Non-canonical mtDNA species in mitochondrial dysfunction and oxidative damage

在我们目前正在进行的DFG资助的项目“线粒体DNA缺失形成的机制-线性线粒体DNA的命运”中，我们能够确定线粒体DNA(MtDNA)降解机制的分子组成。这些是众所周知的线粒体DNA复制机制的组成部分：线粒体5‘-3’外切酶MGME1，线粒体DNA聚合酶Polg的3‘-5’外切酶活性和线粒体DNA解旋酶TWNK。我们最近的实验数据表明，线粒体DNA降解复合体的这些成分的遗传失活导致非规范线粒体DNA物种的大量积累，这些物种作为线粒体DNA复制或修复的中间体，在正常情况下迅速降解。由于目前对这些分子的了解完全缺乏，我们计划在本提案中系统地应用高纯度线粒体DNA的超深度测序，以阐明这些非规范线粒体DNA物种的分子结构。为了避免这些低丰度物种的快速降解，我们将使用从各种遗传失活mtDNA降解途径模型(MGME1敲除的HEK细胞或Polg p.D274A敲除的HEK细胞、Polg p.D257A敲除的小鼠和MGME1敲除的患者成纤维细胞)中分离的mtDNA。为了对非规范mtDNA分子进行全面的分子表征，我们计划将不同处理(天然、T4聚合酶、S1核酸酶、T7内切酶I)后连接的mtDNA的超深度测序与第三代长读PacBio测序相结合，以表征游离DNA末端，以揭示整个mtDNA分子结构。此外，我们计划研究氧化和复制应激对非规范mtDNA产生的影响。对非规范线粒体DNA分子的结构和生成的了解对于更好地理解线粒体DNA复制的分子机制是必不可少的。此外，我们的数据将使我们能够更深入地了解线粒体DNA损伤和修复的过程，这与线粒体DNA突变的产生有关。

In our currently running DFG-funded project ‘Mechanisms of mitochondrial DNA deletion formation – The fate of linear mtDNA’, we were able to identify the molecular components of the mitochondrial DNA (mtDNA) degradation machinery. These are well-known components of the mitochondrial DNA replication machinery: the mitochondrial 5’-3’ exonuclease MGME1, the 3’-5’ exonuclease activity of the mitochondrial DNA polymerase POLG and the mitochondrial DNA helicase TWNK. Our recent experimental data indicate that genetic inactivation of these constituents of the mtDNA degradation complex leads to a substantial accumulation of non-canonical mtDNA species that are, as intermediates of mtDNA replication or repair, under normal circumstances rapidly degraded. Since knowledge about these molecules is presently completely lacking, we plan in the present proposal to systematically apply ultra-deep sequencing of highly purified mtDNA to shed light on the molecular structure of these non-canonical mtDNA species. To avoid the rapid degradation of these low abundant species, we will use mtDNA isolated from various models of the genetically inactivated mtDNA degradation pathway (HEK cells with MGME1 knockout or POLG p.D274A knockin, mice with POLG p.D257A knockin and patient fibroblasts with MGME1 knockout). For a comprehensive molecular characterization of non-canonical mtDNA molecules, we plan to combine ultra-deep sequencing of linker-ligated mtDNA after different pre-treatments (native, T4 polymerase, S1 nuclease, T7 endonuclease I) to characterize free DNA ends with third-generation long-read PacBio sequencing to reveal the structures of entire mtDNA molecules. Furthermore, we plan to study the effects of oxidative and replicative stress on the generation of non-canonical mtDNA. The knowledge about the structure and generation of non-canonical mtDNA molecules is essential for a better understanding of the molecular mechanism of replication of mtDNA. Additionally, our data will allow to obtain a deeper insight into the processes of mtDNA damage and repair, which is relevant for the generation of mtDNA mutations.