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. 2018 Aug 29;13(8):e0203198.
doi: 10.1371/journal.pone.0203198. eCollection 2018.

Characterization of the Human Homozygous R182W POLG2 Mutation in Mitochondrial DNA Depletion Syndrome

Free PMC article

Characterization of the Human Homozygous R182W POLG2 Mutation in Mitochondrial DNA Depletion Syndrome

Kirsten E Hoff et al. PLoS One. .
Free PMC article


Mutations in mitochondrial DNA (mtDNA) have been linked to a variety of metabolic, neurological and muscular diseases which can present at any time throughout life. MtDNA is replicated by DNA polymerase gamma (Pol γ), twinkle helicase and mitochondrial single-stranded binding protein (mtSSB). The Pol γ holoenzyme is a heterotrimer consisting of the p140 catalytic subunit and a p55 homodimeric accessory subunit encoded by the nuclear genes POLG and POLG2, respectively. The accessory subunits enhance DNA binding and promote processive DNA synthesis of the holoenzyme. Mutations in either POLG or POLG2 are linked to disease and adversely affect maintenance of the mitochondrial genome, resulting in depletion, deletions and/or point mutations in mtDNA. A homozygous mutation located at Chr17: 62492543G>A in POLG2, resulting in R182W substitution in p55, was previously identified to cause mtDNA depletion and fatal hepatic liver failure. Here we characterize this homozygous R182W p55 mutation using in vivo cultured cell models and in vitro biochemical assessments. Compared to control fibroblasts, homozygous R182W p55 primary dermal fibroblasts exhibit a two-fold slower doubling time, reduced mtDNA copy number and reduced levels of POLG and POLG2 transcripts correlating with the reported disease state. Expression of R182W p55 in HEK293 cells impairs oxidative-phosphorylation. Biochemically, R182W p55 displays DNA binding and association with p140 similar to WT p55. R182W p55 mimics the ability of WT p55 to stimulate primer extension, support steady-state nucleotide incorporation, and suppress the exonuclease function of Pol γ in vitro. However, R182W p55 has severe defects in protein stability as determined by differential scanning fluorimetry and in stimulating function as determined by thermal inactivation. These data demonstrate that the Chr17: 62492543G>A mutation in POLG2, R182W p55, severely impairs stability of the accessory subunit and is the likely cause of the disease phenotype.

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1
Fig 1. Location of the R182W amino acid substitution on the human apo p55 crystal structure.
(A) Overview of the human apo p55 dimer structure. One monomer has been colored blue while the other monomer is colored green. Highlighted in red spheres is R182. (B) A 90o rotation of the structure. PDB: 1G5H [12].
Fig 2
Fig 2. Patient dermal fibroblasts grow more slowly than control fibroblasts, have reduced mtDNA copy number and reduced POLG/POLG2 transcript levels.
(A) 8-day growth curves comparing control (black lines) and patient (red lines) dermal fibroblasts grown in glucose. Every point on the curves is the average of three independent growths. (B) MtDNA copy number was determined by quantitative PCR of the mitochondrial genes ND1 and ND4 and normalized to the nuclear gene RPPH1, as described in Materials and methods. MtDNA copy number for control fibroblasts (black circles–ND1/black triangles-ND4) or patient dermal fibroblasts (red circles-ND1/red triangles-ND4) are shown. Standard curves of plasmids containing the amplified regions of interest allowed for absolute copy number to be determined by real time PCR. Experiments were done in triplicate, and N = 5 for each data point. (C) Transcript levels for POLG and POLG2 in patient fibroblasts were determined by quantitative PCR and normalized to ACTB as described in Materials and methods. WT p55 (black circles–POLG/black triangles–POLG2) R182W p55 (Red circles–POLG/red triangles–POLG2). Experiments were done in triplicate, and N = 5 for each data point.
Fig 3
Fig 3. Seahorse Flux Analyzer bioenergetics analysis of transfected HEK293 cells illustrates significant reduction in respiration.
(A) Representative graph of the oxygen consumption rate (OCR). ATP-linked oxygen consumption and oxygen consumption associated with proton leakage can be differentiated by inhibiting ATP synthase with oligomycin. Uncoupling electron transport and ATP synthase with 2,4-dinitrophenol (2,4-DNP) reveals the maximum rate of oxygen consumption. The reserve respiratory capacity is the difference between maximal and basal respiration rates. Complete inhibition of respiration using a combination of rotenone/antimycin A determines non-mitochondrial oxygen consumption. The average basal OCR (B), maximal OCR (C), reserve respiratory capacity (D), proton leakage (E) and ATP coupled production (F) for untransfected HEK293 (gray bars), WT POLG2 HEK293 (black bars) and R182W POLG2 HEK293 (red bars) cells are shown. Values are averages ± SEM, N = 3 per group, normalized to cells seeded. * p ≤ 0.05 versus the WT POLG2 transfected HEK293 cells as determined by Students t-test.
Fig 4
Fig 4. Stimulation of Pol γ processive DNA synthesis is similar for WT p55 and R182W p55.
Primer extension reactions were performed and analyzed by denaturing PAGE as described in Materials and methods. Samples with or without p55 were assessed at 0 mM NaCl or at 150 mM NaCl, as indicated. Lanes 1 and 6 exclude enzyme. Lanes 2, 4, 8 and 9 exclude p55. Lanes 3 and 7 include WT p55 while lanes 5 and 10 include R182W p55. Lanes 1–5 have 0 mM NaCl while lanes 6–10 have 150 mM NaCl. The gel is representative of three independent experiments.
Fig 5
Fig 5. R182W p55 has a stability defect that disrupts p140 stimulation.
(A) Differential Scanning Fluorimetry with SYPRO Orange dye. Protein was incubated with SYPRO Orange and warmed from 25°C to 95°C, as described in Materials and methods. Fluorescence of SYPRO Orange increases as more hydrophobic patches become available for binding, thereby reporting the degree of protein denaturation. R182W p55 has a lower melting temperature than WT p55. (B) Thermostability of WT p55 and R182W p55 was assessed in a p140 stimulation assay, as described in Materials and methods. WT p55 and R182W p55 were incubated at 75°C for increasing amounts of time, and holoenzymes were assembled and tested for the ability to incorporate dTTP. The half-life for thermal denaturation of R182W p55 is 70-fold shorter than the half-life for WT p55.

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Grant support

This study was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (ES 065078). M.H. and A.B.N. are supported by NIH/NICHD grant 5P01 HD080642-02. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.