DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion

Abstract

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 generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies.

Therapy Prospects for Mitochondrial DNA Maintenance Disorders

“…For example, the mutator Polg knock in mouse has a dysfunctional proof-reading exonuclease activity and does not present mtDNA depletion [ 183 ], and it is thus not a good model to test whether dN administration enhances the polymerase activity of this enzyme. A recently generated Polg A449T/A449T mouse [ 184 ] (equivalent to the frequent A467T POLG mutation in patients) could be a more adequate model to test this therapy, although its phenotype is also limited (mainly molecular alterations).…”

“…There are several genetically modified mouse strains that aim to model these diseases (reviewed by [ 207 , 208 ] as well as some other vertebrate models [ 206 , 209 ]). Some of the murine models recapitulate, at least partially, the clinical phenotype of the diseases [ 171 , 176 , 187 , 210 , 211 , 212 ], but in some other cases, the phenotype is barely biochemical or molecular, with no or little effect on the functional life of the animals [ 90 , 184 ]. Sometimes, the models can be stressed to show enhanced phenotypes that result as useful to show the efficacy of the experimental therapies [ 188 , 195 , 213 ].…”

Downregulating Mitochondrial DNA Polymerase γ in the Muscle Stimulated Autophagy, Apoptosis, and Muscle Aging-Related Phenotypes in Drosophila Adults

“…PolG1 is broken down by the protease LONP1, and PolG2 protects the catalytic subunit from this degradation. PolG2 knockdown has been shown to reduce PolG1 protein levels in mice [ 46 ]. The amount of Polγ holoenzyme depends on the PolG2 mRNA levels if there is no regulatory mechanism during the translational process.…”

Mitochondrial Neurodegeneration

“…More than 100 mutations in pol-γA have been reported so far (see (accessed on 1 November 2021) for a complete and continuously updated list of POLG mutations associated with different syndromes). Different mutations affecting different domains of pol-γA, including the N-terminal proofreading domain, the C-terminal catalytic polymerase domain, and an intermediate ‘spacer’ region, which binds to pol-γB, thus regulating polymerase processivity, can affect one or more of its enzymatic properties, as shown both in vitro and in model organisms [ 10 , 58 ]. The clinical outcomes vary [ 57 ].…”