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The consequences of DNA lesions for mitochondrial DNA maintenance
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Sjoerd Wanrooij)
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Eukaryotic cells have their own energy-producing organelles called mitochondria. The energy is stored in the adenosine triphosphate (ATP) molecule and is produced via the oxidative phosphorylation process inside the mitochondria. Thirteen of the essential proteins required for this process are encoded on the mitochondrial DNA (mtDNA). To ensure sufficient energy production it is therefore important to maintain mtDNA integrity. MtDNA maintenance is dependent on several factors, which include the replicative DNA polymerase. In humans, the main mitochondrial polymerase is DNA polymerase gamma (Pol γ), whereas in S. cerevisiae the homolog is called Mip1. Defects in the mitochondrial DNA polymerase and mtDNA replication in general cause mitochondrial dysfunction, reduced energy production and, in humans, mitochondrial diseases. 

DNA damage and non-standard nucleotides are frequently forming obstacles to the DNA replication machinery. One of the proteins that assists the nuclear replication machinery in dealing with DNA damage is the primase-polymerase PrimPol, performing either translesion DNA synthesis or alternatively priming replication restart after DNA damage. More recently, PrimPol was also identified inside the mitochondria. We therefore investigated the potential role of PrimPol to assist the mtDNA replication machinery at the site of mtDNA damage. Our results suggest that PrimPol does not work as a conventional translesion DNA polymerase at oxidative damage in the mitochondria, but instead interacts with the mtDNA replication machinery to support restart after replication stalling.

Stalling of DNA replication can also occur at wrongly inserted nucleotides. In this study, we pay extra attention to ribonucleotides, which are non-standard nucleotides in the context of DNA. Ribonucleotides (rNTPs) are normally building blocks for RNA but are occasionally utilized by DNA polymerases during DNA replication. Ribonucleotides are more reactive compared to dNTPs as they have an additional hydroxyl group (-OH). Their presence in the genome can lead to replication stress and genomic instability. In nuclear DNA, ribonucleotides are efficiently removed by the Ribonucleotide Excision Repair (RER) pathway and failure to remove them leads to human disease (e.g., Aicardi-Goutières syndrome). We investigated if ribonucleotides are removed from mtDNA and if not, how the replication machinery can tolerate the presence of ribonucleotides in the mtDNA.  

By using several yeast strains with altered dNTP pools, we found that the RER pathway is not active in mitochondria. Instead, mitochondria have an innate tolerance to ribonucleotide incorporation in mtDNA and under normal cellular conditions mature human mtDNA contains ~50 ribonucleotides per genome. We show that this ribonucleotide tolerance is the result of human Pol γ’s remarkable abilities to 1) efficiently bypass ribonucleotides in the DNA template and 2) proficiently discriminate against the incorporation of free ribonucleotides during mtDNA replication. Pol γ’s discrimination capability against free ribonucleotides comes with a price. In the presence of high rNTP levels, Pol γ is inhibited in DNA synthesis and could eventually lead to frequent replication stalling. Together, these studies are in line with our hypothesis that ribonucleotides in mtDNA can be tolerated, with the consequence that mtDNA replication is in particular vulnerable to imbalances in rNTP/dNTP ratios.

In summary, this study shows that we cannot simply extrapolate our knowledge of nuclear DNA replication stress management to the mtDNA maintenance, highlighting the need to study the molecular mechanism by which the mtDNA replication machinery is able to cope with DNA lesions to prevent loss of mtDNA integrity and disease development. 

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2021. , p. 70
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2133
Keywords [en]
Mitochondria, mitochondrial DNA, DNA replication, DNA lesions, DNA polymerase γ, ribonucleotide incorporation
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-182679ISBN: 978-91-7855-543-7 (electronic)ISBN: 978-91-7855-542-0 (print)OAI: oai:DiVA.org:umu-182679DiVA, id: diva2:1548715
Public defence
2021-05-28, Carl Kempe salen (KB.E3.03), KBC huset, Linnaeus väg 6, 90736, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2021-05-07 Created: 2021-05-03 Last updated: 2024-07-02Bibliographically approved
List of papers
1. Oxidative DNA damage stalls the human mitochondrial replisome
Open this publication in new window or tab >>Oxidative DNA damage stalls the human mitochondrial replisome
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2016 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 6, article id 28942Article in journal (Refereed) Published
Abstract [en]

Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e. DNA polymerase. holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage, and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro, suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-124218 (URN)10.1038/srep28942 (DOI)000378888000001 ()27364318 (PubMedID)2-s2.0-84976902620 (Scopus ID)
Available from: 2016-08-01 Created: 2016-07-28 Last updated: 2024-07-02Bibliographically approved
2. PrimPol is required for replication reinitiation after mtDNA damage
Open this publication in new window or tab >>PrimPol is required for replication reinitiation after mtDNA damage
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2017 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 43, p. 11398-11403Article in journal (Refereed) Published
Abstract [en]

Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins. Our results not only help in the understanding of how mitochondria cope with replicative stress but can also explain some controversial features of the lagging-strand replication.

Place, publisher, year, edition, pages
National Academy of Sciences, 2017
Keywords
DNA repair, fork rescue, mtDNA damage, mtDNA replication
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-141811 (URN)10.1073/pnas.1705367114 (DOI)000413520700056 ()29073063 (PubMedID)2-s2.0-85025835594 (Scopus ID)
Available from: 2017-11-27 Created: 2017-11-27 Last updated: 2023-03-23Bibliographically approved
3. Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired
Open this publication in new window or tab >>Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired
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2017 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 47, p. 12466-12471, article id 201713085Article in journal (Refereed) Published
Abstract [en]

Incorporation of ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove incorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for removal of ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange.

Place, publisher, year, edition, pages
National Academy of Sciences, 2017
Keywords
DNA replication, dNTP, mitochondrial DNA, ribonucleotide excision repair, ribonucleotide incorporation
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-141580 (URN)10.1073/pnas.1713085114 (DOI)000416503700053 ()29109257 (PubMedID)2-s2.0-85034591108 (Scopus ID)
Available from: 2017-11-08 Created: 2017-11-08 Last updated: 2024-07-02Bibliographically approved
4. The presence of rNTPs decreases the speed of mitochondrial DNA replication
Open this publication in new window or tab >>The presence of rNTPs decreases the speed of mitochondrial DNA replication
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2018 (English)In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 14, no 3, article id e1007315Article in journal (Refereed) Published
Abstract [en]

Ribonucleotides (rNMPs) are frequently incorporated during replication or repair by DNA polymerases and failure to remove them leads to instability of nuclear DNA (nDNA). Conversely, rNMPs appear to be relatively well-tolerated in mitochondnal DNA (mtDNA), although the mechanisms behind the tolerance remain unclear. We here show that the human mitochondrial DNA polymerase gamma (Pol gamma) bypasses single rNMPs with an unprecedentedly high fidelity and efficiency. In addition, Pol gamma exhibits a strikingly low frequency of rNMP incorporation, a property, which we find is independent of its exonuclease activity. However, the physiological levels of free rNTPs partially inhibit DNA synthesis by Pol gamma and render the polymerase more sensitive to imbalanced dNTP pools. The characteristics of Pol gamma reported here could have implications for forms of rntDNA depletion syndrome (MDS) that are associated with imbalanced cellular dNTP pools. Our results show that at the rNTPidNIP ratios that are expected to prevail in such disease states, Pol gamma enters a polymerasetexonuclease idling mode that leads to mtDNA replication stalling. This could ultimately lead to mtDNA depletion and, consequently, to mitochondrial disease phenotypes such as those observed in MDS.

Place, publisher, year, edition, pages
Public library science, 2018
National Category
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-146802 (URN)10.1371/journal.pgen.1007315 (DOI)000428840600053 ()29601571 (PubMedID)2-s2.0-85044827320 (Scopus ID)
Available from: 2018-04-26 Created: 2018-04-26 Last updated: 2024-07-02Bibliographically approved

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Forslund, Josefin M. E.

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