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...