Trypanosomatid Mitochondrial DNA Polymerases

锥虫线粒体 DNA 聚合酶

• 批准号: 7104395
• 负责人: Michele M Klingbeil
• 金额: $ 30.62万
• 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/7104395
• 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，可在人类身上引起致命的非洲昏睡病，并在牛身上引起一种相关的疾病，称为nagana。目前的药物治疗不充分，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的不同之处在于一个不同寻常的线粒体基因组，它是一个由上环(数十个)和小环(数千个)组成的连接网络，称为动泡体DNA(KDNA)。在锥虫体内未发现复制型线粒体DNA聚合酶。然而，我们已经鉴定出四种与细菌polI相关的布鲁氏锥虫线粒体蛋白(TbpolIA、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polGamma。我们的长期目标是了解这些多个线粒体极点在维持kDNA方面所起的特殊作用，kDNA是寄生虫生存所必需的。具体的假设是多个PolI样蛋白是kDNA复制酶。我们的假设基于以下观察：1)PolIB和IC位于启动小环复制的kDNA附近，2)单基因RNA干扰(RNAi)实验表明，两者都是原环寄生虫正常生长所必需的，3)沉默IB或IC会导致kDNA网络收缩和小环复制中间产物的积累，但不是完全阻止小环复制。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鉴定线粒体PolI样蛋白的复制特性。我们的三个特定目标集中在PolIB、IC和ID Aim 1。通过使用各种RNA干扰细胞系，包括单个和多个击倒结构，阐明PolI样蛋白的细胞作用(S)。目的2.对重组PolI样蛋白的酶性质进行表征，包括加工性能、保真度和抑制物研究。目的3.确定与特定POL细胞角色有关的相关蛋白质。这些特定目的的完成将提供关于kDNA复制的重要新信息，这一过程保存在锥虫中，并可能提供利用锥虫和哺乳动物线粒体DNA Pols之间的差异来开发药物治疗新策略的机会。布鲁氏锥虫PolI样蛋白家族也提供了独特的机会，可以深入了解用于产生复制聚合酶的进化策略。