| 1. |
- Erdinc, Direnis
(författare)
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Biochemical studies of mitochondrial DNA maintenance and topology
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondria are crucial organelles in eukaryotic cells that produce the majority of adenosine trisphosphate used by cells as an energy currency to drive metabolic processes. Due to the endosymbiotic origin of mitochondria, they have their own genetic material, a small circular double-stranded molecule (mtDNA) of 16.6 kbp in size that encodes 13 essential subunits of the oxidative phosphorylation system. All other mitochondrial proteins are nuclear encoded, including the components of the dedicated enzymatic machineries used to replicate, transcribe, and translate genetic material. The organelle also contains a large number of factors needed to maintain genome integrity and proper topology. In humans, numerous disease-causing mutations affecting factors required for mtDNA maintenance and expression have been identified. These pathological changes impair mitochondrial function and cause a wide variety of symptoms, primarily affecting high-energy demand organs. In this thesis, we use a combination of in vitro biochemistry and cell biology to study the molecular mechanisms underlying mitochondrial disease associated with disturbed mtDNA maintenance. In mitochondria, topoisomerase 3a (TOP3A) is required to separate newly formed genomes after the completion of mtDNA replication, whereas the nuclear isoform of the same enzyme is required to process Holliday junctions. Here, we demonstrate that TOP3A and TOP1MT are the only topoisomerases with clear mitochondrial localization. The two topoisomerases together control the topological states of mtDNA and are required for proper genome maintenance and expression. We also characterize a number of new disease-causing mutations affecting TOP3A and causing adult-onset mitochondrial diseases. Affected patients present with a common set of phenotypes, which are caused by both the nuclear and mitochondrial function of the TOP3A enzyme. We compared the effects of these mutations with mutations in the TOP3A gene, previously shown to cause Bloom syndrome, a genetic disease characterized by short stature and predisposition to cancer. Our findings suggest a model in which the overall severity of the TOP3A mutations determines the clinical presentations. Mutations with milder effects on TOP3A activity cause adult-onset mitochondrial disease, whereas mutations with more severe consequences are responsible for Bloom-like syndromes in combination with early-onset mitochondrial disease. DNA polymerase γ is the sole polymerase required for DNA replication in human mitochondria. The enzyme is a heterotrimer, with one catalytic subunit (POLγA) and two accessory subunits (POLγB). In our work, we characterize a new disease-causing mutation in POLγA (p.F907I). In its homozygous form, this recessive mutation causes severe disease phenotypes and death in infancy. In our work, we demonstrate that the mutation specifically impairs DNA synthesis on double-stranded DNA templates. In combination with structural modeling, we conclude that F907 is required for the coordinated function of POLγ and the replicative DNA helicase TWINKLE at the replication fork. Our study also suggests that direct interactions involving F907 may be formed between these two enzymes at the active replication fork. Finally, we study EXOG, a mitochondrial nuclease with both endo- and exonuclease activities. We find that EXOG is localized to the mitochondrial intermembrane and therefore cannot be an active component of the mtDNA replication machinery, as previously reported. We demonstrate that EXOG primarily acts on RNA substrates and suggest that EXOG might be involved in RNA degradation during the stress response or apoptosis.
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| 2. |
- Erdinc, Direnis, et al.
(författare)
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Pathological variants in TOP3A cause distinct disorders of mitochondrial and nuclear genome stability
- 2023
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Ingår i: Embo Molecular Medicine. - 1757-4676. ; 15:5
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Tidskriftsartikel (refereegranskat)abstract
- Topoisomerase 3 alpha (TOP3A) is an enzyme that removes torsional strain and interlinks between DNA molecules. TOP3A localises to both the nucleus and mitochondria, with the two isoforms playing specialised roles in DNA recombination and replication respectively. Pathogenic variants in TOP3A can cause a disorder similar to Bloom syndrome, which results from bi-allelic pathogenic variants in BLM, encoding a nuclear-binding partner of TOP3A. In this work, we describe 11 individuals from 9 families with an adult-onset mitochondrial disease resulting from bi-allelic TOP3A gene variants. The majority of patients have a consistent clinical phenotype characterised by bilateral ptosis, ophthalmoplegia, myopathy and axonal sensory-motor neuropathy. We present a comprehensive characterisation of the effect of TOP3A variants, from individuals with mitochondrial disease and Bloom-like syndrome, upon mtDNA maintenance and different aspects of enzyme function. Based on these results, we suggest a model whereby the overall severity of the TOP3A catalytic defect determines the clinical outcome, with milder variants causing adult-onset mitochondrial disease and more severe variants causing a Bloom-like syndrome with mitochondrial dysfunction in childhood.
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| 3. |
- Erdinc, Direnis, et al.
(författare)
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The disease-causing mutation p.F907I reveals a novel pathogenic mechanism for POL?-related diseases
- 2023
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Ingår i: Biochimica Et Biophysica Acta-Molecular Basis of Disease. - 0925-4439 .- 1879-260X. ; 1869:7
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in the catalytic domain of mitochondrial DNA polymerase ? (POL?) cause a broad spectrum of clinical conditions. POL? mutations impair mitochondrial DNA replication, thereby causing deletions and/or depletion of mitochondrial DNA, which in turn impair biogenesis of the oxidative phosphorylation system. We here identify a patient with a homozygous p.F907I mutation in POL?, manifesting a severe clinical phenotype with develop-mental arrest and rapid loss of skills from 18 months of age. Magnetic resonance imaging of the brain revealed extensive white matter abnormalities, Southern blot of muscle mtDNA demonstrated depletion of mtDNA and the patient deceased at 23 months of age. Interestingly, the p.F907I mutation does not affect POL? activity on single-stranded DNA or its proofreading activity. Instead, the mutation affects unwinding of parental double-stranded DNA at the replication fork, impairing the ability of the POL? to support leading-strand DNA synthesis with the TWINKLE helicase. Our results thus reveal a novel pathogenic mechanism for POL?-related diseases.
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| 4. |
- Menger, K. E., et al.
(författare)
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Two type I topoisomerases maintain DNA topology in human mitochondria
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 50:19, s. 11154-11174
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Tidskriftsartikel (refereegranskat)abstract
- Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.
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