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De novo dNTP production is essential for normal postnatal murine heart development
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Andrei Chabes)
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Paulina Wanrooij)ORCID iD: 0000-0002-8607-7564
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Andrei Chabes)ORCID iD: 0000-0003-2713-5813
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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. Vol. 394, no 44, p. 15889-15897, article id jbc.RA119.009492
Keywords [en]
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: urn:nbn:se:umu:diva-161767DOI: 10.1074/jbc.RA119.009492ISI: 000499478600002PubMedID: 31300555Scopus ID: 2-s2.0-85074444850OAI: oai:DiVA.org:umu-161767DiVA, id: diva2:1339513
Funder
Swedish Research CouncilSwedish Cancer SocietyAvailable from: 2019-07-30 Created: 2019-07-30 Last updated: 2023-03-24Bibliographically approved
In thesis
1. Pathology of dNTP dysregulation
Open this publication in new window or tab >>Pathology of dNTP dysregulation
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Patologier orsakade av dysfunktionell dNTP-reglering
Abstract [en]

Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac abnormalities and impaired cardiac conduction systems. As a result, they died between two and four weeks after birth. Taken together, our studies provide the first empirical evidence that both the de novo and salvage pathways are essential to keeping the dNTP concentration at an optimal range to prevent mutagenesis, carcinogenesis, and mortality.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2020. p. 43
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2095
Keywords
dNTP metabolism, DNA replication, mutation rate, cancer, heart development, RNR, SAMHD1, Rrm1, Samhd1
National Category
Natural Sciences
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-175260 (URN)978-91-7855-346-4 (ISBN)978-91-7855-345-7 (ISBN)
Public defence
2020-10-28, KB.E3.01, byggnad KBC, Umeå University, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2020-10-07 Created: 2020-09-23 Last updated: 2020-09-28Bibliographically approved

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Tran, PhongWanrooij, Paulina H.Lorenzon, PaoloSharma, SushmaThelander, LarsNilsson, Anna KarinOlofsson, Anna-KarinMedini, Paolovon Hofsten, JonasStål, PerChabes, Andrei

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Tran, PhongWanrooij, Paulina H.Lorenzon, PaoloSharma, SushmaThelander, LarsNilsson, Anna KarinOlofsson, Anna-KarinMedini, Paolovon Hofsten, JonasStål, PerChabes, Andrei
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Department of Medical Biochemistry and BiophysicsDepartment of Integrative Medical Biology (IMB)Umeå Centre for Molecular Medicine (UCMM)Molecular Infection Medicine Sweden (MIMS)
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Journal of Biological Chemistry
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