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

Abstract

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.

Human mitochondrial DNA replication machinery and disease

“…The p140 catalytic subunit harbors active sites for 5′-3′ DNA polymerase, 3′-5′ exonuclease, and 5′ dRP lyase activities [9,10]. The p55 imparts high processivity onto the holoenzyme by increasing the binding affinity to DNA [4,11]. The majority of intermolecular contacts occur between the C-terminal region of the ‘proximal’ p55 monomer (purple in Figure 2) and the AID subdomain (Accessory-Interacting Determinant subdomain that extends an an ‘arm’ around p55) of the p140 catalytic subunit [12–15].…”

“…Because currently identified POLG2 patients harbor heterozygous mutations, and because monomers within the p55 homodimer do not readily dissociate, the patients should harbor a mixture of p55 molecules: 25% WT homodimers, 25% variant homodimers, and 50% heterodimers [4]. Using a tandem affinity strategy and biochemistry to study p55 heterodimers we showed that one p55 disease variant, G451E, is dominant negative and associates with a wild-type p55 monomer in pol γ to poison the enzyme’s activity.…”

“…The remaining 24 genes encode 22 transfer RNAs and 2 ribosomal RNAs required for synthesis of the 13-mitochondrial polypeptides. A cell can contain several thousand copies of mtDNA distributed within hundreds of individual mitochondria [1] or within an elaborate intracellular network of reticular mitochondria Several proteins associate with mtDNA at distinct nucleoid structures on the matrix-side of the inner membrane [2], and such protein-mtDNA nucleoids can be visualized as foci or puncta via immunocytochemistry or live-cell fluorescence microscopy [3,4]. …”

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

“…POLG2 encodes the accessory subunit of DNA polymerase γ, and is required for imparting high processivity to POLG by increasing the affinity of the protein complex to DNA [Lim and others 1999]. Biochemical and functional studies of one pathogenic missense mutation (c.1352G>A; p.G451E) showed decreased processivity of the enzyme complex leading to multiple mitochondrial DNA deletions [Young and others 2015; Young and others 2011]. The second reported heterozygous insertion (c.1207–1208ins24) mutation causes mis-splicing and skipping of exon 7, thereby impairing the C-terminal domain that is required for processivity [Walter and others 2010].…”

Inherited mitochondrial genomic instability and chemical exposures

“…Mutations in POLG2 that cause disease have been shown to be defective in their ability to bind the catalytic subunit, bind to double stranded DNA, and/or self-dimerize (Craig et al 2012; Longley et al 2006; Young et al 2011). Cell and biochemical studies by Young et al showed that mutant POLG2 heterodimers can either poison the enzyme activity of the mtDNA polymerase or fail to bind nucleoids, ultimately leading to failed mtDNA replication, decreased bioenergetics and contributes to mitochondrial disease seen in patients (Young et al 2015). The TWNK mtDNA helicase, also known as Twinkle, is essential for mtDNA replication as it is needed to unwind the double helix ahead of the replication fork, as well as synthesis of nascent D-loop strands (Milenkovic et al 2013).…”