MITOCHONDRIAL DNA REPLICATION FIDELITY & CARDIAC DISEASE

这项建议的长期目标是澄清 动物线粒体的复制机制及其调控。 拟议的工作侧重于两个主要目标。第一个是 从力学角度评价线粒体DNA与DNA之间的关系 复制保真度和改变形式的线粒体DNA 在正常老化和患病的心脏中积累。两种可能 线粒体DNA产生的复制机制 具有缺失/复制的分子将在体外反应中进行测试 利用两种Kepy复制蛋白，线粒体DNA 聚合酶和线粒体单链DNA结合蛋白。 要检查的模型是暂时性链未对齐和 DNA合成过程中模板DNA链的切换。第二，一个 用于产生碱基替换的容易出错的旁路机制 DNA合成过程中经过DNA损伤位置的错误将是 测试过。除了提供确凿的生化DNA 这些模型对线粒体DNA突变的支持 重要的目标将是将改变的形式重新引入动物体内 这两个关键蛋白质之间建立了因果关系 线粒体DNA突变与心脏病。 第二个主要目标是识别新的线粒体DNA 在复制叉处起作用的复制蛋白质。在这两个地方 原核和真核系统，DNA复制的保真度可能是 受辅助蛋白影响较大。蛋白质亲和力 将使用特征层析技术来识别相互作用的蛋白质 物理上与线粒体单链DNA结合蛋白。 将同时开发生化分析来鉴定蛋白质。 与线粒体DNA聚合酶和线粒体DNA聚合酶协同工作 复制分叉处的单链DNA结合蛋白。 线粒体DNA的改变形式会导致人类疾病。到目前为止，几乎没有 机制信息可以用来证明突变体是如何 将生成表单。许多线粒体DNA疾病显示出明显的心脏 主要表现为心脏病，在几种情况下，心脏病是主要表现。 此外，大量的临床数据现在表明，心脏组织在 否则，健康的心脏病患者会承受广泛的心脏损害 与不同病理类型的线粒体DNA疾病的类型相同。这 工作将有助于建立参与的基本生化机制 产生这些改变的形式，还提供了一个动物模型 确定线粒体DNA突变是原因还是原因的系统 心脏病的后果。

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.