Trypanosomatid Mitochondrial DNA Polymerases

锥虫线粒体 DNA 聚合酶

• 批准号: 7172328
• 负责人: Michele M Klingbeil
• 金额: $ 29.77万
• 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/7172328
• 关键词: African Trypanosomiasis; Biochemistry; Cattle; Cell Line; DNA biosynthesis; DNA-Directed DNA Polymerase; Disease; Enzymes; Eukaryota; Eukaryotic Cell; Genes; Genetic; Goals; Growth; Human; Kinetoplast DNA; Localized; Mitochondria; Mitochondrial DNA; Mitochondrial Proteins; Molecular Biology; Parasites; Pharmaceutical Preparations; Play; Polymerase; Process; Property; Protein Family; Proteins; RNA Interference; Recombinants; Role; Trypanosoma brucei brucei; Vaccines; Vertebrates; Yeasts; base; inhibitor/antagonist; insight; kDNA Minicircles; mitochondrial genome; nagana; pol genes

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）：Brucei锥虫是一种寄生虫，会导致人类致命的非洲卧铺疾病，并且是一种称为Nagana的牛的相关疾病。当前的药物治疗不足，经常有毒，没有疫苗可用。 T. brucei和其他锥形植物与所有其他真核生物通过不寻常的线粒体基因组区分开来，该基因组是最大圆形（TENS）的catenated网络（TENS）和称为动型成体DNA（KDNA）的小圆形网络（TENS）和小圆形网络（千圈）。未从锥虫剂中鉴定出复制性线粒体DNA聚合酶（POL）。但是，我们已经确定了与细菌pol有关的四种t. brucei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仅利用单个线粒体复制酶Pol Gamma。我们的长期目标是了解这些多个线粒体pol在维持kDNA中发挥作用的特定作用，这对于寄生虫可行性至关重要。特定的假设是多种pol I样蛋白是KDNA复制酶。 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微圆复制。我们将使用包括遗传学，分子生物学和生物化学在内的多方面方法来识别线粒体pol I样蛋白的复制特性。我们的三个特定目的集中在POL IB，IC和ID AIM 1上。通过使用各种RNAi细胞系，包括单个和多个敲低构建体，阐明了Pol I样蛋白的细胞作用。 AIM 2。表征重组pol I样蛋白的酶促特性，包括加工性，保真度和抑制剂研究。目标3。确定相关的蛋白质，这些蛋白有助于特定的pol细胞作用。这些特定目标的完成将提供有关KDNA复制的重要新信息，在锥形剂中保守的过程以及可能利用锥虫和哺乳动物线粒体DNA POR之间的差异的机会，以开发新的药物治疗策略。 T. brucei pol I样蛋白质家族还提供了独特的机会，可以洞悉用于生产复制聚合酶的进化策略。