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

• Applicants: Professor Dr. Wolfram S. Kunz; Dr. Gábor Zsurka
• Subject Area: Molecular and Cellular Neurology and Neuropathology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2014 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 252501411

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.

• DFG Programme: Research Grants