MAMMALIAN MITOCHONDRIAL DNA DOUBLE-STRAND-BREAK-REPAIR

最近的数据表明，线粒体DNA（mtDNA）的累积损伤可能在心肌病，中风和其他年龄相关的病理学，如神经退行性疾病中发挥重要作用。事实上，已经有人提出，mtDNA损伤的积累在正常衰老中起着重要作用。此外，最近还描述了一种人类遗传性疾病（常染色体显性进行性眼外肌麻痹，adPEO），其中mtDNA不稳定可导致过早死亡。这些观察结果突出了mtDNA损伤对健康的有害后果。然而，对哺乳动物细胞中mtDNA的DNA修复知之甚少。最近的一系列观察提供了关于这一过程的新信息：（1）已经鉴定出一种新型线粒体特异性DNA连接酶，（2）从多种哺乳动物细胞中制备的线粒体蛋白提取物具有强大的DNA末端连接活性，（3）这种末端连接活性的产物与体内观察到的突变线粒体DNA分子具有惊人的相似之处，（4）在线粒体蛋白提取物中鉴定出DNA末端结合活性。基于这些观察，我们假设哺乳动物线粒体具有非同源末端连接DNA修复途径，类似于在细胞核中的功能。本提案中描述的实验旨在检验这一假设。具体而言，将使用基因靶向来修饰线粒体DNA连接酶基因，并评估“敲除”细胞的mtDNA修复/稳定性表型。此外，还将克隆线粒体DNA末端结合基因。基因靶向将用于创建突变细胞系，其中该基因已失活，并将确定这些细胞的mtDNA修复和稳定性表型。基于本提案中提出的初步结果，可以合理地预测上述敲除细胞系可能具有mtDNA突变子表型。如果这是事实，未来的研究可以致力于在小鼠中创建类似的基因失活，从而允许对累积的mtDNA损伤影响衰老的假设进行测试。

Recent data indicate that accumulate damage to mitochondrial DNA (mtDNA) may play an important role in cardiac myopathy, stroke , and other age-related pathologies such as neurodegeneration. In fact, it has been proposed that the accumulation of mtDNA damage plays a fundamental role in normal aging. In addition, a human genetic disease (autosomal dominant progressive external ophthalmoplegia, adPEO) has recently been described in which mtDNA instability can lead to premature death. These observations highlight the deleterious health consequences of damage to mtDNA. Yet comparatively little is known about DNA repair of mtDNA in mammalian cells. A series of recent observations have provided new information about this process: (1) A novel mitochondrial-specific DNA ligase has been identified, (2) Mitochondrial protein extracts prepared from a variety of mammalian cells possess potent DNA end-joining activity, (3) The products of this end-joining activity bear a striking resemblance to mutant mitochondrial DNA molecules observed in vivo, and (4) A DNA end- binding activity has been identified in mitochondrial protein extracts. Based on these observations, we have hypothesized that mammalian mitochondrial possess a non-homologous end-joining DNA repair pathway, analogous to that which functions in the nucleus. The experiments described in this proposal are designed to test this hypothesis. Specifically, gene targeting will be used to inactivate the mitochondrial DNA ligase gene, and the mtDNA repair/stability phenotype of 'knockout' cells evaluated. In addition, the mitochondrial DNA end-binding gene will be cloned. Gene targeting will be used to create mutant cell lines in which this gene has been inactivated, and the mtDNA repair and stability phenotype of these cells will be determined. Based on preliminary results presented in this proposal, it is reasonable to predict that the knockout cell lines described above could possess a mtDNA mutator phenotype. If this provides to be the case, future studies could be devoted to the creation of similar gene inactivations in mice, thereby permitting a test of the hypothesis that accumulated mtDNA damage influences aging.