We here reconstitute a minimal mammalian mitochondrial DNA (mtDNA) replisome in vitro. The mtDNA polymerase (POLc) cannot use double-stranded DNA (dsDNA) as template for DNA synthesis. Similarly, the TWINKLE DNA helicase is unable to unwind longer stretches of dsDNA. In combination, POLc and TWINKLE form a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The addition of the mitochondrial ssDNA-binding protein stimulates the reaction further, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. The observed DNA synthesis rate is 180 base pairs (bp)/min, corresponding closely to the previously calculated value of 270 bp/min for in vivo DNA replication. Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. Furthermore, mutations in TWINKLE and POLc cause autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with deletions in mitochondrial DNA. The functional interactions between TWINKLE and POLc thus explain why mutations in these two proteins cause an identical syndrome.

Regulation of mammalian mtDNA gene expression is critical for altering oxidative phosphorylation capacity in response to physiological demands and disease processes. The basal machinery for initiation of mtDNA transcription has been molecularly defined, but the mechanisms regulating its activity are poorly understood. In this study, we show that MTERF3 is a negative regulator of mtDNA transcription initiation. The MTERF3 gene is essential because homozygous knockout mouse embryos die in midgestation. Tissue-specific inactivation of MTERF3 in the heart causes aberrant mtDNA transcription and severe respiratory chain deficiency. MTERF3 binds the mtDNA promoter region and depletion of MTERF3 increases transcription initiation on both mtDNA strands. This increased transcription initiation leads to decreased expression of critical promoter-distal tRNA genes, which is possibly explained by transcriptional collision on the circular mtDNA molecule. To our knowledge, MTERF3 is the first example of a mitochondrial protein that acts as a specific repressor of mammalian mtDNA transcription initiation in vivo.

The human mitochondrial transcription termination factor (mTERF) is involved in the regulation of transcription of the mitochondrial genome. Similarity searches and phylogenetic analysis demonstrate that mTERF is a member of large and complex protein family (the MTERF family) shared amongst metazoans and plants. Interestingly, we identify three novel MTERF genes in vertebrates, which all encode proteins with predicted mitochondrial localization. Members of the MTERF family have so far not been detected in fungi, supporting the notion that mitochondrial transcription regulation may have evolved separately in yeast and animal cells.

A deficient activity of one or more of the mitochondrial oxidative phosphorylation (OXPHOS) enzyme complexes leads to devastating diseases, with high unmet medical needs. Mitochondria, and more specifically the OXPHOS system, are the main cellular production sites of Reactive Oxygen Species (ROS). Increased ROS production, ultimately leading to irreversible oxidative damage of macromolecules or to more selective and reversible redox modulation of cell signalling, is a causative hallmark of mitochondrial diseases. Here we report on the development of a new clinical-stage drug KH176 acting as a ROS-Redox modulator. Patient-derived primary skin fibroblasts were used to assess the potency of a new library of chromanyl-based compounds to reduce ROS levels and protect cells against redox-stress. The lead compound KH176 was studied in cell-based and enzymatic assays and in silico. Additionally, the metabolism, pharmacokinetics and toxicokinetics of KH176 were assessed in vivo in different animal species. We demonstrate that KH176 can effectively reduce increased cellular ROS levels and protect OXPHOS deficient primary cells against redox perturbation by targeting the Thioredoxin/Peroxiredoxin system. Due to its dual activity as antioxidant and redox modulator, KH176 offers a novel approach to the treatment of mitochondrial (-related) diseases. KH176 efficacy and safety are currently being evaluated in a Phase 2 clinical trial.