In vivo and in vitro mechanistic characterization of a clinically relevant PolγA mutation

Mutations in POLG , encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG -related diseases is poorly understood and efficient treatments are missing. Here, we generated a POLGA449T/A449T mouse model, which reproduces the most common human recessive mutation of POLG , encoding the A467T change, and dissected the mechanisms underlying pathogenicity. We show that the A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ in vivo and in vitro . Interestingly, the A467T mutation also strongly impairs interactions with POLγB, the homodimeric accessory subunit of holo-POLγ. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA, which in turn exacerbates the molecular phenotypes of PolgA449T/A449T mice. Importantly, we validated this mechanism for other mutations affecting the interaction between the two POLγ subunits. We suggest that LONP1 dependent degradation of POLγA can be exploited as a target for the development of future therapies.