Mitochondrial DNA Fate and Deletion Formation Following Double-Strand Breaks

Mitochondrial DNA (mtDNA) deletions have been identified in patients with bona-fide mitochondrial disorders as well as implicated in normal aging. The mechanism of how these deletions form is still debated – some possibilities include formation as a result of errors in replication, double-strand breaks (DSBs), or during mtDNA repair. Due to the multi-copy nature of mtDNA, usually following DSB, there is a rapid degradation of linear mtDNA fragments instead of repair. The goals of this study were to first understand which nucleases play a role in mtDNA degradation following DSBs and second to generate and characterize murine cellular models harboring large mtDNA deletions. For the first aim we used mitochondrially-targeted restriction endonucleases to create DSBs in the mtDNA in ex vivo and in vivo mouse models of exonuclease-deficient polymerase gamma (Polg), known as the mutator mouse. Although there was a rapid degradation of linear mtDNA fragments in the wild-type samples, this degradation was impaired in the mutator samples. This impaired degradation was only found in exonuclease-deficient POLG, but not polymerase-deficient POLG. One consequence of the persistence of these linear mtDNA fragments was the formation of mtDNA rearrangements or deletions, which amplified when the partially-deleted mtDNA contained the origins of replication. For the second aim, we fused synaptosomes from the cortex of a mouse expressing the mitochondrial-targeted restriction endonuclease PstI with mouse ρ0 cells to generate cell lines with high levels of mtDNA deletions. Clones harboring the “PstI Deletion” had decreased cell growth, decreased steady-state mitochondrial protein levels, altered supercomplex formation, decreased protein synthesis, and decreased respiration. Additionally, there was a heteroplasmy-dependence to the measured parameters. To use these cell lines for the development of a genetic therapy we generated a mitoTALEN which was able to shift mtDNA heteroplasmy in the PstI Deletion clones, and this shift was stable over time. Together, these studies give a new role to POLG in degrading linear mtDNA fragments following DSBs and provide us with a novel tool for studying mtDNA deletions