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Correlating defects in mitochondrial DNA replication to physiology

将线粒体 DNA 复制缺陷与生理学相关联

基本信息

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
项目类别：
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&apos 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

项目摘要

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.