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- Pontarin, Giovanna, et al.
(författare)
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Deoxyribonucleotide metabolism in cycling and resting human fibroblasts with a missense mutation in p53R2, a subunit of ribonucleotide reductase.
- 2011
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Ingår i: The Journal of biological chemistry. - 1083-351X.
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reduction provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and DNA repair. In cycling mammalian cells the reaction is catalyzed by two proteins, R1 and R2. A third protein, p53R2, with the same function as R2 occurs in minute amounts. In quiescent cells, p53R2 replaces the absent R2. In humans genetic inactivation of p53R2 causes early death with mtDNA depletion, especially in muscle. We found that cycling fibroblasts from a patient with a lethal mutation in p53R2 contained a normal amount of mtDNA and showed normal growth, ribonucleotide reduction and dNTP pools. However, when made quiescent by prolonged serum starvation the mutant cells strongly down-regulated ribonucleotide reduction, decreased their dCTP and dGTP pools and virtually abolished the catabolism of dCTP in substrate cycles. mtDNA was not affected. Also nuclear DNA synthesis and the cell-cycle regulated enzymes R2 and thymidine kinase 1 decreased strongly, but the mutant cell populations retained unexpectedly larger amounts of the two enzymes than the controls. This difference was probably due to their slightly larger fraction of S-phase cells and therefore not related to the absence of p53R2 activity. We conclude that loss of p53R2 affects ribonucleotide reduction only in resting cells and there leads to a decrease of dNTP catabolism by substrate cycles that counterweighs the loss of anabolic activity. We speculate that this compensatory mechanism suffices to maintain mtDNA in fibroblasts but not in muscle cells with a larger content of mtDNA necessary for their high energy requirements.
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- Pontarin, Giovanna, et al.
(författare)
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Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage.
- 2008
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:46, s. 17801-17806
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and repair. The mammalian enzyme consists of a catalytic (R1) and a radical-generating (R2 or p53R2) subunit. During S-phase, a R1/R2 complex is the major provider of deoxynucleotides. p53R2 is induced by p53 after DNA damage and was proposed to supply deoxynucleotides for DNA repair after translocating from the cytosol to the cell nucleus. Similarly R1 and R2 were claimed to move to the nucleus during S-phase to provide deoxynucleotides for DNA replication. These models suggest translocation of ribonucleotide reductase subunits as a regulatory mechanism. In quiescent cells that are devoid of R2, R1/p53R2 synthesizes deoxynucleotides also in the absence of DNA damage. Mutations in human p53R2 cause severe mitochondrial DNA depletion demonstrating a vital function for p53R2 different from DNA repair and cast doubt on a nuclear localization of the protein. Here we use three independent methods to localize R1, R2, and p53R2 in fibroblasts during cell proliferation and after DNA damage: Western blotting after separation of cytosol and nuclei; immunofluorescence in intact cells; and transfection with proteins carrying fluorescent tags. We thoroughly validate each method, especially the specificity of antibodies. We find in all cases that ribonucleotide reductase resides in the cytosol suggesting that the deoxynucleotides produced by the enzyme diffuse into the nucleus or are transported into mitochondria and supporting a primary function of p53R2 for mitochondrial DNA replication.
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