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Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Andrei Chabes)ORCID iD: 0000-0002-8607-7564
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Sjoerd Wanrooij)
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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. Vol. 114, no 47, p. 12466-12471, article id 201713085
Keywords [en]
DNA replication, dNTP, mitochondrial DNA, ribonucleotide excision repair, ribonucleotide incorporation
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-141580DOI: 10.1073/pnas.1713085114ISI: 000416503700053PubMedID: 29109257Scopus ID: 2-s2.0-85034591108OAI: oai:DiVA.org:umu-141580DiVA, id: diva2:1155535
Available from: 2017-11-08 Created: 2017-11-08 Last updated: 2024-07-02Bibliographically approved
In thesis
1. The consequences of DNA lesions for mitochondrial DNA maintenance
Open this publication in new window or tab >>The consequences of DNA lesions for mitochondrial DNA maintenance
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
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:nbn:se:umu:diva-182679 (URN)978-91-7855-543-7 (ISBN)978-91-7855-542-0 (ISBN)
Public defence
2021-05-28, Carl Kempe salen (KB.E3.03), KBC huset, Linnaeus väg 6, 90736, Umeå, 09:00 (English)
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Available from: 2021-05-07 Created: 2021-05-03 Last updated: 2024-07-02Bibliographically approved

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Wanrooij, Paulina H.Forslund, Josefin M. E.Nilsson, Anna KarinWanrooij, SjoerdChabes, Andrei

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Wanrooij, Paulina H.Forslund, Josefin M. E.Nilsson, Anna KarinWanrooij, SjoerdChabes, Andrei
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Department of Medical Biochemistry and BiophysicsMolecular Infection Medicine Sweden (MIMS)
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Proceedings of the National Academy of Sciences of the United States of America
Cell and Molecular Biology

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