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Mechanisms of mitochondrial DNA deletion formation - The fate of linear mtDNA

线粒体DNA缺失形成的机制——线性线粒体DNA的命运

基本信息

批准号：
252501411
负责人：
Professor Dr. Wolfram S. Kunz
金额：
--
依托单位：
Klinik für Epileptologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2014
资助国家：
德国
起止时间：
2013-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/252501411?language=en
关键词：
Mechanisms mitochondrial DNA deletion formation

项目摘要

Deletions of the mitochondrial DNA (mtDNA) are often the cause of insufficient mitochondrial ATP synthesis and, at the same time, they are important indicators of mitochondrial damage. Present hypotheses suggest that the processing of free ends of linear mtDNA species (created by different types of mtDNA damage such as oxidative lesions or replication stalling) is a major source of mtDNA deletions. However, no molecular details of the underlying processes have been revealed till date. In order to investigate in detail the molecular mechanisms that link linear mtDNA processing with generation of deletions, we established cell culture models of controlled mtDNA double-strand break formation by inducible expression of mitochondria-targeted restriction endonucleases. Through the tight control of timing and site of the double-strand break, these cell lines are especially suitable to investigate the initial steps of linear mtDNA end processing and deletion generation. Our preliminary data demonstrate that linearized mtDNA is rapidly degraded from the ends, suggesting the role of exonuclease activities. Knocking down the expression of the mitochondrial exonuclease MGME1 by siRNA treatment or knocking out the MGME1 gene by CRISPR/Cas9 technology substantially delays the degradation of linear mtDNA, thus, suggesting that MGME1 is a major actor in this process. We hypothesize that the persistence of linear mtDNA due to deficient degradation has direct implications for mtDNA deletion generation. In the proposed project, we will use siRNA and CRISPR/Cas9 techniques to manipulate candidate constituents of the degradation machinery, in order to identify proteins that cooperate with MGME1 in performing end processing, bulk degradation of linear mtDNA, and formation of deletions. The high-throughput experimental methods, including ultra-deep sequencing of the mitochondrial genome, that we established to identify mtDNA alterations in human tissues will be essential for the characterization of time-dependent changes in our cellular models of mtDNA double-strand breaks. We will compare our findings in the mitochondria-targeted endonuclease model of mtDNA damage to other (oxidative and replicative) damage conditions. Understanding the molecular details that determine the fate of damaged DNA in mitochondria is inevitable for developing strategies to minimize long-term mitochondrial dysfunction caused by accumulation of mtDNA deletions.

线粒体DNA（mtDNA）的缺失通常是线粒体ATP合成不足的原因，同时，它们是线粒体损伤的重要指标。目前的假设表明，线性mtDNA种类的自由端的加工（由不同类型的mtDNA损伤，如氧化损伤或复制停滞产生）是mtDNA缺失的主要来源。然而，到目前为止，还没有揭示潜在过程的分子细节。为了详细研究线性mtDNA加工与缺失产生之间的分子机制，我们建立了通过诱导表达靶向限制性内切酶来控制mtDNA双链断裂形成的细胞培养模型。通过严格控制双链断裂的时间和位点，这些细胞系特别适合于研究线性mtDNA末端加工和缺失产生的初始步骤。我们的初步数据表明，线性化的mtDNA从末端迅速降解，这表明核酸外切酶活性的作用。通过siRNA处理敲除线粒体核酸外切酶MGME 1的表达或通过CRISPR/Cas9技术敲除MGME 1基因实质上延迟了线性mtDNA的降解，因此，表明MGME 1是该过程中的主要参与者。我们推测，线性mtDNA的持久性，由于缺乏降解有直接的影响mtDNA缺失一代。在拟议的项目中，我们将使用siRNA和CRISPR/Cas9技术来操纵降解机制的候选成分，以鉴定与MGME 1合作进行末端加工、线性mtDNA的大量降解和缺失形成的蛋白质。高通量实验方法，包括线粒体基因组的超深度测序，我们建立了识别人体组织中mtDNA的改变，这将是必不可少的，在我们的细胞模型的mtDNA双链断裂的时间依赖性变化的特征。我们将比较我们的研究结果在线粒体DNA损伤的靶向核酸内切酶模型的其他（氧化和复制）损伤条件。了解决定线粒体中受损DNA命运的分子细节对于制定策略以最大限度地减少由mtDNA缺失积累引起的长期线粒体功能障碍是不可避免的。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Replication fork rescue in mammalian mitochondria

哺乳动物线粒体中的复制叉救援

DOI：
10.1038/s41598-019-45244-6
发表时间：
2019
期刊：
Scientific Reports
影响因子：
4.6
作者：
Torregrosa-Muñumer R;Hangas A;Goffart S;Blei D;Zsurka G;Griffith J;Kunz WS;Pohjoismäki JLO
通讯作者：
Pohjoismäki JLO

Is There Still Any Role for Oxidative Stress in Mitochondrial DNA-Dependent Aging?