The maintenance of mitochondrial DNA after oxidative stress

Abstract

Mitochondria are vital within eukaryotic cells and the maintenance of mitochondrial DNA (mtDNA) is therefore crucial to maintain mitochondria function. mtDNA has been shown to be particularly vulnerable to oxidative damage due to its proximity to reactive oxygen species (ROS) generated by oxidative phosphorylation (OXPHOS) and the electron trans-port chain in generating energy for cells in the form in ATP (Alencar et al., 2019). The hydroxyl radical produced via the Fenton reaction with free iron in the mitochondrial matrix has been shown to generate oxidative damage in mtDNA (Petrat et al., 2002). Utilizing Southern blotting and ultra-deep sequencing, this thesis aims to determine a paradigm for the damage that occurs to mtDNA after a transient H₂O₂ pulse, and then to elucidate when this damage is repaired versus when these damaged molecules are degraded and mtDNA is repopulated by replication with intact species, and what proteins are involved in this process. CRISPR/Cas9 technology was employed to generate knock-out HEK293 cell lines of genes known to be involved in the maintenance of mtDNA: MGME1 and POLGexo known to degrade linear mtDNA (Peeva et al., 2018), and APEX1, POLG, EXOG, and LIG3 known to be involved in base excision repair (BER) (Alencar et al., 2019). The strong 1 mM H₂O₂ pulse results in sugar-backbone damage in the form of single-strand breaks (SSBs) and double-strand breaks (DSBs) in mtDNA, which are eliminated in the control cells that have recovery of the native supercoiled mtDNA species within a period of hours. SSB repair is responsible for the fast recovery of mtDNA seen in the control; increased replication is not responsible for this recovery because BrdU incorporation indicating de novo mtDNA was not increased in the presence of H₂O₂ (Trombly et al., 2023). Impaired recovery of supercoiled mtDNA in APEX1^-/- and LIG3^-/- demonstrates the importance of SSB repair and subsequent replication to repopulate intact mtDNA. Additionally, efficient degradation and the clearance of damaged templates is also necessary for the recovery of mtDNA after H₂O₂ oxidative damage (Trombly et al., 2023), as damage in MGME1^-/- and POLGexo^-/- cells persists and impairs supercoiled mtDNA recovery. Taken together, the results of this thesis have helped to elucidate the type of mtDNA damage that results from H₂O₂, with repair responsible for mtDNA recovery in the short time scale and degra-dation with <em>de novo</em> synthesis of mtDNA also playing an important role for the recovery of intact mtDNA species.