MAMMALIAN MITOCHONDRIAL DNA DOUBLE-STRAND-BREAK-REPAIR

哺乳动物线粒体 DNA 双链断裂修复

• 批准号: 6509636
• 负责人: COLIN R CAMPBELL
• 金额: $ 17.15万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-15 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6509636
• 关键词: CHO cells; DNA binding protein; DNA damage; DNA repair; DNA replication; alleles; chickens; complementary DNA; enzyme activity; gel mobility shift assay; gene expression; gene targeting; mitochondrial DNA; molecular cloning; mutant; nucleoproteins; phenotype; phosphoester ligase; polymerase chain reaction; protein structure function; western blottings

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.

最近的数据表明，对线粒体DNA（MTDNA）累积损害可能在心脏肌病，中风和其他与年龄相关的病理（如神经变性病）中起重要作用。实际上，已经提出，mtDNA损伤的积累在正常衰老中起着基本作用。此外，最近已经描述了人类遗传疾病（常染色体显性进行性外科眼科，ADPEO），其中mtDNA不稳定性会导致过早死亡。这些观察结果凸显了对mtDNA损害的有害健康后果。然而，关于哺乳动物细胞中mtDNA的DNA修复知之甚少。 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）在线粒体蛋白提取物中已经鉴定出DNA末端结合活性。基于这些观察结果，我们假设哺乳动物线粒体具有非同源的末端连接DNA修复途径，类似于在细胞核中起作用的DNA修复途径。本提案中描述的实验旨在检验该假设。具体而言，基因靶向将用于灭活线粒体DNA连接酶基因，并评估了“基因敲除”细胞的mtDNA修复/稳定性表型。此外，将克隆线粒体DNA末端结合基因。基因靶向将用于创建该基因已失活的突变细胞系，并确定这些细胞的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细胞系可能具有mtDNA突变器表型。如果是这样的，则可以将未来的研究致力于在小鼠中产生相似的基因失活，从而允许对积累mtDNA损伤影响衰老的假设进行检验。