No abstract

The outbreak of the COVID‐19 pandemic prompted the search for effective antivirals. Remdesivir (Gilead Sciences) was the first nucleoside analogue approved by the FDA for COVID‐19 treatment, and currently the FDA considers authorization for the use of another analogue, molnupiravir (Merck). Mitochondrial toxicity, resulting from the interference with mitochondrial DNA (mtDNA) replication, is the most common side effect of nucleoside analogues treatment. In addition, defects to the mtDNA synthesis may give rise to heritable mutations, which may be carried undetected for generations before populating mitochondria to the level that onsets a mitochondrial disease. Therefore, nucleoside analogues need to be thoroughly investigated for their impact on the maintenance and integrity of the mitochondrial genome. We recently assessed the effect of remdesivir on the maintenance of the mitochondrial genome. We observed that in in vitro DNA synthesis assays, its triphosphate metabolite may impede the synthetic activity of the mitochondrial replicative polymerase, Pol γ, when in excess over nucleotides. Prompted by this finding, we tested the effect of the nucleoside metabolite of remdesivir on the maintenance and integrity of the mitochondrial genome in proliferating fibroblasts. However, we found no evidence for deleterious effects on the integrity of the mitochondrial genome. We are currently performing an analogical assessment of the effects of molnupiravir on the integrity of the mitochondrial genome. Thus far, we have observed that, unlike remdesivir, molnupiravir is cytotoxic for proliferating fibroblasts and results in a decrease in mtDNA copy number. We are currently testing the impact of molnupiravir on the activity of Pol γ in vitro, and its potential to elicit mutations in the mitochondrial genome in vivo.

Mitochondrial DNA (mtDNA) encodes thirteen essential proteins of the oxidative phosphorylation system, responsible for the major production of ATP in the cell. Therefore, damages to the mitochondrial genome result in energy deprivation, which may in turn onset human diseases. Notably, due to its proximity to the electron transport chain, mtDNA remains exposed to damage by reactive oxygen species, thus the maintenance of its integrity requires a robust repair system. Until recently, DNA polymerase gamma (Pol γ) has been the only polymerase identified in mitochondria, bearing responsibility for efficient replication as well as post‐replication repair of the genome. We have previously suggested that the division of the roles of Pol γ may be controlled by the association of its catalytic subunit, Pol γA, with the accessory subunit Pol γB, such that the holoenzyme is engaged in the processive mtDNA replication, whereas, alone, Pol γA may serve the repair processes. Recently, the major repair polymerase of the nucleus, Pol β, has been discovered to also localize in mitochondria, which raises the question for its competition or cooperation with Pol γ in the mtDNA repair processes. To address this, we have tested in vitro the efficiency of DNA synthesis by the two polymerases, separately and in combination, using various DNA substrates. In agreement with previous reports, we did not observe any indication of a functional interaction between the Pol γ holoenzyme and Pol β. We did, however, observe a cooperative activity of Pol β with the Pol γA subunit. In conclusion, our results suggest that the repair of mtDNA may entail a synergistic activity of the catalytic subunit of Pol γ and Pol β.