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.

该提议的长期目标是阐明 复制机理及其在动物线粒体中的调节。 拟议的工作人员在两个主要目标上。 首先是 从机械上评估线粒体DNA之间的关系 复制保真度和改变形式的线粒体DNA 积聚在正常的衰老和患病的心脏中。 两个可能 生成线粒体DNA的复制机制 具有缺失/重复的分子将在体外反应测试 使用两个Kepy复制蛋白，线粒体DNA 聚合酶和线粒体单链DNA结合蛋白。 要检查的模型是瞬态链错位和 DNA合成过程中的模板DNA链切换。 第二，一个 基本替换生成错误的旁路机制 DNA合成期间的错误过去的DNA损伤部位将是 测试。 除了提供结论性的生化DNA之外 这些模型用于线粒体DNA诱变，一种 重要目标是重新引入动物的改变形式 这两个关键蛋白质，建立 线粒体DNA突变和心脏病。 第二个主要目标是识别新的线粒体DNA 复制蛋白在复制叉处发挥作用。 在这两个中 丙酸和念珠菌系统，DNA复制保真度可能是 受辅助蛋白的影响很大。 蛋白质亲和力 炭疽菌将被用来识别相互作用的蛋白质 用线粒体单链DNA结合蛋白进行物理。 生化测定将平行开发以鉴定蛋白质 该功能与线粒体DNA聚合酶和 复制叉处的单链DNA结合蛋白。 mtDNA的改变形式引起人类疾病。 迄今为止，很少 机械信息可以证明突变体如何 形式是生成的。 许多mtDNA疾病表现出明显的心脏 表现形式，在几种中，心脏病是主要发现。 现在，大量的临床数据表明，心脏组织 否则，健康的心脏病患者对 与具有可变病理的mtDNA疾病相同的类型。 这 工作将有助于建立涉及的基本生化机制 产生这些改变的形式，还提供动物模型 确定mtDNA突变是原因还是 心脏病的结果。