Publication Date




Embargo Period


Degree Type


Degree Name

Doctor of Philosophy (PHD)


Neuroscience (Medicine)

Date of Defense


First Committee Member

Carlos T. Moraes

Second Committee Member

Alejandro Caicedo

Third Committee Member

Antonio Barrientos

Fourth Committee Member

Miguel Perez-Pinzon

Fifth Committee Member

William C. Copeland


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.


mitochondria; mitochondrial DNA; mtDNA; mtDNA deletions; polymerase gamma; double-strand breaks

Available for download on Thursday, November 26, 2020