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Mitochondrial deoxyribonucleotides, pool sizes, synthesis, and regulation
Full text linkWe quantify cytosolic and mitochondrial deoxyribonucleoside triphosphates (dNTPs) from four established cell lines using a recently described method for the separation of cytosolic and mitochondrial (mt) dNTPs from as little as 10 million cells in culture (Pontarin, G., Gallinaro, L., Ferraro, P., Reichard, P., and Bianchi, V. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 12159-12164). In cycling cells the concentrations of the phosphates of thymidine, deoxycytidine, and deoxyadenosine (combining mono-, di-, and triphosphates in each case) did not differ significantly between mitochondria and cytosol, whereas deoxyguanosine phosphates were concentrated to mitochondria. We study the source and regulation of the mt dTTP pool as an example of mt dNTPs. We suggest two pathways as sources for mt dTTP: (i) import from the cytosol of thymidine diphosphate by a deoxynucleotide transporter, predominantly in cells involved in DNA replication with an active synthesis of deoxynucleotides and (ii) import of thymidine followed by phosphorylation by the mt thymidine kinase, predominantly in resting cells. Here we demonstrate that the second pathway is regulated by a mt 5'-deoxyribonucleotidase (mdN). We modify the in situ activity of mdN and measure the transfer of radioactivity from [(3)H]thymidine to mt thymidine phosphates. In cycling cells lacking the cytosolic thymidine kinase, a 30-fold overproduction of mdN decreases the specific radioactivity of mt dTTP to 25%, and an 80% decrease of mdN by RNA interference increases the specific radioactivity 2-fold. These results suggest that mdN modulates the synthesis of mt dTTP by counteracting in a substrate cycle the phosphorylation of thymidine by the mt thymidine kinase
Interrelations between substrate cycles and de novo synthesis of pyrimidine deoxyribonucleoside triphosphates in 3T6 cells.
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Changes of deoxyribonucleoside triphosphate pools induced by hydroxyurea and their relation to DNA synthesis.
No full textRegulation of pyrimidine deoxyribonucleotide metabolism by substrate cycles in dCMP deaminase-deficient V79 hamster cells.
No full text5'-Nucleotidases: specific assays for five different enzymes in cell extracts
No full textSeveral mammalian 5'-nucleotidases (5-NTs), attached to membranes or present in the cytosol or in mitochondria, remove the phosphate from ribo- and deoxyribonucleotides with different specificities for the sugar and base moieties. Some enzymes probably participate in signaling functions by producing adenosine from AMP. A more general function may be to prevent overproduction of deoxyribonucleotides. 5-NTs may affect the pharmacological activity of nucleoside analogs and also be involved in their mitochondrial toxicity. Here we describe for five cloned 5-NT specific assays that largely rely on new inhibitors for some of the enzymes. The assays can be used to quantitate each enzyme in crude cell extracts. To ascertain their validity we applied each assay to extracts from genetically modified cells that overproduce separately each of the five enzymes. The methodology should be useful in further studies of the physiological function of 5-NTs and their influence on the clinical use of nucleoside analogs
Interruption of the ferredoxin (flavodoxin) NADP+ oxidoreductase gene of Escherichia coli does not affect anaerobic growth but increases sensitivity to paraquat.
No full textOrigins of mitochondrial thymidine triphosphate: dynamic relations to cytosolic pools
No full textNuclear and mitochondrial (mt) DNA replication occur within two physically separated compartments and on different time scales. Both require a balanced supply of dNTPs. During S phase, dNTPs for nuclear DNA are synthesized de novo from ribonucleotides and by salvage of thymidine in the cytosol. Mitochondria contain specific kinases for salvage of deoxyribonucleosides that may provide a compartmentalized synthesis of dNTPs. Here we investigate the source of intra-mt thymidine phosphates and their relationship to cytosolic pools by isotope-flow experiments with [3H]thymidine in cultured human and mouse cells by using a rapid method for the clean separation of mt and cytosolic dNTPs. In the absence of the cytosolic thymidine kinase, the cells (i) phosphorylate labeled thymidine exclusively by the intra-mt kinase, (ii) export thymidine phosphates rapidly to the cytosol, and (iii) use the labeled dTTP for nuclear DNA synthesis. The specific radioactivity of dTTP is highly diluted, suggesting that cytosolic de novo synthesis is the major source of mt dTTP. In the presence of cytosolic thymidine kinase dilution is 100-fold less, and mitochondria contain dTTP with high specific radioactivity. The rapid mixing of the cytosolic and mt pools was not expected from earlier data. We propose that in proliferating cells dNTPs for mtDNA come largely from import of cytosolic nucleotides, whereas intra-mt salvage of deoxyribonucleosides provides dNTPs in resting cells. Our results are relevant for an understanding of certain genetic mitochondrial diseases
Effects of mutational loss of nucleoside kinases on deoxyadenosine 5'-phosphate/deoxyadenosine substrate cycle in cultured CEM and V79 cells.
No full textMammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells.
No full textIn postmitotic mammalian cells, protein p53R2 substitutes for protein R2 as a subunit of ribonucleotide reductase. In human patients with mutations in RRM2B, the gene for p53R2, mitochondrial (mt) DNA synthesis is defective, and skeletal muscle presents severe mtDNA depletion. Skin fibroblasts isolated from a patient with a lethal homozygous missense mutation of p53R2 grow normally in culture with an unchanged complement of mtDNA. During active growth, the four dNTP pools do not differ in size from normal controls, whereas during quiescence, the dCTP and dGTP pools decrease to 50% of the control. We investigate the ability of these mutated fibroblasts to synthesize mtDNA and repair DNA after exposure to UV irradiation. Ethidium bromide depleted both mutant and normal cells of mtDNA. On withdrawal of the drug, mtDNA recovered equally well in cycling mutant and control cells, whereas during quiescence, the mutant fibroblasts remained deficient. Addition of deoxynucleosides to the medium increased intracellular dNTP pools and normalized mtDNA synthesis. Quiescent mutant fibroblasts were also deficient in the repair of UV-induced DNA damage, as indicated by delayed recovery of dsDNA analyzed by fluorometric analysis of DNA unwinding and the more extensive and prolonged phosphorylation of histone H2AX after irradiation. Supplementation by deoxynucleosides improved DNA repair. Our results show that in nontransformed cells only during quiescence, protein p53R2 is required for maintenance of mtDNA and for optimal DNA repair after UV damage
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