POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and
Matthew J Young1, Margaret M Humble1, Karen L DeBalsi1
1Genome Integrity and Structural Biology Laboratory, NIEHS, National Institutes of Health, DHHS, Research Triangle Park, NC 27709, USA.
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.
Insights
Mitochondrial DNA polymerase gamma (polγ) accessory subunit (p55) variants cause POLG2-related disorders. Heterodimers of wild-type and mutant p55 impair polγ function, leading to mitochondrial dysfunction.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Human mitochondrial DNA (mtDNA) replication and repair rely on mtDNA polymerase gamma (polγ).
- Polγ comprises a catalytic subunit (p140) and a homodimeric accessory subunit (p55), encoded by POLG and POLG2, respectively.
- Mutations in POLG or POLG2 are linked to specific human disorders.
Purpose of the Study:
- To investigate the functional consequences of POLG2 mutations, particularly in heterozygous individuals.
- To develop a method for isolating and analyzing p55 heterodimers, which are present in heterozygotes.
- To elucidate the subcellular effects and impact on mitochondrial function of disease-associated p55 variants.
Main Methods:
- A tandem affinity strategy was employed to isolate p55 heterodimers.
- In vitro assays were used to assess the impact of heterodimers on polγ activity.
- HEK293 cells were transfected with plasmids encoding GFP-tagged p55 variants to analyze subcellular localization and mitochondrial function.
Main Results:
- The wild-type/G451E p55 heterodimer demonstrated impaired polγ function in vitro, identifying the p.G451E mutation as dominant-negative.
- p55 disease variants (P205R, L475DfsX2) exhibited abnormal diffuse mitochondrial localization and failed to form puncta associated with mtDNA nucleoids.
- Aberrant multimer formation was observed for P205R and L475DfsX2 p55 variants, and cells overexpressing these variants showed impaired mitochondrial reserve capacity.
Conclusions:
- POLG2 mutations can result in dominant-negative p55 variants, impacting polγ function through heterodimer formation.
- Abnormal protein folding, aggregation, and impaired mitochondrial bioenergetics contribute to the pathophysiology of POLG2-related disorders.
- These findings highlight the critical role of p55 subunit integrity and function in maintaining mitochondrial health.
More Related Videos
09:34Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Published on: April 4, 2018
08:56Modeling Mitochondrial Disease Using Brain Organoids: A Focus on Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes
Published on: October 10, 2025
Related Concept Videos
01:43Incomplete Dominance
01:16Pharmacogenetics of Drug Transporters: P-Glycoprotein and Solute Carrier Transporters
01:31Translation
Translation Produces the Building Blocks of Life
Proteins are...
01:31Translation
Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes the final step in the expression of genes. This process is carried out by ribosomes, complexes of protein and specialized RNA molecules. Ribosomes, transfer RNA (tRNA), and other proteins produce a chain of amino acids—the polypeptide—as the end product of translation.
Translation Produces the Building Blocks of...
01:46Electron Transport Chain: Complex I and II
ROS generation is regulated and maintained at moderate levels necessary...
01:48ATP Synthase: Mechanism