POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity
Open accessAbstract
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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The accessory subunit of Pol γ is required for enhanced DNA processivity by augmenting the affinity of the catalytic subunit for DNA.
“The accessory subunit is required for enhanced DNA processivity by augmenting the affinity of the catalytic subunit for DNA (Lim et al., 1999; Young et al., 2015).”
From Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis, aging and age-related diseases (2017) · cites this paper as Young et al., 2015
The WT/G451E heterodimer has significantly reduced stimulation of PolG processivity due to weaker binding to both PolG and DNA.
“Only heterozygotes have been clinically identified, and recent biochemical characterization of heterodimers has found that the WT/G451E heterodimer has significantly reduced stimulation of PolG processivity due to weaker binding to both PolG and DNA (Young et al., 2015).”
From Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis, aging and age-related diseases (2017) · cites this paper as Young et al., 2015
The WT/R369G heterodimer functions like the wild-type enzyme in vitro.
“The WT/R369G heterodimer functions like the wild-type enzyme in vitro.”
From Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis, aging and age-related diseases (2017) · cites this paper as Young et al., 2015
Both WT/G451E and WT/R369G mutant cell lines have significantly reduced reserve capacity in bioenergetic analysis of transfected HEK-293 cells.
“Though WT/G451E and WT/R369G localize to mtDNA like WT PolG2, bioenergetic analysis of transfected HEK-293 cells finds that both mutant cell lines have significantly reduced reserve capacity (Young et al., 2015).”
From Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis, aging and age-related diseases (2017) · cites this paper as Young et al., 2015
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- [4]Cohen B.H., Chinnery P.F., Copeland W.C. (2010) *POLG*-Related Disorders, *GeneReviews at GeneTests: Medical Genetics Information Resource [database online]* *.* *Copyright**,* University of Washington, Seattle, 1997–2010. Available at <http://www.genetests.org>. [[PubMed](https://pubmed.ncbi.nlm.nih.gov/20301791/)] [[Google Scholar](https://scholar.google.com/scholar_lookup?Cohen%20B.H.,%20Chinnery%20P.F.,%20Copeland%20W.C.%20(2010)%20POLG-Related%20Disorders,%20GeneReviews%20at%20GeneTests:%20Medical%20Genetics%20Information%20Resource%20[database%20online]%20.%20Copyright,%20University%20of%20Washington,%20Seattle,%201997%E2%80%932010.%20Available%20at%20http://www.genetests.org.)]
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- Authors
- Young, M. J., Humble, M. M., DeBalsi, K. L., Sun, K. Y., & Copeland, W. C.
- Affiliations
- National Institutes of Health (US)
- Corresponding author
- Copeland, William C.
- Venue
- Human Molecular Genetics
- Publisher
- Oxford University Press (OUP)
- Published
- 2015-06-29
- Volume
- 24
- Issue
- 18
- Pages
- 5184-5197
- ISSN
- 0964-6906 (ISSN-L)
- 1460-2083
- PMC
- PMC4550827
- OpenAlex
- W2129196926
- Cited by
- 32
- References
- 42
- Citations by year
- 2026: 2 · 2025: 3 · 2024: 3 · 2023: 1 · 2022: 6 · 2021: 1 · 2020: 1 · 2019: 3 · 2018: 3 · 2017: 4 · 2016: 5
- FWCI
- 1.74
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- public-domain
- Locations
- Human Molecular Genetics (publishedVersion, public-domain)
- Europe PMC (PubMed Central) (publishedVersion, public-domain)
- PubMed Central (publishedVersion, public-domain)
- PubMed (publishedVersion)
- Funders
- NIH (1K99ES022638-01)
- National Institute of Environmental Health Sciences (065078)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
- Keywords
- POLG2, mitochondrial DNA polymerase gamma, dominant negative effect, heterodimer, mitochondrial DNA replication, mitochondrial DNA repair, mitochondrial localization, protein aggregation, mitochondrial bioenergetics, tandem affinity purification
- MeSH terms
- Carrier Proteins, Cell Line, DNA, DNA-Directed DNA Polymerase, DNA, Mitochondrial, Genes, Dominant, Humans, Mitochondria, Protein Binding, Recombinant Fusion Proteins, Gene Expression, Cell Respiration, Protein Subunits, Protein Transport, Protein Multimerization
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