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: 8860390
• 负责人: KENNETH ALLEN JOHNSON
• 金额: $ 38.08万
• 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/8860390
• 关键词: Active Sites; Address; Adverse effects; Affect; Age; Age of Onset; Alleles; Alpers' Syndrome; Biochemical; Biochemistry; Biological Assay; Biological Models; Biomedical Research; Birth; Catalytic Domain; Cessation of life; Chemicals; Clinical; Complex; Coupled; DNA; DNA Double Strand Break; 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; Heterozygote; Human; Impairment; In Vitro; Kinetics; Knowledge; Lead; Learning; Literature; Liver diseases; Malignant Neoplasms; Measurement; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Modeling; Molecular; Monitor; Mutation; Myopathy; Nucleotides; Patients; Peripheral Nervous System Diseases; Phenotype; Physiological; Physiology; Point Mutation; Polymerase; Publishing; Reaction; Refractory; Research; Role; SS DNA BP; Seizures; Severities; Severity of illness; Site-Directed Mutagenesis; Skeletal Muscle; Speed; Structure; Structure-Activity Relationship; Symptoms; Syndrome; System; Toxic effect; Virus Diseases; Work; Yeast Model System; Yeasts; accurate diagnosis; base; clinical effect; clinical phenotype; effective therapy; helicase; human DNA sequencing; human disease; mutant; nucleoside analog; oxidative damage; pol Gene Products; polymerization; public health relevance; reconstitution

 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.

 描述（由适用提供）：生物医学研究的一个重要目标是建立疾病的分子基础，以便可以设计出更有效的疗法。将可遗传点突变与其身体和临床后果相关的生化效应是实现这一目标的挑战，但重要的一步。人线粒体DNA（mtDNA）聚合酶的突变已与各种线粒体疾病有关，包括mtDNA部署综合征，Alpers综合征和渐进式外科治疗层（PEO）。 Alpers综合征的症状包括肝病和难治性癫痫发作，而PEO患者出现外科肌肉的逐渐弱点和骨骼肌病。许多用于治疗病毒感染的核类似物由于抑制mtDNA复制而具有毒性副作用，首先被视为周围神经病。线粒体DNA复制是通过完成核编码的DNA聚合酶，加工性因子，单链DNA结合蛋白（MTSSSB）和DNA解旋酶的复制体进行的。解释MTDNA聚合酶中突变的临床作用的主要挑战在于了解症状缓慢发作的分子基础。像线粒体基因的其他遗传疾病以及用于治疗艾滋病毒感染的核外侧类似物的毒性副作用一样，mtDNA聚合酶中的突变导致疾病通常导致疾病，通常是由于mtDNA缺陷的积累而引起的缓慢发作的特征，尽管某些突变导致某些严重的症状导致了两年的死亡。了解mtDNA聚合酶中点突变的临床后果需要精确，准确的测量和严格的数据分析。我们将使用位置定向的诱变和全面的动力学分析来评估突变对MTDNA聚合酶体外的影响。此外，我们将致力于将聚合物的结构和功能变化与表达人类mtDNA聚合酶的人性化酵母模型系统中可观察到的这些突变的物理后果相关联，这似乎是预测人类突变的长期后果的良好模型系统。我们将使用单个周转率快速动力学研究直接检查在活性部位发生的反应，以量化密钥 控制DNA复制的动力学参数。我们还将努力研究mtDNA解旋酶和mTSSB在协调的DNA解开和领先链合成中的作用。 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 nuclearotide discrimination by the human mtDNA polymerase, the physical basis for the toxicity of nuclear lateral analogs used to treat HIV infections, and the role of mtDNA polymerase and helicase mutations in heritable disorders.