MITOCHONDRIAL DNA REPLICATION FIDELITY & CARDIAC DISEASE

线粒体 DNA 复制保真度

• 批准号: 2771634
• 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/2771634
• 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.

该提案的长期目标是阐明 动物线粒体的复制机制及其调控。 拟议的工作重点关注两个主要目标。 第一个是 机械地评估线粒体 DNA 之间的关系 复制保真度和线粒体 DNA 的改变形式 积累在正常衰老和患病的心脏中。 两种可能 线粒体 DNA 生成的复制机制 具有缺失/重复的分子将在体外反应中进行测试 使用两种复制蛋白，即线粒体 DNA 聚合酶和线粒体单链 DNA 结合蛋白。 要检查的模型是瞬时链错位和 DNA 合成过程中模板 DNA 链的转换。 其次，一个 用于生成碱基替换的容易出错的旁路机制 DNA 合成过程中 DNA 损伤位点的错误将是 已测试。 除了提供确凿的生化 DNA 之外， 这些模型对线粒体 DNA 突变的支持， 重要的目标是将改变形式的病毒重新引入动物体内 这两个关键蛋白质，以建立之间的因果关系 线粒体 DNA 突变与心脏病。 第二个主要目标是鉴定新的线粒体 DNA 复制蛋白在复制叉上发挥作用。 在两者中 原核和真核系统，DNA复制保真度可能是 受辅助蛋白影响较大。 蛋白质亲和力 将采用色谱法来鉴定相互作用的蛋白质 物理上与线粒体单链 DNA 结合蛋白。 将同时开发生化测定法来鉴定蛋白质 与线粒体 DNA 聚合酶协同作用， 复制叉上的单链 DNA 结合蛋白。 线粒体 DNA 的改变形式会导致人类疾病。 迄今为止，很少 机制信息可用于证明突变体如何 表格已生成。 许多 mtDNA 疾病表现出明显的心脏病症状 表现，在某些情况下，心脏病是主要发现。 此外，现在大量的临床数据表明，心脏组织 原本健康的心脏病患者承受着广泛的脑损伤 与具有不同病理学的 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 生成这些改变的形式，并提供动物模型 确定 mtDNA 突变是原因还是原因的系统 心脏病的后果。