Loss-of-function mutations in MGME1 impair mtDNA replication and cause multisystemic mitochondrial disease

Journal name:
Nature Genetics
Year published:
Published online

Known disease mechanisms in mitochondrial DNA (mtDNA) maintenance disorders alter either the mitochondrial replication machinery (POLG, POLG2 and C10orf2)1, 2, 3 or the biosynthesis pathways of deoxyribonucleoside 5′-triphosphates for mtDNA synthesis4, 5, 6, 7, 8, 9, 10, 11. However, in many of these disorders, the underlying genetic defect has yet to be discovered. Here, we identify 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 show that MGME1 cleaves single-stranded DNA and processes DNA flap substrates. Fibroblasts from affected individuals do not repopulate after chemically induced mtDNA depletion. They also accumulate intermediates of stalled replication and show increased levels of 7S DNA, as do MGME1-depleted cells. Thus, we show that MGME1-mediated mtDNA processing is essential for mitochondrial genome maintenance.

At a glance


  1. Protein sequence, domain architecture, disease-causing alterations and subcellular localization of MGME1.
    Figure 1: Protein sequence, domain architecture, disease-causing alterations and subcellular localization of MGME1.

    (a) Domain architecture of MGME1 (top) indicating the sequence alterations identified in affected individuals. The key motifs of the PD−(D/E)XK nuclease superfamily are indicated with Roman numerals, and the sequence details for the motifs characteristic of the RecB-type subgroup are shown as colored boxes below the schematic. MTS indicates the predicted mitochondrial targeting signal. An alignment of protein sequences encoded by the orthologs of MGME1 and RecB-type nucleases is shown below. The c.698A>G (p.Tyr233Cys) mutation is highlighted in black. Colons denote chemical similarity between the sequences; asterisks indicate identical residues. The GenBank accession numbers of the sequences used in the alignment are as follows: human, NP_443097; mouse, NP_083260; chicken, XP_415017; green anole lizard, XP_003219931; Xenopus tropicalis, NP_001120532; zebrafish, NP_001008640; lancelet, XP_002610511; sea squirt, XP_002119404; Cyanobacteria, YP_002377694; Bacillus subtilis AddA, YP_004207075; E. coli RecB, EGB85545. (b) Predicted active site structural motif of MGME1 based on the known crystal structure of the homologous RecB-type nucleases. Structure prediction was made using the 3D-Jury algorithm and modeled using MODELLER software. The active site structure of E. coli RecB is provided for comparison. The colors used are as in a. (c) Protein blot showing the amounts of MGME1 protein in fibroblasts from P1976 (FB1976) and control fibroblasts. β-actin was used as a loading control. MW, molecular weight. (d) Cellular localization of the GFP-tagged variant of MGME1 (green) in human fibroblasts. mtSSBP was used as a mitochondrial marker (red). Nuclei were stained with DAPI (blue). Colocalization of the green and red signals appears yellow in digitally overlaid images. Scale bars, 10 μm.

  2. Loss of MGME1 leads to mtDNA depletion and higher levels of 7S DNA.
    Figure 2: Loss of MGME1 leads to mtDNA depletion and higher levels of 7S DNA.

    (a) Relative mtDNA copy numbers and 7S DNA/mtDNA ratios in skeletal muscle from individuals with MGME1 mutations. Data are shown as the averages ± s.d. from three independent quantitative PCR (qPCR) determinations and were normalized to corresponding control values, which are the averages ± s.d. from 11 skeletal muscle biopsy samples (age range of 18–33 years; mtDNA copy-number range of 8,146–11,416 molecules per nucleus). *P < 0.05, **P < 0.01; two-tailed Student's t test. a.u., arbitrary units. Ages at biopsy are indicated. (b) Total DNA from control or mutant (FB1976) fibroblasts (cell passage 15–21) and from untransfected HeLa cells or HeLa cells transfected with siRNA to GFP or MGME1 for 6 d analyzed by one-dimensional Southern blotting with a radioactive probe specific for the non-coding region in human mtDNA (14,258–4,121) followed by a probe specific for 18S ribosomal DNA. Relative DNA levels were obtained by quantifying PhosphorImager scans of Southern blots using ImageQuant software and normalizing for the values obtained for control fibroblasts or untransfected HeLa cells (bottom). *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student's t test; n = 3; error bars = 1 s.d. (c) Relative mtDNA copy numbers and 7S DNA/mtDNA ratios in fibroblasts from an individual with a MGME1-null mutation (P1976; cell passage 6–10) and a control. Where indicated, fibroblasts were transduced with low (+) or high (+++) titer of a lentivirus encoding MGME1. *P < 0.05, **P < 0.01; two-tailed Student's t test; n = 3; error bars = 1 s.d.

  3. Characterization of the enzymatic activity of MGME1.
    Figure 3: Characterization of the enzymatic activity of MGME1.

    (a) DNase activity of MGME1 and its preference for ssDNA versus dsDNA. Left, 1 pmol of radioactively labeled substrates (asterisks in the schematics indicate the label) were incubated for 30 min with increasing concentrations (0.125, 0.25, 0.5, 1, 2, 4 pmol) of purified MGME1 or 4 pmol of the Lys253Ala mutant (K253A). Reaction products were subjected to denaturing PAGE and autoradiography and were quantified. Right, substrate band intensity plotted against enzyme concentration. (b,c) MGME1 activity on 5′-displaced (b) and 3′-displaced (c) splayed-arm and flap-like DNA structures. Reaction conditions were the same as in a. (d) Comparison of MGME1 processing efficiency on the 5′-displaced (5′-flap) and 3′-displaced (3′-flap) flap-like DNA substrates with mapping of the radioactive products. Signal intensities of processed and partially processed reaction products were quantified and plotted as in a. (e) MGME1 activity on RNA-DNA chimeric substrates that resemble Okazaki fragments. Increasing concentrations of purified MGME1 were incubated with 5′ radioactively end-labeled RNA-DNA substrates. Numbers on the top indicate oligonucleotide lengths. SA, splayed-arm structure. Quantification of the reactions with PAGE and autoradiography is shown to the right.

  4. Perturbed mitochondrial replication in mutant fibroblasts and MGME1-depleted cells.
    Figure 4: Perturbed mitochondrial replication in mutant fibroblasts and MGME1-depleted cells.

    (a) mtDNA copy number during induced depletion and repopulation of human control fibroblasts and MGME1-null fibroblasts (FB1976). Depletion of mtDNA was achieved by the addition of 20 μM ddC to the culture medium for 12 d. mtDNA copy numbers were determined by qPCR using the mitochondrial primers MT3922F25 and MT4036R26. Data points for FB1976 represent the mean values from three determinations ± s.d. The data for control fibroblasts are shown as the averages ± s.d. of mtDNA depletion-repopulation experiments with two separate cell lines. *P < 0.05, **P < 0.01; two-tailed Student's t test. (b,c) mtDNA replication in MGME1-null fibroblasts and MGME1-depleted cells. Total DNA from control or FB1976 fibroblasts (b) and HeLa cells transfected with siRNA to GFP or MGME1 for 6 d (c) was subjected to two-dimensional neutral-neutral agarose gel electrophoresis followed by Southern blotting. Restriction enzymes and probes used (gray bars) are indicated to the left. The black bar indicates the non-coding region, and OH marks the origin of H-strand replication. The interpretation based on previous work23, 28 is provided at the right. 1n, non-replicating fragment. (d) Schematic showing the proposed involvement of MGME1 in mtDNA maintenance. Mutation or siRNA knockdown of MGME1 results in accumulation of replication intermediates followed by mtDNA depletion and 7S DNA accumulation (right black arrow up). In contrast, overexpression of MGME1 causes promiscuous degradation of mtDNA (including 7S DNA), resulting in mtDNA depletion (right black arrow down).


  1. Van Goethem, G. et al. Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat. Genet. 28, 211212 (2001).
  2. Longley, M.J. et al. Mutant POLG2 disrupts DNA polymerase γ subunits and causes progressive external ophthalmoplegia. Am. J. Hum. Genet. 78, 10261034 (2006).
  3. Spelbrink, J.N. et al. Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4–like protein localized in mitochondria. Nat. Genet. 28, 223231 (2001).
  4. Nishino, I., Spinazzola, A. & Hirano, M. Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder. Science 283, 689692 (1999).
  5. Kaukonen, J. et al. Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science 289, 782785 (2000).
  6. Mandel, H. et al. The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA. Nat. Genet. 29, 337341 (2001).
  7. Saada, A. et al. Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat. Genet. 29, 342344 (2001).
  8. Elpeleg, O. et al. Deficiency of the ADP-forming succinyl-CoA synthase activity is associated with encephalomyopathy and mitochondrial DNA depletion. Am. J. Hum. Genet. 76, 10811086 (2005).
  9. Spinazzola, A. et al. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion. Nat. Genet. 38, 570575 (2006).
  10. Bourdon, A. et al. Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nat. Genet. 39, 776780 (2007).
  11. Ostergaard, E. et al. Deficiency of the α subunit of succinate–coenzyme A ligase causes fatal infantile lactic acidosis with mitochondrial DNA depletion. Am. J. Hum. Genet. 81, 383387 (2007).
  12. Haack, T.B. et al. Molecular diagnosis in mitochondrial complex I deficiency using exome sequencing. J. Med. Genet. 49, 277283 (2012).
  13. Elstner, M. et al. MitoP2: an integrative tool for the analysis of the mitochondrial proteome. Mol. Biotechnol. 40, 306315 (2008).
  14. Calvo, S.E. et al. Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing. Sci. Transl. Med. 4, 118ra10 (2012).
  15. Pagliarini, D.J. et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, 112123 (2008).
  16. Steczkiewicz, K. et al. Sequence, structure and functional diversity of PD-(D/E)XK phosphodiesterase superfamily. Nucleic Acids Res. 40, 70167045 (2012).
  17. Aravind, L. et al. Holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories. Nucleic Acids Res. 28, 34173432 (2000).
  18. Singleton, M.R. et al. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432, 187193 (2004).
  19. Holt, I.J. et al. Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100, 515524 (2000).
  20. Liu, P. et al. Removal of oxidative DNA damage via FEN1-dependent long-patch base excision repair in human cell mitochondria. Mol. Cell. Biol. 28, 49754987 (2008).
  21. Brown, T.A. & Clayton, D.A. Release of replication termination controls mitochondrial DNA copy number after depletion with 2′,3′-dideoxycytidine. Nucleic Acids Res. 30, 20042010 (2002).
  22. Stewart, J.D. et al. POLG mutations cause decreased mitochondrial DNA repopulation rates following induced depletion in human fibroblasts. Biochim. Biophys. Acta 1812, 321325 (2011).
  23. Wanrooij, S. et al. Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes. Nucleic Acids Res. 35, 32383251 (2007).
  24. Copeland, W.C. & Longley, M.J. DNA2 resolves expanding flap in mitochondrial base excision repair. Mol. Cell 32, 457458 (2008).
  25. Zheng, L. et al. Human DNA2 is a mitochondrial nuclease/helicase for efficient processing of DNA replication and repair intermediates. Mol. Cell 32, 325336 (2008).
  26. Duxin, J.P. et al. Human Dna2 is a nuclear and mitochondrial DNA maintenance protein. Mol. Cell. Biol. 29, 42744282 (2009).
  27. Tann, A.W. et al. Apoptosis induced by persistent single-strand breaks in mitochondrial genome: critical role of EXOG (5′-EXO/endonuclease) in their repair. J. Biol. Chem. 286, 3197531983 (2011).
  28. Pohjoismäki, J.L. et al. Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid. J. Mol. Biol. 397, 11441155 (2010).
  29. McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 12971303 (2010).
  30. Zsurka, G. et al. Clonally expanded mitochondrial DNA mutations in epileptic individuals with mutated DNA polymerase γ. J. Neuropathol. Exp. Neurol. 67, 857866 (2008).
  31. Danhauser, K. et al. Cellular rescue-assay aids verification of causative DNA-variants in mitochondrial complex I deficiency. Mol. Genet. Metab. 103, 161166 (2011).
  32. Rorbach, J. et al. PDE12 removes mitochondrial RNA poly(A) tails and controls translation in human mitochondria. Nucleic Acids Res. 39, 77507763 (2011).
  33. Reyes, A. et al. Analysis of replicating mitochondrial DNA by two-dimensional agarose gel electrophoresis. Methods Mol. Biol. 372, 219232 (2007).

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Author information

  1. These authors contributed equally to this work.

    • Cornelia Kornblum,
    • Thomas J Nicholls &
    • Tobias B Haack


  1. Department of Neurology, University of Bonn Medical Center, Bonn, Germany.

    • Cornelia Kornblum
  2. Medical Research Council (MRC) Mitochondrial Biology Unit, Cambridge, UK.

    • Thomas J Nicholls,
    • Joanna Rorbach &
    • Michal Minczuk
  3. Institute of Human Genetics, Technische Universität München and Helmholtz Zentrum München–German Research Center for Environmental Health, Munich, Germany.

    • Tobias B Haack,
    • Katharina Danhauser,
    • Arcangela Iuso,
    • Thomas Wieland,
    • Tim M Strom,
    • Thomas Meitinger &
    • Holger Prokisch
  4. Department of Epileptology, University of Bonn Medical Center, Bonn, Germany.

    • Susanne Schöler,
    • Viktoriya Peeva,
    • Kerstin Hallmann,
    • Gábor Zsurka &
    • Wolfram S Kunz
  5. Life and Brain Center, University of Bonn Medical Center, Bonn, Germany.

    • Susanne Schöler,
    • Viktoriya Peeva,
    • Kerstin Hallmann,
    • Gábor Zsurka &
    • Wolfram S Kunz
  6. Neuromuscular Unit, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ca' Granda, Ospedale Maggiore Policlinico, Centro Dino Ferrari, University of Milan, Milan, Italy.

    • Monica Sciacco &
    • Maurizio Moggio
  7. Neurology Unit, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Centro Dino Ferrari, University of Milan, Milan, Italy.

    • Dario Ronchi &
    • Giacomo P Comi
  8. Department of Neurology, Columbia University Medical Center, New York, New York, USA.

    • Catarina M Quinzii &
    • Salvatore DiMauro
  9. Department of Molecular Biology and Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Sarah E Calvo &
    • Vamsi K Mootha
  10. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Sarah E Calvo &
    • Vamsi K Mootha
  11. Broad Institute, Cambridge, Massachusetts, USA.

    • Sarah E Calvo &
    • Vamsi K Mootha
  12. Department of Neurology, Friedrich-Baur-Institute, Ludwig-Maximilians-Universität München, Munich, Germany.

    • Thomas Klopstock


C.K. identified, clinically characterized, collected samples and histochemically analyzed skeletal muscle biopsies from family I and the sporadic case and obtained fibroblasts from P1976. M.S., D.R., G.P.C., M. Moggio, C.M.Q. and S.D. identified, clinically characterized, collected samples and histochemically analyzed skeletal muscle biopsies from family II and obtained fibroblasts from P4050 and P4052. T.B.H., T.W., T.M.S., T.M. and H.P. performed exome sequencing and analysis of family I. S.E.C. and V.K.M. performed targeted mitochondrial exome sequencing and analysis of family II. T.J.N., G.Z. and M. Minczuk performed the computational analysis. T.J.N. analyzed protein amounts, performed subcellular localization studies, purified and characterized recombinant MGME1 and analyzed the cells with siRNA knockdown and P1976 fibroblasts. T.B.H., K.D., A.I. and H.P. performed subcellular localization and complementation experiments. S.S. performed the mtDNA repopulation experiments. V.P. performed copy-number and deletion quantification. K.H. screened PEO samples for MGME1 mutations and identified P931. J.R. contributed to the characterization of fibroblasts from P1976. T.K. and T.M. provided samples and coordinated the German network of mitochondrial disorders. C.K., G.Z., M. Minczuk, W.S.K. and H.P. planned the project and wrote the manuscript.

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