Mutant POLG2 disrupts DNA polymerase gamma subunits and causes progressive external ophthalmoplegia

Abstract

DNA polymerase gamma (pol gamma ) is required to maintain the genetic integrity of the 16,569-bp human mitochondrial genome (mtDNA). Mutation of the nuclear gene for the catalytic subunit of pol gamma (POLG) has been linked to a wide range of mitochondrial diseases involving mutation, deletion, and depletion of mtDNA. We describe a heterozygous dominant mutation (c.1352G-->A/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol gamma , that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)-deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype.

Figure 1

Skeletal muscle histochemistry and mtDNA analysis. A, Dual COX-SDH histochemistry (see the “Subjects and Methods” section) showing a mosaic distribution of COX-deficient muscle fibers (blue) among fibers with normal COX activity (brown). B, Long-range PCR of skeletal muscle mtDNA (extracted from a tissue homogenate). Lane 1, 1-kb ladder; lane 2, young control muscle; lane 3, age-matched control muscle; lane 4, patient muscle showing multiple mtDNA deletions. C, Real-time PCR analysis of COX-negative muscle fibers. The majority of fibers contain high-percentage levels of mtDNA deletions that remove the ND4 region (see the “Subjects and Methods” section).

Figure 2

A novel mutation in POLG2. A, Pedigree of the proband (II-2, arrow) and her unaffected siblings. B, Sequence traces showing a heterozygous base substitution in the proband, which is absent in her unaffected sisters. C, DHPLC traces for an exon 8 amplicon from the proband (upper) and control individual (lower). D, Amino acid conservation across species in exon 8 of POLG2, with codon 451 identified in red.

Figure 3

Effects of WT p55 and G451E p55 on pol γ activity. DNA polymerase activity was measured on poly(rA)-oligo(dT), as described in the “Subjects and Methods” section. Reactions contained 12 ng (88 fmol) p140, either alone (squares) or with 9.6 ng (178 fmol) of WT p55 (triangles) or G451E p55 (circles), and the indicated amounts of NaCl. Values are the averages of two independent measurements.

Figure 4

Analysis of pol γ subunits by analytical single-stranded DNA–cellulose chromatography. Purified recombinant p140, WT p55, and G451E p55 were resolved by single-stranded DNA–cellulose chromatography, as described in the “Subjects and Methods” section. Elution of proteins from the column was monitored by absorbance at 280 nm. A, WT p55 (dashed line), G451E p55 (solid line), or p140 (dotted line) were applied separately and eluted with an NaCl gradient. B, The p140 catalytic subunit (29 μg) was mixed with 31 μg WT p55 (dashed line) or 27 μg G451E p55 (solid line) and was eluted as before. Samples of the indicated fractions of the p140-WT p55 profile (C) and the p140-G451E p55 profile (D) were resolved by SDS-PAGE and were stained with Coomassie Brilliant Blue, as described in the “Subjects and Methods” section. Arrows indicate the individual migration positions of p140 and p55 (C and D).

Figure 5

G451E p55 only partially protects p140 against inactivation by NEM. DNA polymerase activity was measured as described in the “Subjects and Methods” section, except that 2-mercaptoethanol was excluded. Reactions contained 75 mM NaCl, 8.0 ng (58 fmol) p140—either alone (squares) or in the presence of 6.3 ng (116 fmol) WT p55 (triangles) or G451E p55 (circles)—and the indicated amounts of NEM. Values are the average of at least two independent measurements.

Figure 6

G451E p55 fails to coimmunoprecipitate with p140. The p140-dependent coimmunoprecipitation of WT p55 or G451E p55 with immobilized anti-p140 rabbit antibodies was assayed as described in the “Subjects and Methods” section. Samples contained WT p55 alone (lane 1), p140 catalytic subunit and WT p55 (lane 2), G451E mutant p55 (lane 3), p140 and G451E mutant p55 (lane 4), or 0.2 μg each of p140 and p55 loaded directly as positive controls for the western blot (lane 5). The positions of p140 and p55 standards are indicated with arrows.

Figure 7

G451E p55 does not enhance processive DNA synthesis of pol γ. Primer-extension reactions were performed as described in the “Subjects and Methods” section. Reactions contained p140 catalytic subunit (lanes 1–6), WT p55 (lanes 3 and 4), G451E p55 (lanes 5 and 6), and singly primed M13 DNA. Activity was measured at 0 mM NaCl (odd-numbered lanes) or 150 mM NaCl (even-numbered lanes). Lane 0 had no enzyme. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis (A) and alkaline agarose gel electrophoresis (B). The arrows in panels A and B mark the unextended 35-mer primer. Markers indicate the number of nucleotides synthesized past the primer.

Figure 8

Homologous position of the G451E side chain on the ribbon structure of the homodimeric mouse accessory subunit. The ribbon drawing was generated with Swiss PDB viewer from Protein Data Bank file 1G5H. Each monomer is colored either blue or red. Amino acids in domains 1 and 2 constitute the dimer interfaces, which are far removed from the homologous position of the G451E substitution in human PEO (arrows). Helices A–M are explained in the “Discussion” section.