Non-canonical mtDNA species in mitochondrial dysfunction and oxidative damage

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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.