MITOCHONDRIAL DNA REPLICATION FIDELITY & CARDIAC DISEASE

线粒体 DNA 复制保真度

• 批准号: 2469853
• 负责人: LAURIE SIMON KAGUNI
• 金额: $ 11.03万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2469853
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA replication; Drosophilidae; affinity chromatography; arthropod genetics; gel electrophoresis; gene mutation; genetic regulation; genetically modified animals; heart disorder; helicase; mitochondrial DNA; nucleic acid chemical synthesis; nucleic acid sequence; polymerase chain reaction; site directed mutagenesis

The long-term objective of this proposal is to elucidate the mechanism of replication and its regulation in animal mitochondria. The proposed workfocuses on two major objectives. The first is to evaluate mechanistically the relationship between mitochondrial DNA replication fidelity and altered forms of mitochondrial DNA that accumulate in normal aging and diseased hearts. Two possible replicative mechanisms for generation of mitochondrial DNA molecules with deletions/duplications will be tested in vitro reactions employing two kepy replicative proteins, mitochondrial DNA polymerase and mitochondrial single-stranded DNA-binding protein. The models to be examined are transient strand misalignment and template DNA strand switching during DNA synthesis. Second, an error-prone bypass mechanism for generation of base substitution errors during DNA synthesis past sites of DNA damage will be tested. In addition to providing conclusive biochemical DNA is support of these models for mitochondrial DNA mutagenesis, an important goal will be to reintroduce into animals altered forms of these two key proteins, to establish a causal relationship between mitochondrial DNA mutations and cardiac disease. The second major objective is to identify new mitochondrial DNA replication proteins functioning at the replication fork. In both procaryotic and eucaryotic systems, DNA replication fidelity may be influenced greatly by auxiliary proteins. Protein affinity charomatography will be employed to identify proteins that interact physically with mitochondrial single-stranded DNA-binding protein. Biochemical assays will be developed in parallel to identify proteins that function in concert with mitochondrial DNA polymerase and single-stranded DNA-binding protein at the replication fork. Altered forms of mtDNA cause human disease. To date, very little mechanistic information is available to demonstrate how the mutant forms are generated. Many mtDNA diseases show significant cardiac manifestations and in several, cardiac disease is the major finding. Furhter, substantial clinical data now show that the heart tissue in otherwise healthy cardiac patients bears extensive damage of the same types as in mtDNA diseases with variable pathologies. This work will help establish the basic biochemical mechanisms involved in generating these altered forms, and also provide an animal model system to determine whether mtDNA mutations are a cause or a consequence of cardiac disease.

本建议的长远目标是阐明 复制机制及其在动物线粒体中的调节。 拟议的工作集中在两个主要目标。 一是 从机制上评估线粒体DNA 复制保真度和改变形式的线粒体DNA， 在正常衰老和患病的心脏中积累。 两个可能 线粒体DNA的复制机制 将在体外反应中检测具有缺失/重复的分子 使用两种krna复制蛋白，线粒体DNA， 聚合酶和线粒体单链DNA结合蛋白。 要检查的模型是瞬时股线错位和 DNA合成过程中的模板DNA链转换。 二是 用于产生碱基置换的易错旁路机制 在DNA合成过程中的错误过去的DNA损伤位点将是 测试. 除了提供决定性的生化DNA外， 支持这些模型的线粒体DNA诱变， 一个重要的目标将是重新引入动物的改变形式， 这两个关键蛋白质，以建立因果关系， 线粒体DNA突变和心脏病。 第二个主要目标是鉴定新的线粒体DNA 复制蛋白在复制叉处发挥作用。 无论是 在原核和真核系统中，DNA复制保真度可能是 受辅助蛋白影响较大。 蛋白亲和 将采用特征图谱法来鉴定相互作用的蛋白质， 与线粒体单链DNA结合蛋白物理结合。 生化分析将平行发展，以确定蛋白质 与线粒体DNA聚合酶协同作用， 复制叉处的单链DNA结合蛋白。 线粒体DNA的改变会导致人类疾病。 迄今为止， 机械信息可用于证明突变体如何 形式产生。 许多线粒体DNA疾病表现出显著的心脏 临床表现和在一些，心脏病是主要的发现。 此外，大量的临床数据显示， 否则健康的心脏病患者会承受广泛的心脏损伤， 与不同病理的线粒体DNA疾病类型相同。 这 这项工作将有助于建立基本的生化机制， 产生这些改变的形式，并提供动物模型 系统来确定mtDNA突变是一个原因还是一个 心脏病的后果。