Mitochondrial Disorders Associated with the Mitochondrial DNA Polymerase g: A Focus on Intersubunit Interactions

Young, Matthew J.; Copeland, William C.

doi:10.1007/978-1-4614-3722-2_3

Cited by 8 publications

(8 citation statements)

References 65 publications

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“…Evidence supporting tight association of p55 monomers within the homodimer comes from studies demonstrating that the affinity between monomers is extremely high with an estimated K d of <0.1 nM (22) and the structure of the dimer interface is extensive at about 4000 Å 2 (21). In the absence of genetic linkage and cosegregation analyses, four nuclear mutations encoding p55 disease variants that are biochemically defective at stimulating polγ activity have previously been identified, P205R, R369G, G451E and L475DfsX2 p55 (16,(23)(24)(25) and Table 3. We predict that if homodimeric wild-type (WT) p55 molecules and defective variant p55 molecules could readily dissociate and re-associate, then polγ DNA synthesis reactions would fail or become defective.…”

Section: Introductionmentioning

confidence: 99%

“…Because all currently identified patients with POLG2-disorders harbor heterozygous POLG2 mutations that alter evolutionary conserved residues (23,25), we hypothesize that affected individuals would harbor mixtures of variant and WT p55 molecules. As previous biochemical studies suggest that monomers within the homodimer do not readily dissociate and because we expect both POLG2 alleles to be expressed equally, p55 should occur as 50% heterodimers, 25% WT homodimers and 25% variant homodimers in vivo.…”

Section: Introductionmentioning

confidence: 99%

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POLG2disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity

et al. 2015

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Human mitochondrial DNA (mtDNA) is replicated and repaired by the mtDNA polymerase gamma, polγ. Polγ is composed of three subunits encoded by two nuclear genes: (1) POLG codes for the 140-kilodalton (kDa) catalytic subunit, p140 and (2) POLG2 encodes the ∼110-kDa homodimeric accessory subunit, p55. Specific mutations are associated with POLG- or POLG2-related disorders. During DNA replication the p55 accessory subunit binds to p140 and increases processivity by preventing polγ's dissociation from the template. To date, studies have demonstrated that homodimeric p55 disease variants are deficient in the ability to stimulate p140; however, all patients currently identified with POLG2-related disorders are heterozygotes. In these patients, we expect p55 to occur as 25% wild-type (WT) homodimers, 25% variant homodimers and 50% heterodimers. We report the development of a tandem affinity strategy to isolate p55 heterodimers. The WT/G451E p55 heterodimer impairs polγ function in vitro, demonstrating that the POLG2 c.1352G>A/p.G451E mutation encodes a dominant negative protein. To analyze the subcellular consequence of disease mutations in HEK293 cells, we designed plasmids encoding p55 disease variants tagged with green fluorescent protein (GFP). P205R and L475DfsX2 p55 variants exhibit irregular diffuse mitochondrial fluorescence and unlike WT p55, they fail to form distinct puncta associated with mtDNA nucleoids. Furthermore, homogenous preparations of P205R and L475DfsX2 p55 form aberrant reducible multimers. We predict that abnormal protein folding or aggregation or both contribute to the pathophysiology of these disorders. Examination of mitochondrial bioenergetics in stable cell lines overexpressing GFP-tagged p55 variants revealed impaired mitochondrial reserve capacity.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

POLG2disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity

et al. 2015

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“…The Arg182 residue in POLG2 is conserved across vertebrates (Fig. 4D), where it exists as a dimer [Young and Copeland 2013], but is not conserved in insects, in which POLG2 exist as monomers. Thus the Arg182Trp substitution may disrupt POLG2 dimerization.…”

Section: Resultsmentioning

confidence: 99%

Whole exome sequencing identifies a homozygous POLG2 missense variant in an infant with fulminant hepatic failure and mitochondrial DNA depletion

Varma

Faust

Iglesias

et al. 2016

European Journal of Medical Genetics

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Mitochondrial DNA (mtDNA) depletion syndrome manifests as diverse early-onset diseases that affect skeletal muscle, brain and liver function. Mutations in several nuclear DNA-encoded genes cause mtDNA depletion. We report on a patient, a 3-month-old boy who presented with hepatic failure, and was found to have severe mtDNA depletion in liver and muscle. Whole-exome sequencing identified a homozygous missense variant (c.544C>T, p.R182W) in the accessory subunit of mitochondrial DNA polymerase gamma (POLG2), which is required for mitochondrial DNA replication. This variant is predicted to disrupt a critical region needed for homodimerization of the POLG2 protein and cause loss of processive DNA synthesis. Both parents were phenotypically normal and heterozygous for this variant. Heterozygous mutations in POLG2 were previously associated with progressive external ophthalmoplegia and mtDNA deletions. This is the first report of a patient with a homozygous mutation in POLG2 and with a clinical presentation of severe hepatic failure and mitochondrial depletion.

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“…(ii) Null mutations in Drosophila melanogaster POLG2 cause lethality in the early pupal stage [ 27 ] and (iii) oocyte maturation is affected in a porcine POLG2 knockdown model [ 28 ]. Until recently, the clinically identified mutations in POLG2 were all heterozygous mutations [ 13 , 22 – 24 ]. The Chr17: 62492543G>A POLG2 mutation (R182W p55) was the first homozygous POLG2 mutation identified, and the affected infant presented with liver failure at 3 months of age and subsequently died at 9 months [ 29 ].…”

Section: Discussionmentioning

confidence: 99%

“…A small collection of heterozygous POLG2 mutations has also been identified and analyzed both biochemically and in vivo . These include P205R, R369G, G451E and L475DfsX2, which were originally characterized as mutant homodimers [ 13 , 22 – 24 ]. Recent work characterizing heterodimers with one WT and one mutant copy of p55 implicate the G451E as a strong dominant negative mutation [ 25 ].…”

Section: Introductionmentioning

confidence: 99%

Characterization of the human homozygous R182W POLG2 mutation in mitochondrial DNA depletion syndrome

et al. 2018

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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.

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Mitochondrial Disorders Associated with the Mitochondrial DNA Polymerase g: A Focus on Intersubunit Interactions

Cited by 8 publications

References 65 publications

POLG2disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity

POLG2disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity

Whole exome sequencing identifies a homozygous POLG2 missense variant in an infant with fulminant hepatic failure and mitochondrial DNA depletion

Characterization of the human homozygous R182W POLG2 mutation in mitochondrial DNA depletion syndrome

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