Correlating defects in mitochondrial DNA replication to physiology

将线粒体 DNA 复制缺陷与生理学相关联

• 批准号: 9206171
• 负责人: KENNETH ALLEN JOHNSON
• 金额: $ 35.94万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9206171
• 关键词: Active Sites; Address; Adverse effects; Affect; Age of Onset; Aging; Alleles; Alpers' Syndrome; Biochemical; Biochemistry; Biological Assay; Biological Models; Biomedical Research; Birth; Catalytic Domain; Cessation of life; Chemicals; Clinical; Complex; Coupled; DNA; DNA Sequence; DNA biosynthesis; DNA copy number; DNA-Directed DNA Polymerase; Data Analyses; Defect; Diagnosis; Discrimination; Disease; Enzymes; Excision; Exonuclease; Frequencies; Genes; Genetic; Goals; Growth; Guidelines; HIV Infections; Heritability; Heterozygote; Human; Impairment; In Vitro; Kinetics; Knowledge; Lead; Learning; Literature; Liver diseases; Malignant Neoplasms; Measurement; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Modeling; Molecular; Monitor; Muscle; Mutation; Myopathy; Nuclear; Nucleotides; Patients; Peripheral Nervous System Diseases; Phenotype; Physiological; Physiology; Point Mutation; Polymerase; Proteins; Publishing; Reaction; Refractory; Research; Role; SS DNA BP; Seizures; Severities; Severity of illness; Site-Directed Mutagenesis; Speed; Structure; Structure-Activity Relationship; Symptoms; Syndrome; System; Toxic effect; Virus Diseases; Work; Yeast Model System; Yeasts; accurate diagnosis; base; clinical effect; clinical phenotype; clinical predictors; effective therapy; helicase; human DNA; human disease; mutant; nucleoside analog; oxidative damage; polymerization; public health relevance; reconstitution; skeletal

 DESCRIPTION (provided by applicant): An important goal of biomedical research is to establish the molecular basis for disease so that more effective therapies can be devised. Relating the biochemical effects of heritable point mutations to their physiological and clinical consequences is a challenging but important step toward reaching this goal. Mutations in the human mitochondrial DNA (mtDNA) polymerase have been correlated with various mitochondrial disorders, including mtDNA depletion syndrome, Alpers Syndrome, and progressive external opthalmoplegia (PEO). Symptoms of Alpers Syndrome include liver disease and refractory seizures, while patients with PEO present with progressive weakness of the external ocular muscles and skeletal myopathy. Many of the nucleoside analogs used to treat viral infections have toxic side effects due to inhibition of mtDNA replication, which are seen first as peripheral neuropathy. Mitochondrial DNA replication is performed by a replisome comprised of a nuclearly-encoded DNA polymerase, processivity factor, single-stranded DNA binding protein (mtSSB), and DNA helicase. The major challenge in interpreting the clinical effects of mutations in the mtDNA polymerase lies in understanding the molecular basis for the slow onset of the symptoms. Like other heritable disorders of the mitochondrial genes and the toxic side effects of nucleoside analogs used to treat HIV infection, mutations in the mtDNA polymerase lead to diseases often characterized by slow onset due to the accumulation of mtDNA defects and oxidative damage, although certain mutations lead to more severe symptoms resulting in death within one to two years of birth. Understanding the clinical consequences of point mutations in the mtDNA polymerase requires precise and accurate measurements and rigorous data analysis. We will use site-directed mutagenesis and comprehensive kinetic analysis to evaluate the effects of mutations on the mtDNA polymerase in vitro. In addition, we will work to correlate changes in structure and function of the polymeras to the physiological consequences of these mutations observable in a humanized yeast model system expressing the human mtDNA polymerase, which appears to be a good model system to predict the long term consequences of mutations in humans. We will use single turnover rapid kinetic studies to directly examine reactions occurring at the active site in order to quantify key kinetic parameters governing DNA replication. We will also work to examine the role of the mtDNA helicase and mtSSB in the coordinated DNA unwinding and leading strand synthesis. This research will provide a better understanding of the role of the mtDNA polymerase in diseases related to mitochondrial function, and will provide new information to define the molecular basis for nucleotide discrimination by the human mtDNA polymerase, the physiological basis for the toxicity of nucleoside analogs used to treat HIV infections, and the role of mtDNA polymerase and helicase mutations in heritable disorders.

 生物医学研究的一个重要目标是建立疾病的分子基础，以便设计出更有效的治疗方法。将遗传性点突变的生化效应与其生理和临床后果联系起来是实现这一目标的一个具有挑战性但重要的一步。人类线粒体DNA（mtDNA）聚合酶的突变与多种线粒体疾病相关，包括mtDNA耗竭综合征、阿尔珀斯综合征和进行性眼外肌麻痹（PEO）。Alpers综合征的症状包括肝脏疾病和难治性癫痫发作，而PEO患者表现为进行性眼外肌无力和骨骼肌病。许多用于治疗病毒感染的核苷类似物由于抑制mtDNA复制而具有毒副作用，其首先被视为周围神经病变。线粒体DNA复制是由一个由核编码的DNA聚合酶，持续合成因子，单链DNA结合蛋白（mtSSB）和DNA解旋酶组成的复制体进行的。解释mtDNA聚合酶突变的临床效应的主要挑战在于理解症状缓慢发作的分子基础。与其他线粒体基因的遗传性疾病和用于治疗HIV感染的核苷类似物的毒副作用一样，mtDNA聚合酶的突变导致的疾病通常以由于mtDNA缺陷和氧化损伤的积累而缓慢发作为特征，尽管某些突变导致更严重的症状，导致出生后一到两年内死亡。理解mtDNA聚合酶点突变的临床后果需要精确和准确的测量和严格的数据分析。我们将使用定点突变和综合动力学分析来评估突变对mtDNA聚合酶的影响。此外，我们将致力于将polymeras的结构和功能的变化与在表达人mtDNA聚合酶的人源化酵母模型系统中观察到的这些突变的生理后果相关联，这似乎是预测人类突变的长期后果的良好模型系统。我们将使用单周转快速动力学研究，直接检查活性位点发生的反应，以量化关键 控制DNA复制的动力学参数。我们还将研究mtDNA解旋酶和mtSSB在协调DNA解旋和前导链合成中的作用。这项研究将提供一个更好的理解的线粒体DNA聚合酶在疾病中的作用与线粒体功能，并将提供新的信息，以确定核苷酸歧视的分子基础上的人类线粒体DNA聚合酶，用于治疗艾滋病毒感染的核苷类似物的毒性的生理基础，和线粒体DNA聚合酶和解旋酶突变在遗传性疾病中的作用。