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Mitochondria are essential organelles in eukaryotic cells. They contain their own genome that encodes proteins of the OXPHOS complexes essential for cellular ATP production. Failure to maintain mitochondrial DNA (mtDNA) integrity, therefore, can impair energy production and lead to mitochondrial dysfunction, which consequently results in a wide range of diseases, including rare genetic mitochondrial disorders and neurodegeneration. 

In human mitochondria, the genome is replicated by a unique enzymatic machinery including the mitochondrial replicative DNA Polymerase Gamma (Polγ). Polγ is a holoenzyme consisting of a catalytic Polγ α subunit and two accessory Polγ β subunits. The catalytic subunit Polγ α has both DNA polymerase and exonuclease activities that are required for high-fidelity mtDNA replication. A precise cooperation of the two activities of Polγ during mtDNA replication is therefore critical to ensure proper mtDNA integrity. In our study, we investigated the mechanism by which the pathogenic mutation Y951N induces replication stalling and a loss of mtDNA in patient cells. Our findings showed that the Y951N mutation in the polymerase domain disrupts Polγ’s ability to switch between its polymerase and exonuclease activities, leading to severe mtDNA replication stalling and eventually mtDNA depletion. Identifying Polγ residues critical for this intramolecular switching mechanism provides insights into how various pathogenic mutations affect the maintenance of the mitochondrial genome. 

In response to mitochondrial dysfunction that alters the cellular energy state, particularly in the context of mitochondrial DNA depletion, AMP-activated protein kinase (AMPK), a key energy sensor and metabolic regulator can be activated to restore energy homeostasis. However, the degree of severity of mitochondrial dysfunction required to induce AMPK activation, as well as how mitochondrial biogenesis can be restored following stimulation of AMPK activity, remains to be elucidated. To address this, we used a cell model of mtDNA depletion syndromes (MDS) in which the expression of a pathogenic Polγ variant causes severe mtDNA loss, leading to progressive mitochondrial dysfunction. We observed that the activation of mitochondria-associated AMPK occurs during the early stages of advancing mitochondrial dysfunction. Moreover, our results showed that stimulation of AMPK activity using a specific agonist, A-769662, can mitigate impaired mitochondrial phenotypes and partially restore mtDNA levels. These findings contribute to our understanding of the impacts of specific activators of AMPK on mitochondrial and cellular function as well as their potential applications in mitochondrial diseases. 

In addition to defects in nuclear genes involved in mtDNA replication, mutations in the mitochondrial genome that arise from errors during mtDNA replication or from repair of damaged mtDNA can also result in a loss of mitochondrial genetic integrity, e.g. due to an accumulation of large-scale mtDNA deletions. Many of these mtDNA deletion breakpoints were recently suggested to occur at sequence motifs with potential to form secondary DNA structures, G-quadruplexes (G4s). In our study, by developing a novel tool for mapping G4s in living cells, we were able to determine mtDNA G4 formation in human cells under different cellular conditions. Our results indicated that replication stalling enhances G4 formation, which in turn blocks the replication fork progression and causes mtDNA loss, potentially leading to mitochondrial disease. The new mtG4-ChiP tool will enable future research to further investigate the factors involved in G4 formation and resolution, as well as the mechanistic roles of G4s in the generation of pathogenic mtDNA deletions. 

In parallel with studying mechanisms of mtDNA deletions and depletion, we investigated how cells tolerate mitochondrial genome damage to preserve mtDNA integrity. PrimPol, a primase–polymerase that can restart stalled mtDNA replication, requires stimulation by Polymerase δ–interacting protein 2 (PolDIP2) for efficient DNA synthesis. While characterizing this interaction, we unexpectedly found that PolDIP2 forms disulfide-linked homodimers, a process that is enhanced under oxidative stress. Notably, PolDIP2 interacts with coiled-coil-helix-coiled-coil-helix domain–containing protein 2 (CHCHD2), a mitochondrial protein implicated in maintaining cristae structure and dynamics. Our findings suggest that redox-sensitive PolDIP2 dimerization may influence mitochondrial function by modulating its interaction with CHCHD2 during oxidative stress. 

In summary, the findings presented here provided valuable insights into molecular mechanisms of mitochondrial genome instability caused by pathogenic mutations and cellular responses to mitochondrial dysfunctions, as well as the involvement of potential factors in mitochondrial DNA maintenance.