Respiring mitochondria maintain a membrane potential (⌬⌿) 1 of Ϫ150 to Ϫ180 mV (⌬⌿, inside negative). This high ⌬⌿ constitutes a large driving force for the electrophoretic influx of cations, either through specific channels or by diffusion through the membrane. Several cation channels have been characterized physiologically (reviewed in Refs. 1 and 2), and recently, a single one has been identified molecularly (3). These transport systems seem to have intrinsic control mechanisms which ensure that the matrix cation concentrations stay within physiological ranges, far below chemical equilibrium.Diffusive permeability of the inner mitochondrial membrane to ions is generally low but physiologically significant, as it lowers the pH gradient and membrane potential. Moreover, if not counteracted by extrusion, steadily increasing concentrations of matrix cations (and of compensating anions) will lead to an imbalance of osmotic pressure across the inner mitochondrial membrane. As a consequence, water will pass through the membrane, causing excessive swelling and eventual rupture of the organelle (1, 2, 4).As first proposed by P. Mitchell (5), mitochondria have carrier systems allowing the electroneutral exchange of cations against H ϩ (and anions against OH Ϫ ). These exchangers counteract the ⌬⌿-driven cation leakage of the membrane and also cation imbalances due to changes in mitochondrial physiology. Mitochondrial cation distribution is, therefore, a steady state, in which the accumulation ratio is modulated by the relative rates of cation influx and efflux by means of separate pathways.Many physiological studies have been devoted to cation/H ϩ exchange systems (reviewed in Ref.

Known disease mechanisms in mitochondrial DNA (mtDNA) maintenance disorders alter either the mitochondrial replication machinery (POLG1, POLG22 and C10orf23) or the biosynthesis pathways of deoxyribonucleoside 5′-triphosphates for mtDNA synthesis4–11. However, in many of these disorders, the underlying genetic defect has not yet been discovered. Here, we identified homozygous nonsense and missense mutations in the orphan gene C20orf72 in three families with a mitochondrial syndrome characterized by external ophthalmoplegia, emaciation, and respiratory failure. Muscle biopsies showed mtDNA depletion and multiple mtDNA deletions. C20orf72, hereafter MGME1 (mitochondrial genome maintenance exonuclease 1), encodes a mitochondrial RecB-type exonuclease belonging to the PD-(D/E)XK nuclease superfamily. We demonstrate that MGME1 cleaves single-stranded DNA and processes DNA flap substrates. Upon chemically induced mtDNA depletion, patient fibroblasts fail to repopulate. They also accumulate intermediates of stalled replication and show increased levels of 7S DNA, as do MGME1-depleted cells. Hence, we show that MGME1-mediated mtDNA processing is essential for mitochondrial genome maintenance.

Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown. Here, we show that, in cellular models of restriction endonuclease-induced mtDNA double-strand breaks, linear mtDNA is eliminated within hours by exonucleolytic activities. Inactivation of the mitochondrial 5′-3′exonuclease MGME1, elimination of the 3′-5′exonuclease activity of the mitochondrial DNA polymerase POLG by introducing the p.D274A mutation, or knockdown of the mitochondrial DNA helicase TWNK leads to severe impediment of mtDNA degradation. We do not observe similar effects when inactivating other known mitochondrial nucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, or RBBP8). Our data suggest that rapid degradation of linearized mtDNA is performed by the same machinery that is responsible for mtDNA replication, thus proposing novel roles for the participating enzymes POLG, TWNK, and MGME1.

Sequencing-based studies have identified novel risk genes associated with severe epilepsies and revealed an excess of rare deleterious variation in less-severe forms of epilepsy. To identify the shared and distinct ultra-rare genetic risk factors for different types of epilepsies, we performed a whole-exome sequencing (WES) analysis of 9,170 epilepsy-affected individuals and 8,436 controls of European ancestry. We focused on three phenotypic groups: severe developmental and epileptic encephalopathies (DEEs), genetic generalized epilepsy (GGE), and non-acquired focal epilepsy (NAFE). We observed that compared to controls, individuals with any type of epilepsy carried an excess of ultra-rare, deleterious variants in constrained genes and in genes previously associated with epilepsy; we saw the strongest enrichment in individuals with DEEs and the least strong in individuals with NAFE. Moreover, we found that inhibitory GABA A receptor genes were enriched for missense variants across all three classes of epilepsy, whereas no enrichment was seen in excitatory receptor genes. The larger gene groups for the GABAergic pathway or cation channels also showed a significant mutational burden in DEEs and GGE. Although no single gene surpassed exome-wide significance among individuals with GGE or NAFE, highly constrained genes and genes encoding ion channels were among the lead associations; such genes included CACNA1G, EEF1A2, and GABRG2 for GGE and LGI1, TRIM3, and GABRG2 for NAFE. Our study, the largest epilepsy WES study to date, confirms a convergence in the genetics of severe and less-severe epilepsies associated with ultra-rare coding variation, and it highlights a ubiquitous role for GABAergic inhibition in epilepsy etiology.

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MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5′ ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA.

Perfect direct repeats and, in particular, the prominent 13-bp repeat, are thought to cause mitochondrial DNA (mtDNA) deletions, which have been associated with the aging process. Accordingly, individuals lacking the 13-bp repeat are highly prevalent among centenarians and the number of repeats negatively correlates with mammalian longevity. However, detailed examination of the distribution of mtDNA deletions challenges the role of the 13-bp repeat and other perfect repeats in generating mtDNA deletions. Instead, deletions appear to depend on long and stable, albeit imperfect, duplexes between distant mtDNA segments. Furthermore, significant dissimilarities in breakpoint distributions suggest that multiple mechanisms are involved in creating mtDNA deletions. Direct repeats in the mitochondrial genome and longevityThe premise that accumulation of mtDNA mutations [1] and, in particular, of large-scale deletions in mtDNA is one of the possible causes of aging has received substantial support from biochemical and longevity studies [2], [3], [4], [5]. mtDNA deletions are usually flanked by direct repeats, implying that these repeats are involved in the generation of deletions. Recombination [6], [7], slip-replication [8], and double-stranded break repair [9] have been suggested as potential alternative mechanisms involving direct repeats. In corroboration of the connection between mtDNA deletions and aging, the number of direct repeats in mtDNA of various mammal species is inversely correlated with longevity [10], [11]. Of particular interest is the so-called "common deletion" [6], the deletion most frequently detected in humans, which is flanked by a prominent 13-bp perfect direct repeat.Corresponding author: Konstantin Khrapko (kkhrapko@gmail.com). * XG and KP equally contributed to the study Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Interestingly, the carriers of the well studied D4a mitochondrial haplogroup, who are significantly enriched among Japanese centenarians [12], lack the 13-bp direct repeat in their mtDNA, and thus presumably lack the common deletion, which seems to support the premise that deletions are involved in the aging process [13]. It should be noted, however, that although the "common" deletion is the most abundant mtDNA deletion, it typically constitutes no more than 10% of all deletions in aging tissues [5]. Therefore D4a individuals would have at most 10% fewer deletions, which perhaps is too moderate a change to affect longevity. There is another possibility, though [13]. According to an elegant hypothesis by Samuels, Schon and Chinnery...

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