充分的，往往是有毒的，而且没有疫苗可用。布鲁氏锥虫和其他锥虫与所有其他真核生物不同的是，线粒体基因组是一个由大圆环（数十个）和小圆环（数千个）组成的链状网络，称为着丝体DNA （kDNA）。在锥虫中未发现复制线粒体DNA聚合酶（pol）。然而，我们已经确定了四种与细菌pol i相关的布鲁氏T.线粒体蛋白（Tbpol IA， IB， IC和ID）。这是独一无二的，因为酵母和脊椎动物只利用一种线粒体复制酶，pol γ。我们的长期目标是了解这些多线粒体pol在维持对寄生虫生存至关重要的kDNA中所起的特殊作用。具体的假设是多种pol - i样蛋白是kDNA复制酶。我们的假设基于以下观察结果：1)pol IB和IC定位于启动小环复制的kDNA附近；2)单基因RNA干扰（RNAi）实验表明，它们是原环寄生虫正常生长所必需的；3)IB或IC的沉默导致kDNA网络收缩和小环复制中间物的积累，但不会完全阻断小环复制。我们将使用多方面的方法，包括遗传学，分子生物学和生物化学来鉴定线粒体pol - i样蛋白的复制特性。我们的三个具体目标集中在IB， IC和ID目标1。通过使用不同的RNAi细胞系，包括单个和多个敲低结构，阐明pol - i样蛋白的细胞作用。目标2。表征重组pol i样蛋白的酶特性，包括加工性、保真度和抑制剂研究。目标3。鉴定参与特定pol细胞作用的相关蛋白。这些特定目标的完成将提供关于锥虫中保守的kDNA复制过程的重要新信息，并可能有机会利用锥虫和哺乳动物线粒体DNA pols之间的差异来开发新的药物治疗策略。布鲁氏T. pori样蛋白家族也提供了一个独特的机会来揭示用于产生复制聚合酶的进化策略。