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  • 1. Al-Behadili, Ali
    et al.
    Uhler, Jay P.
    Berglund, Anna-Karin
    Peter, Bradley
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Reyes, Aurelio
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zeviani, Massimo
    Falkenberg, Maria
    A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 18, p. 9471-9483Article in journal (Refereed)
    Abstract [en]

    The role of Ribonuclease H1 (RNase H1) during primer removal and ligation at the mitochondrial origin of light-strand DNA synthesis (OriL) is a key, yet poorly understood, step in mitochondrial DNA maintenance. Here, we reconstitute the replication cycle of L-strand synthesis in vitro using recombinant mitochondrial proteins and model OriL substrates. The process begins with initiation of DNA replication at OriL and ends with primer removal and ligation. We find that RNase H1 partially removes the primer, leaving behind the last one to three ribonucleotides. These 5′-end ribonucleotides disturb ligation, a conclusion which is supported by analysis of RNase H1-deficient patient cells. A second nuclease is therefore required to remove the last ribonucleotides and we demonstrate that Flap endonuclease 1 (FEN1) can execute this function in vitro. Removal of RNA primers at OriL thus depends on a two-nuclease model, which in addition to RNase H1 requires FEN1 or a FEN1-like activity. These findings define the role of RNase H1 at OriL and help to explain the pathogenic consequences of disease causing mutations in RNase H1.

  • 2.
    Al-Furoukh, Natalie
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ianni, Alessandro
    Nolte, Hendrik
    Hölper, Soraya
    Krüger, Marcus
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Braun, Thomas
    ClpX stimulates the mitochondrial unfolded protein response (UPRmt) in mammalian cells2015In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1853, no 10, p. 2580-2591Article in journal (Refereed)
    Abstract [en]

    Proteostasis is crucial for life and maintained by cellular chaperones and proteases. One major mitochondrial protease is the ClpXP complex, which is comprised of a catalytic ClpX subunit and a proteolytic ClpP subunit. Based on two separate observations, we hypothesized that ClpX may play a leading role in the cellular function of ClpXP. Therefore, we analyzed the effect of ClpX overexpression on a myoblast proteome by quantitative proteomics. ClpX overexpression results in the upregulation of markers of the mitochondria( proteostasis pathway, known as the "mitochondrial unfolded protein response" (UPRmt). Although this pathway is described in detail in Caenorhabditis elegans, it is not clear whether it is conserved in mammals. Therefore, we compared features of the classical nematode UPRmt with our mammalian ClpX-triggered UPRmt dataset. We show that they share the same retrograde mitochondria-to-nucleus signaling pathway that involves the key UPRmt transcription factor CHOP (also known as Ddit3, CEBPZ or GADD153). In conclusion, our data confirm the existence of a mammalian UPRmt that has great similarity to the C elegans pathway. Furthermore, our results illustrate that ClpX overexpression is a good and simple model to study the underlying mechanisms of the UPRmt in mammalian cells.

  • 3. Boldinova, Elizaveta O.
    et al.
    Stojkovic, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Khairullin, Rafil
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Kazan Volga Reg Fed Univ, Russia.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Makarova, Alena V.
    Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 9, article id e0184489Article in journal (Refereed)
    Abstract [en]

    Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6-6.5, low salt concentrations and 3 mM Mg2+ or 0.3-3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37 degrees C. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.

  • 4. Boldinova, Elizaveta O.
    et al.
    Wanrooij, Paulina H.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shilkin, Evgeniy S.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Makarova, Alena V.
    DNA Damage Tolerance by Eukaryotic DNA Polymerase and Primase PrimPol2017In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 18, no 7, article id 1584Article, review/survey (Refereed)
    Abstract [en]

    PrimPol is a human deoxyribonucleic acid (DNA) polymerase that also possesses primase activity and is involved in DNA damage tolerance, the prevention of genome instability and mitochondrial DNA maintenance. In this review, we focus on recent advances in biochemical and crystallographic studies of PrimPol, as well as in identification of new protein-protein interaction partners. Furthermore, we discuss the possible functions of PrimPol in both the nucleus and the mitochondria.

  • 5.
    Forslund, Josefin M. E.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pfeiffer, Annika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Stojkovič, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wanrooij, Pauline H.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The presence of rNTPs decreases the speed of mitochondrial DNA replication2018In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 14, no 3, article id e1007315Article in journal (Refereed)
    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.

  • 6. Miralles Fusté, Javier
    et al.
    Shi, Yonghong
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Zhu, Xuefeng
    Jemt, Elisabeth
    Persson, Orjan
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gustafsson, Claes M
    Falkenberg, Maria
    In vivo occupancy of mitochondrial single-stranded DNA binding protein supports the strand displacement mode of DNA replication2014In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 12, p. e1004832-Article in journal (Refereed)
    Abstract [en]

    Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.

  • 7.
    Moodie, Lindon W. K.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hubert, Madlen
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Zhou, Xin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Albers, Michael Franz
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hedberg, Christian
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Photoactivated Colibactin Probes Induce Cellular DNA Damage2019In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 58, no 5, p. 1417-1421Article in journal (Refereed)
    Abstract [en]

    Colibactin is a small molecule produced by certain bacterial species of the human microbiota that harbour the pks genomic island. Pks(+) bacteria induce a genotoxic phenotype in eukaryotic cells and have been linked with colorectal cancer progression. Colibactin is produced in a benign, prodrug form which, prior to export, is enzymatically matured by the producing bacteria to its active form. Although the complete structure of colibactin has not been determined, key structural features have been described including an electrophilic cyclopropane motif, which is believed to alkylate DNA. To investigate the influence of the putative "warhead" and the prodrug strategy on genotoxicity, a series of photolabile colibactin probes were prepared that upon irradiation induced a pks(+) like phenotype in HeLa cells. Furthermore, results from DNA cross-linking and imaging studies of clickable analogues enforce the hypothesis that colibactin effects its genotoxicity by directly targeting DNA.

  • 8. Pohjoismäki, Jaakko L. O.
    et al.
    Forslund, Josefin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Goffart, Steffi
    Torregrosa-Muñumer, Rubén
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Known Unknowns of Mammalian Mitochondrial DNA Maintenance2018In: Bioessays, ISSN 0265-9247, E-ISSN 1521-1878, Vol. 40, no 9, article id 1800102Article in journal (Refereed)
    Abstract [en]

    Mammalian mitochondrial DNA (mtDNA) replication and repair have been studied intensively for the last 50 years. Although recently advances in elucidating the molecular mechanisms of mtDNA maintenance and the proteins involved in these have been made, there are disturbing gaps between the existing theoretical models and experimental observations. Conflicting data and hypotheses exist about the role of RNA and ribonucleotides in mtDNA replication, but also about the priming of replication and the formation of pathological rearrangements. In the presented review, we have attempted to match these loose ends and draft consensus where it can be found, while identifying outstanding issues for future research.

  • 9.
    Stojkovic, Gorazd
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Makarova, Alena V.
    Wanrooij, Paulina H.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
    Forslund, Josefin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Burgers, Peter M.
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
    Oxidative DNA damage stalls the human mitochondrial replisome2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 28942Article in journal (Refereed)
    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.

  • 10. Torregrosa-Muñumer, Rubén
    et al.
    Forslund, Josefin M. E.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Goffart, Steffi
    Pfeiffer, Annika
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Stojkovič, Gorazd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Carvalho, Gustavo
    Al-Furoukh, Natalie
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Blanco, Luis
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pohjoismäki, Jaakko L. O.
    PrimPol is required for replication reinitiation after mtDNA damage2017In: 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)
    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.

  • 11.
    Wanrooij, Paulina H.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Engqvist, Martin K. M.
    Forslund, Josefin M. E.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Navarrete, Clara
    Nilsson, Anna Karin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sedman, Juhan
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Clausen, Anders R.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired2017In: 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)
    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.

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