Trypanosomatid Mitochondrial DNA Polymerases

描述（由申请方提供）：布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nagana。目前的药物治疗是不够的，往往有毒，没有疫苗可用。T.布氏锥虫和其它锥虫与所有其它真核生物的区别在于一种不寻常的线粒体基因组，该线粒体基因组是称为动基体DNA（kDNA）的大环（数十个）和小环（数千个）的链状网络。没有复制型线粒体DNA聚合酶（pol）已确定锥虫。但是，我们已经确定了四个T。与细菌pol I相关的布氏杆菌线粒体蛋白（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单一的线粒体复制酶，聚γ。我们的长期目标是了解这些多个线粒体pols在维持kDNA中发挥的特殊作用，这对寄生虫的生存能力至关重要。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：1）pol IB和IC定位在小环复制起始的kDNA附近，2）单基因RNA干扰（RNAi）实验表明这两者都是前环寄生虫正常生长所需的，3）IB或IC的沉默导致kDNA网络收缩和小环复制中间体的积累，但不是小环复制的完全阻断。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，以确定线粒体pol I样蛋白的复制特性。我们的三个具体目标集中在pol IB，IC和ID Aim 1。通过使用各种RNAi细胞系（包括单个和多个敲低构建体）阐明pol I样蛋白的细胞作用。目标2.表征重组pol I样蛋白的酶性质，包括持续合成能力、保真度和抑制剂研究。目标3。鉴定有助于特定pol细胞作用的相关蛋白质。完成这些特定的目标将提供重要的新信息kDNA复制，锥虫中保守的过程，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，开发新的药物治疗策略。霸王布氏杆菌pol I样蛋白家族还提供了独特的机会来揭示用于产生复制聚合酶的进化策略。

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.