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Wanrooij, Paulina H.ORCID iD iconorcid.org/0000-0002-8607-7564
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Publications (10 of 18) Show all publications
Breidenstein, A., Lamy, A., Bader P.J., C., Sun, W.-S., Wanrooij, P. H. & Berntsson, R.-A. P. A. (2024). PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs. Life Science Alliance, 7(8), Article ID e202402693.
Open this publication in new window or tab >>PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs
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2024 (English)In: Life Science Alliance, E-ISSN 2575-1077, Vol. 7, no 8, article id e202402693Article in journal (Refereed) Published
Abstract [en]

A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded type IV secretion systems (T4SSs). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many, their function remains unclear. Here, we characterize PrgE, a proposed SSB from the Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very unusual DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string supported by its N-terminal tail. In vitro studies highlight the plasticity of PrgE oligomerization and confirm the importance of the N-terminus. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from eukaryotic SSBs.

Place, publisher, year, edition, pages
Life Science Alliance, 2024
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-225963 (URN)10.26508/lsa.202402693 (DOI)38811160 (PubMedID)2-s2.0-85194886099 (Scopus ID)
Funder
Swedish Research Council, 2016-03599Swedish Research Council, 2023-02423Swedish Research Council, 2019-01874Knut and Alice Wallenberg FoundationThe Kempe Foundations, SMK-1762The Kempe Foundations, SMK-1869
Available from: 2024-06-11 Created: 2024-06-11 Last updated: 2024-06-11Bibliographically approved
Gorospe, C. M., Repolês, B. M. & Wanrooij, P. H. (2023). Determination of the ribonucleotide content of mtDNA using alkaline gels. In: Thomas J. Nicholls; Jay P. Uhler; Maria Falkenberg (Ed.), Mitochondrial DNA: methods and protocols (pp. 293-314). New York: Humana Press, 2615
Open this publication in new window or tab >>Determination of the ribonucleotide content of mtDNA using alkaline gels
2023 (English)In: Mitochondrial DNA: methods and protocols / [ed] Thomas J. Nicholls; Jay P. Uhler; Maria Falkenberg, New York: Humana Press, 2023, Vol. 2615, p. 293-314Chapter in book (Refereed)
Abstract [en]

Impaired mitochondrial DNA (mtDNA) maintenance, due to, e.g., defects in the replication machinery or an insufficient dNTP supply, underlies a number of mitochondrial disorders. The normal process of mtDNA replication leads to the incorporation of multiple single ribonucleotides (rNMPs) per mtDNA molecule. Given that embedded rNMPs alter the stability and properties of the DNA, they may have consequences for mtDNA maintenance and thereby for mitochondrial disease. They also serve as a readout of the intramitochondrial NTP/dNTP ratios. In this chapter, we describe a method for the determination of mtDNA rNMP content using alkaline gel electrophoresis and Southern blotting. This procedure is suited for the analysis of mtDNA in total genomic DNA preparations as well as in purified form. Moreover, it can be performed using equipment found in most biomedical laboratories, allows the simultaneous analysis of 10-20 samples depending on the gel system employed, and can be modified for the analysis of other mtDNA modifications.

Place, publisher, year, edition, pages
New York: Humana Press, 2023
Series
Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029 ; 2615
Keywords
Alkaline gels, Alkaline hydrolysis, Denaturing gels, Ribonucleotides, rNMPs, Southern blot
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-205504 (URN)10.1007/978-1-0716-2922-2_21 (DOI)36807800 (PubMedID)2-s2.0-85148677775 (Scopus ID)9781071629215 (ISBN)9781071629222 (ISBN)
Available from: 2023-03-15 Created: 2023-03-15 Last updated: 2023-07-11Bibliographically approved
Gorospe, C. M., Carvalho, G., Herrera Curbelo, A., Marchhart, L., Mendes, I., Niedźwiecka, K. & Wanrooij, P. H. (2023). Mitochondrial membrane potential acts as a retrograde signal to regulate cell cycle progression. Life Science Alliance, 6(12), Article ID e202302091.
Open this publication in new window or tab >>Mitochondrial membrane potential acts as a retrograde signal to regulate cell cycle progression
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2023 (English)In: Life Science Alliance, E-ISSN 2575-1077, Vol. 6, no 12, article id e202302091Article in journal (Refereed) Published
Abstract [en]

Mitochondria are central to numerous metabolic pathways whereby mitochondrial dysfunction has a profound impact and can manifest in disease. The consequences of mitochondrial dysfunction can be ameliorated by adaptive responses that rely on crosstalk from the mitochondria to the rest of the cell. Such mito-cellular signalling slows cell cycle progression in mitochondrial DNA-deficient (ρ0) Saccharomyces cerevisiae cells, but the initial trigger of the response has not been thoroughly studied. Here, we show that decreased mitochondrial membrane potential (ΔΨm) acts as the initial signal of mitochondrial stress that delays G1-to-S phase transition in both ρ0 and control cells containing mtDNA. Accordingly, experimentally increasing ΔΨm was sufficient to restore timely cell cycle progression in ρ0 cells. In contrast, cellular levels of oxidative stress did not correlate with the G1-to-S delay. Restored G1-to-S transition in ρ0 cells with a recovered ΔΨm is likely attributable to larger cell size, whereas the timing of G1/S transcription remained delayed. The identification of ΔΨm as a regulator of cell cycle progression may have implications for disease states involving mitochondrial dysfunction.

Place, publisher, year, edition, pages
Life Science Alliance, LLC, 2023
National Category
Cell Biology Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-214616 (URN)10.26508/lsa.202302091 (DOI)37696576 (PubMedID)2-s2.0-85170626490 (Scopus ID)
Funder
Swedish Cancer Society, 190022JIASwedish Cancer Society, 190098PjSwedish Research Council, 2019-01874Swedish Society for Medical Research (SSMF), S17-0023The Kempe Foundations, JCK-1830Åke Wiberg Foundation, M20-0132
Available from: 2023-09-27 Created: 2023-09-27 Last updated: 2023-09-27Bibliographically approved
Wanrooij, P. H. & Chabes, A. (2023). NME6: ribonucleotide salvage sustains mitochondrial transcription. EMBO Journal, 42(18), Article ID e114990.
Open this publication in new window or tab >>NME6: ribonucleotide salvage sustains mitochondrial transcription
2023 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 42, no 18, article id e114990Article in journal (Refereed) Published
Abstract [en]

The building blocks for RNA and DNA are made in the cytosol, meaning mitochondria depend on the import and salvage of ribonucleoside triphosphates (rNTPs) and deoxyribonucleoside triphosphates (dNTPs) for the synthesis of their own genetic material. While extensive research has focused on mitochondrial dNTP homeostasis due to its defects being associated with various mitochondrial DNA (mtDNA) depletion and deletion syndromes, the investigation of mitochondrial rNTP homeostasis has received relatively little attention. In this issue of the EMBO Journal, Grotehans et al provide compelling evidence of a major role for NME6, a mitochondrial nucleoside diphosphate kinase, in the conversion of pyrimidine ribonucleoside diphosphates into the corresponding triphosphates. These data also suggest a significant physiological role for NME6, as its absence results in the depletion of mitochondrial transcripts and destabilization of the electron transport chain (Grotehans et al, 2023).

Place, publisher, year, edition, pages
John Wiley & Sons, 2023
National Category
Biochemistry and Molecular Biology Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-212988 (URN)10.15252/embj.2023114990 (DOI)001043742100001 ()37548337 (PubMedID)2-s2.0-85167349571 (Scopus ID)
Funder
Knut and Alice Wallenberg FoundationSwedish Cancer SocietySwedish Research Council
Available from: 2023-08-21 Created: 2023-08-21 Last updated: 2024-01-05Bibliographically approved
Carvalho, G., Repolês, B. M., Mendes, I. & Wanrooij, P. H. (2022). Mitochondrial DNA Instability in Mammalian Cells. Antioxidants and Redox Signaling, 36(13-15), 885-905
Open this publication in new window or tab >>Mitochondrial DNA Instability in Mammalian Cells
2022 (English)In: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 36, no 13-15, p. 885-905Article, review/survey (Refereed) Published
Abstract [en]

Significance: The small, multicopy mitochondrial genome (mitochondrial DNA [mtDNA]) is essential for efficient energy production, as alterations in its coding information or a decrease in its copy number disrupt mitochondrial ATP synthesis. However, the mitochondrial replication machinery encounters numerous challenges that may limit its ability to duplicate this important genome and that jeopardize mtDNA stability, including various lesions in the DNA template, topological stress, and an insufficient nucleotide supply.

Recent Advances: An ever-growing array of DNA repair or maintenance factors are being reported to localize to the mitochondria. We review current knowledge regarding the mitochondrial factors that may contribute to the tolerance or repair of various types of changes in the mitochondrial genome, such as base damage, incorporated ribonucleotides, and strand breaks. We also discuss the newly discovered link between mtDNA instability and activation of the innate immune response.

Critical Issues: By which mechanisms do mitochondria respond to challenges that threaten mtDNA maintenance? What types of mtDNA damage are repaired, and when are the affected molecules degraded instead? And, finally, which forms of mtDNA instability trigger an immune response, and how?

Future Directions: Further work is required to understand the contribution of the DNA repair and damage-tolerance factors present in the mitochondrial compartment, as well as the balance between mtDNA repair and degradation. Finally, efforts to understand the events underlying mtDNA release into the cytosol are warranted. Pursuing these and many related avenues can improve our understanding of what goes wrong in mitochondrial disease.

Place, publisher, year, edition, pages
Mary Ann Liebert, 2022
Keywords
mitochondrial DNA, genome instability, DNA replication
National Category
Cell and Molecular Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-187332 (URN)10.1089/ars.2021.0091 (DOI)000669978100001 ()34015960 (PubMedID)2-s2.0-85130003932 (Scopus ID)
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Society for Medical Research (SSMF)Åke Wiberg Foundation
Available from: 2021-09-08 Created: 2021-09-08 Last updated: 2022-06-09Bibliographically approved
Repolês, B. M., Gorospe, C. M., Tran, P., Nilsson, A. K. & Wanrooij, P. H. (2021). The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method. Mitochondrion (Amsterdam. Print), 61, 179-187
Open this publication in new window or tab >>The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method
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2021 (English)In: Mitochondrion (Amsterdam. Print), ISSN 1567-7249, E-ISSN 1872-8278, Vol. 61, p. 179-187Article in journal (Refereed) Published
Abstract [en]

The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.

Place, publisher, year, edition, pages
Elsevier, 2021
Keywords
DNA integrity, Long-range PCR, Mitochondrial DNA, mtDNA, Nuclease activity
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-189552 (URN)10.1016/j.mito.2021.10.005 (DOI)000717836700001 ()2-s2.0-85118478892 (Scopus ID)
Funder
Åke Wiberg Foundation, M20-0132Swedish Cancer Society, 19 0022 JIA, 190098 Pj 01 HSwedish Society for Medical Research (SSMF), S17-0023Swedish Research Council, 2019-01874
Available from: 2021-11-16 Created: 2021-11-16 Last updated: 2023-09-05Bibliographically approved
Wanrooij, P. H., Tran, P., Thompson, L. J., Carvalho, G., Sharma, S., Kreisel, K., . . . Chabes, A. (2020). Elimination of rNMPs from mitochondrial DNA has no effect on its stability. Proceedings of the National Academy of Sciences of the United States of America, 117(25), 14306-14313
Open this publication in new window or tab >>Elimination of rNMPs from mitochondrial DNA has no effect on its stability
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2020 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 117, no 25, p. 14306-14313Article in journal (Refereed) Published
Abstract [en]

Ribonucleotides (rNMPs) incorporated in the nuclear genome are a well-established threat to genome stability and can result in DNA strand breaks when not removed in a timely manner. However, the presence of a certain level of rNMPs is tolerated in mitochondrial DNA (mtDNA) although aberrant mtDNA rNMP content has been identified in disease models. We investigated the effect of incorporated rNMPs on mtDNA stability over the mouse life span and found that the mtDNA rNMP content increased during early life. The rNMP content of mtDNA varied greatly across different tissues and was defined by the rNTP/dNTP ratio of the tissue. Accordingly, mtDNA rNMPs were nearly absent in SAMHD1 -/- mice that have increased dNTP pools. The near absence of rNMPs did not, however, appreciably affect mtDNA copy number or the levels of mtDNA molecules with deletions or strand breaks in aged animals near the end of their life span. The physiological rNMP load therefore does not contribute to the progressive loss of mtDNA quality that occurs as mice age.

Place, publisher, year, edition, pages
National Academy of Sciences, 2020
Keywords
SAMHD1, dNTP pool, mitochondrial DNA, mtDNA, ribonucleotide incorporation
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-171765 (URN)10.1073/pnas.1916851117 (DOI)000546764000005 ()32513727 (PubMedID)2-s2.0-85087094387 (Scopus ID)
Funder
Swedish Research CouncilSwedish Research CouncilSwedish Cancer SocietySwedish Foundation for Strategic Research Lars Hierta Memorial FoundationThe Kempe Foundations
Available from: 2020-06-10 Created: 2020-06-10 Last updated: 2023-03-24Bibliographically approved
Doimo, M., Pfeiffer, A., Wanrooij, P. H. & Wanrooij, S. (2020). MtDNA replication, maintenance, and nucleoid organization. In: Giuseppe Gasparre; Anna Maria Porcelli (Ed.), The human mitochondrial genome: from basic biology to disease (pp. 3-33). Academic Press
Open this publication in new window or tab >>MtDNA replication, maintenance, and nucleoid organization
2020 (English)In: The human mitochondrial genome: from basic biology to disease / [ed] Giuseppe Gasparre; Anna Maria Porcelli, Academic Press, 2020, p. 3-33Chapter in book (Refereed)
Abstract [en]

Part of the genetic information in human cells resides in the mitochondria. Faithful maintenance of mitochondrial deoxyribonucleic acid (mtDNA) is crucial for the oxidative phosphorylation system that produces the majority of the cellular ATP, and therefore to life. This chapter provides an introduction into the characteristics of human mtDNA and summarizes the processes and factors required for the replication and maintenance of this small but essential genome. We also describe the organization of mtDNA in specialized nucleoprotein structures called nucleoids. Where applicable, we refer to human disease states that are caused by defects in the described factors or processes.

Place, publisher, year, edition, pages
Academic Press, 2020
Keywords
Mitochondrial DNA, mtDNA
National Category
Biochemistry and Molecular Biology Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-182657 (URN)10.1016/B978-0-12-819656-4.00001-2 (DOI)2-s2.0-85124853615 (Scopus ID)9780128196564 (ISBN)
Available from: 2021-04-29 Created: 2021-04-29 Last updated: 2022-02-28Bibliographically approved
Tran, P., Wanrooij, P. H., Lorenzon, P., Sharma, S., Thelander, L., Nilsson, A. K., . . . Chabes, A. (2019). De novo dNTP production is essential for normal postnatal murine heart development. Journal of Biological Chemistry, 394(44), 15889-15897, Article ID jbc.RA119.009492.
Open this publication in new window or tab >>De novo dNTP production is essential for normal postnatal murine heart development
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2019 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 394, no 44, p. 15889-15897, article id jbc.RA119.009492Article in journal (Refereed) Published
Abstract [en]

The building blocks of DNA, dNTPs, can be produced de novo or can be salvaged from deoxyribonucleosides. However, to what extent the absence of de novo dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated de novo dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the de novo production of dNTPs, at embryonic day 13. All other tissues had normal de novo dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in wild-type postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for de novo dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in wildtype hearts.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2019
Keywords
cardiac function, cardiac muscle, dNTP metabolism, dNTP salvage, deoxyribonucleoside kinases, desmin, heart development, nucleoside/nucleotide biosynthesis, nucleoside/nucleotide metabolism, ribonucleotide reductase
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-161767 (URN)10.1074/jbc.RA119.009492 (DOI)000499478600002 ()31300555 (PubMedID)2-s2.0-85074444850 (Scopus ID)
Funder
Swedish Research CouncilSwedish Cancer Society
Available from: 2019-07-30 Created: 2019-07-30 Last updated: 2023-03-24Bibliographically approved
Wanrooij, P. H. & Chabes, A. (2019). Ribonucleotides in mitochondrial DNA. FEBS Letters, 593(13), 1554-1565
Open this publication in new window or tab >>Ribonucleotides in mitochondrial DNA
2019 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 593, no 13, p. 1554-1565Article, review/survey (Refereed) Published
Abstract [en]

The incorporation of ribonucleotides (rNMPs) into DNA during genome replication has gained substantial attention in recent years and has been shown to be a significant source of genomic instability. Studies in yeast and mammals have shown that the two genomes, the nuclear DNA (nDNA) and the mitochondrial DNA (mtDNA), differ with regard to their rNMP content. This is largely due to differences in rNMP repair - whereas rNMPs are efficiently removed from the nuclear genome, mitochondria lack robust mechanisms for removal of single rNMPs incorporated during DNA replication. In this minireview, we describe the processes that determine the frequency of rNMPs in the mitochondrial genome and summarise recent findings regarding the effect of incorporated rNMPs on mtDNA stability and function.

Place, publisher, year, edition, pages
John Wiley & Sons, 2019
Keywords
dNTP, genome stability, mitochondrial DNA, ribonucleotides
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-159088 (URN)10.1002/1873-3468.13440 (DOI)000477916000012 ()31093968 (PubMedID)2-s2.0-85066896219 (Scopus ID)
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Society for Medical Research (SSMF)
Note

Special Issue: Krakow Special Issue

Available from: 2019-05-17 Created: 2019-05-17 Last updated: 2024-07-02Bibliographically approved
Projects
Checkpoint mechanisms that protect the human mitochondrial genome [2019-01974_VR]; Umeå University
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-8607-7564

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