Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is a triphosphohydrolase converting deoxynucleoside triphosphates (dNTPs) to deoxynucleosides. The enzyme was recently identified as a component of the human innate immune system that restricts HIV-1 infection by removing dNTPs required for viral DNA synthesis. SAMHD1 has deep evolutionary roots and is ubiquitous in human organs. Here we identify a general function of SAMHD1 in the regulation of dNTP pools in cultured human cells. The protein was nuclear and variably expressed during the cell cycle, maximally during quiescence and minimally during S-phase. Treatment of lung or skin fibroblasts with specific siRNAs resulted in the disappearence of SAMHD1 accompanied by loss of the cell-cycle regulation of dNTP pool sizes and dNTP imbalance. Cells accumulated in G1 phase with oversized pools and stopped growing. Following removal of the siRNA, the pools were normalized and cell growth restarted, but only after SAMHD1 had reappeared. In quiescent cultures SAMHD1 down-regulation leads to a marked expansion of dNTP pools. In all cases the largest effect was on dGTP, the preferred substrate of SAMHD1. Ribonucleotide reductase, responsible for the de novo synthesis of dNTPs, is a cytosolic enzyme maximally induced in S-phase cells. Thus, in mammalian cells the cell cycle regulation of the two main enzymes controlling dNTP pool sizes is adjusted to the requirements of DNA replication. Synthesis by the reductase peaks during S-phase, and catabolism by SAMHD1 is maximal during G1 phase when large dNTP pools would prevent cells from preparing for a new round of DNA replication. dNTP regulation | cell cycle arrest | dGTP pool

Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP.

In 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.DNA precursors | dNTP de novo synthesis | cell cycle | mitochondrial disease D NA replication and repair require the continued synthesis of the four dNTPs. They are synthesized by evolutionary-related ribonucleotide reductases operating with slightly different mechanisms in aerobic and anaerobic organisms (1). Each ribonucleotide reductase provides the required amounts of all four dNTPs. A similar allosteric mechanism, maintained throughout evolution, regulates both the enzyme's activity and its substrate specificity. Cells contain small dNTP pools of similar sizes, approximately 10-fold larger during DNA replication than during quiescence. Regulation of pool sizes by ribonucleotide reductases is of great importance for correct DNA replication, and changes in the actual sizes or in their balance lead to increased mutation rates (2). For mammalian cells, the induction of mutations by pool imbalances has been described in detail, along with possible mechanisms (3). In yeast, a recent elegant study (4) linked specific amino acid substitutions in the catalytic subunit of ribonucleotide reductase to defined pool imbalances, which result in increased mutation rates.In mammalian cells, the canonical ribonucleotide reductase is a complex between two proteins: the large catalytic protein R1 that contains the allosteric sites and the smaller protein R2 that contributes a stable tyrosyl free radical during the reaction (1). Both proteins are transcriptionally activated during early S-phase (5) a...

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Nuclear 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 [ 3 H]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.M itochondria contain two separate potential pathways to provide dTTP for mitochondrial (mt) DNA replication ( Fig. 1): (i) deoxynucleotide transporters in the membrane introduce nucleotides from the cytosol (1, 2), and (ii) thymidine kinase 2 (TK2) in the mt matrix phosphorylates thymidine to dTMP (3-6), which is further phosphorylated by nucleotide kinases to dTTP. Preliminary evidence for a third pathway via an intra-mt ribonucleotide reductase (7) has not been followed up, nor could we confirm it. We do not further consider it here.The first pathway in Fig. 1 relies mainly on de novo synthesis of deoxyribonucleoside diphosphates by ribonucleotide reductase in the cytosol (8) and to a minor extent on the activity of the cytosolic thymidine kinase 1 (TK1) (9). Both enzymes are active only during the S phase of the cell cycle (10, 11). The first pathway is therefore absent from terminally differentiated cells. The second pathway in Fig. 1 uses thymidine imported from the extracellular milieu and is active also outside S phase because TK2 is not cell-cycle regulated. Regulation of this pathway may occur by an intra-mt substrate cycle involving TK2 and 5Ј(3Ј)-deoxyribonucleotidase 2 (dNT-2) (12), similar to the cytosolic substrate cycle between TK1 and deoxyribonucleotidase 1 (13). Several genetic diseases affecting mtDNA replication arise from malfunction of enzymes in either mt pathway (14, 15). Also, the genetic loss of the cytosolic thymidine phosphorylase results in mutatio...

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.DNA precursors ͉ immunofluorescence ͉ mitochondrial DNA ͉ p53R2 ͉ subcellular localization D NA replication and repair require a balanced supply of the four common deoxynucleoside triphosphates (dNTPs). In mammalian cells DNA synthesis occurs in two separate compartments: nucleus and mitochondria. The complete nuclear DNA is replicated only in cycling cells during S-phase, whereas cycling and quiescent cells replicate mitochondrial DNA and repair damaged DNA during their whole existence. Thus cycling cells require during a limited period a large supply of dNTPs in the nucleus. Outside S-phase cells consume much smaller amounts of dNTPs, mainly in the cytosol for mitochondrial (mt) DNA replication. In all cells the major supply of dNTPs comes from the de novo reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates by the enzyme ribonucleotide reductase (RNR) (1).In cycling cells, the dominant form of mammalian RNR consists of two proteins called R1 and R2. The activity of the R1/R2 enzyme is exquisitely regulated by allosteric mechanisms involving nucleoside triphosphates and also by S-phase-specific transcription and proteasome-mediated degradation of R2 in late mitosis (2). Thus postmitotic cells are completely devoid of protein R2. How do these cells synthesize dNTPs for mitochondrial DNA replication and DNA repair? Until recently the answer to this question was by salvage of deoxynucleosides but the picture changed su...

Human fibroblasts in culture obtain deoxynucleotides by de novo ribonucleotide reduction or by salvage of deoxynucleosides. In cycling cells the de novo pathway dominates, but in quiescent cells the salvage pathway becomes important. Two forms of active mammalian ribonucleotide reductases are known. Each form contains the catalytic R1 protein, but the two differ with respect to the second protein (R2 or p53R2). R2 is cell cycle-regulated, degraded during mitosis, and absent from quiescent cells. The recently discovered p53-inducible p53R2 was proposed to be linked to DNA repair processes. During quiescence, incorporation of deoxynucleotides into DNA was very low. Deoxynucleotides were instead degraded to deoxynucleosides and exported into the medium as deoxycytidine, deoxyuridine, and thymidine. The rate of export was surprisingly high, 25% of that in cycling cells. Total ribonucleotide reduction in quiescent cells amounted to only 2-3% of cycling cells. We suggest that in quiescent cells an important function of p53R2 is to provide deoxynucleotides for mitochondrial DNA replication.The synthesis of dNTPs occurs both in the cytosol and in mitochondria. In the cytosol a de novo pathway starting from small molecules involves the enzymes ribonucleotide reductase (1), dCMP deaminase (2), and thymidylate synthase (2) as the major players. An auxiliary salvage pathway in the cytosol starting from preformed deoxynucleosides involves thymidine kinase 1 (3) and deoxycytidine kinase (4). Deoxynucleotides formed in the cytosol are imported into mitochondria by specific transporters (5, 6). Salvage of deoxynucleosides occurs also inside mitochondria by two intra-mt 2 enzymes, thymidine kinase 2 (7) and deoxyguanosine kinase (8). These two kinases can between themselves phosphorylate all four canonical deoxyribosides, and because of the presence of appropriate nucleotide kinases the resulting monophosphates are transformed to dNTPs to satisfy the requirement for building blocks for mtDNA replication.The classical ribonucleotide reductase of the de novo pathway consists of two proteins, R1 and R2 (1). Of these, R2 is absent from quiescent cells (9). Also the cytosolic thymidine kinase 1 is S-phase-specific (10). It would therefore appear that the cytosolic pathways cannot provide all required dNTPs for mtDNA replication and repair in quiescent cells. These would therefore be expected to depend on the two intra-mt kinases. In agreement, in humans a genetic deficiency of either kinase (11, 12) depletes mtDNA in organs with terminally differentiated cells causing severe disease. In cultured cells kinetic experiments with labeled thymidine supported the existence of the two pathways for the synthesis of mt dNTPs and the predominance of the salvage pathway in quiescent cells (13).However, it was recently found (14) that quiescent mouse 3T3 fibroblasts obtained by serum starvation contained p53R2 (15, 16), a protein that can substitute for R2 to form an active enzyme together with R1, the second subunit of ribonucleotide reductase ...

Mitochondrial (mt) neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease associated with depletion, deletions, and point mutations of mtDNA. Patients lack a functional thymidine phosphorylase and their plasma contains high concentrations of thymidine and deoxyuridine; elevation of the corresponding triphosphates probably impairs normal mtDNA replication and repair. To study metabolic events leading to MNGIE we used as model systems skin and lung fibroblasts cultured in the presence of thymidine and/or deoxyuridine at concentrations close to those in the plasma of the patients, a more than 100-fold excess relative to controls. The two deoxynucleosides increased the mt and cytosolic dTTP pools of skin fibroblasts almost 2-fold in cycling cells and 8-fold in quiescent cells. During up to a two-month incubation of quiescent fibroblasts with thymidine (but not with deoxyuridine), mtDNA decreased to ϳ50% without showing deletions or point mutations. When we removed thymidine, but maintained the quiescent state, mtDNA recovered rapidly. With thymidine in the medium, the dTTP pool of quiescent cells turned over rapidly at a rate depending on the concentration of thymidine, due to increased degradation and resynthesis of dTMP in a substrate (‫؍‬futile) cycle between thymidine kinase and 5-deoxyribonucleotidase. The cycle limited the expansion of the dTTP pool at the expense of ATP hydrolysis. We propose that the substrate cycle represents a regulatory mechanism to protect cells from harmful increases of dTTP. Thus MNGIE patients may increase their consumption of ATP to counteract an unlimited expansion of the dTTP pool caused by circulating thymidine. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)2 is an autosomal recessive human mitochondrial (mt) disease, caused by the defective function of the cytosolic enzyme thymidine phosphorylase (1) and is associated with depletion (2), multiple deletions (2, 3), and point mutations of mtDNA (4). Thymidine phosphorylase converts the pyrimidine deoxynucleosides thymidine and deoxyuridine to free bases and deoxyribose 1-phosphate. Human blood from healthy individuals contains Ͻ0.05 M of the two deoxynucleosides, whereas both thymidine and deoxyuridine are found at 10 -20 M concentrations in plasma from MNGIE patients (5, 6). It has been proposed that this large increase in circulating deoxynucleosides results in a similar increase in the intracellular concentration of their triphosphates disrupting the normal balance of the 4 dNTPs and thereby interfering with the replication of mtDNA (4, 6). Experimental evidence supports this hypothesis. Addition of thymidine to the medium of cultured, rapidly growing cells lead to an increase in the dTTP pool and a rapid cessation of nuclear (and probably also mt) DNA replication caused by a depletion of the dCTP pool (7). Depletion of dCTP, as well as the concomitant increase of dGTP and dATP were secondary to the allosteric effects of the large dTTP pool on the enzyme ribonucleotide reductase. In that...