The deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G1, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3-4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent Km for dATP at site 2 is ?10 ?m for mouse and 1 ?m for human SAMHD1, for dTTP the corresponding values are 50 and 2 ?m. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower Km constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells.
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.
During myogenesis, myoblasts fuse into multinucleated myotubes that acquire the contractile fibrils and accessory structures typical of striated skeletal muscle fibers. To support the high energy requirements of muscle contraction, myogenesis entails an increase in mitochondrial (mt) mass with stimulation of mtDNA synthesis and consumption of DNA precursors (dNTPs). Myotubes are quiescent cells and as such down-regulate dNTP production despite a high demand for dNTPs. Although myogenesis has been studied extensively, changes in dNTP metabolism have not been examined specifically. In differentiating cultures of C2C12 myoblasts and purified myotubes, we analyzed expression and activities of enzymes of dNTP biosynthesis, dNTP pools, and the expansion of mtDNA. Myotubes exibited pronounced post-mitotic modifications of dNTP synthesis with a particularly marked down-regulation of de novo thymidylate synthesis. Expression profiling revealed the same pattern of enzyme down-regulation in adult murine muscles. The mtDNA increased steadily after myoblast fusion, turning over rapidly, as revealed after treatment with ethidium bromide. We individually down-regulated p53R2 ribonucleotide reductase, thymidine kinase 2, and deoxyguanosine kinase by siRNA transfection to examine how a further reduction of these synthetic enzymes impacted myotube development. Silencing of p53R2 had little effect, but silencing of either mt kinase caused 50% mtDNA depletion and an unexpected decrease of all four dNTP pools independently of the kinase specificity. We suggest that during development of myotubes the shortage of even a single dNTP may affect all four pools through dysregulation of ribonucleotide reduction and/or dissipation of the non-limiting dNTPs during unproductive elongation of new DNA chains.
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 deoxynucleoside triphosphate (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 induced by the absence of p53R2 activity. We conclude that loss of p53R2 affects ribonucleotide reduction only in resting cells and leads to a decrease of dNTP catabolism by substrate cycles that counterweigh 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.
Deoxyribonucleoside triphosphates (dNTPs) are the precursors used by DNA polymerases for replication and repair of nuclear and mitochondrial DNA in animal cells. Accurate DNA synthesis requires adequate amounts of each dNTP and appropriately balanced dNTP pools. Total cellular pool sizes are in the range of 10-100pmoles of each dNTP/million cells during S phase, with mitochondrial pools representing at most 10% of the total. In quiescent or differentiated cells pools are about 10-fold lower both in the cytosol and mitochondria. Contrary to what may be expected on the basis of the roughly equimolar abundance of the 4 nitrogen bases in DNA, the four dNTPs are present in the pools in different ratios, with pyrimidines often exceeding purines. Individual cell lines may exhibit different pool compositions even if they are derived from the same animal species. It has been known for several decades that imbalance of dNTP pools has mutagenic and cytotoxic effects, and leads to "mutator" phenotypes characterized by increased mutation frequencies. Until 10 years ago this phenomenon was considered to affect exclusively the nuclear genome. With the discovery that thymidine phosphorylase deficiency causes destabilization of mitochondrial DNA and a severe multisystemic syndrome the importance of dNTP pool balance was extended to mitochondria. Following that first discovery, mutations in other genes coding for mitochondrial or cytosolic enzymes of dNTP metabolism have been associated with mitochondrial DNA depletion syndromes. Both excess and deficiency of one dNTP may be detrimental. We study the mechanisms that in mammalian cells keep the dNTP pools in balance, and are particularly interested in the enzymes that, similar to thymidine phosphorylase, contribute to pool regulation by degrading dNTP precursors. The role of some relevant enzymes is illustrated with data obtained by chemical or genetic manipulation of their expression in cultured mammalian cells.
Mitochondrial thymidine kinase (TK2) catalyzes the phosphorylation of thymidine in mitochondria. Its function becomes essential for dTTP synthesis in noncycling cells, where cytosolic dTTP synthesis via R1/R2 ribonucleotide reductase and thymidine kinase 1 is turned down. Mutations in the nuclear gene for TK2 cause a fatal mtDNA depletion syndrome. Only selected cell types are affected, suggesting that the other cells compensate for the TK2 deficiency by adapting the enzyme network that regulates dTTP synthesis outside S-phase. Here we looked for such metabolic adaptation in quiescent cultures of fibroblasts from two TK2-deficient patients with a slow-progressing syndrome. In cell extracts, we measured the activities of TK2, deoxycytidine kinase, thymidine phosphorylase, deoxynucleotidases and the amounts of the three ribonucleotide reductase subunits. Patient cells contained 40% or 5% TK2 activity and unchanged activities of the other enzymes. However, their mitochondrial and cytosolic dTTP pools were unchanged, and also the overall composition of the dNTP pools was normal. TK2-dependent phosphorylation of [(3)H]thymidine in intact cells and the turnover of the dTTP pool showed that even the fibroblasts with 5% residual TK2 activity synthesized dTTP at an almost normal rate. Normal fibroblasts apparently contain more TK2 than needed to maintain dTTP during quiescence, which would explain why TK2-mutated fibroblasts do not manifest mtDNA depletion despite their reduced TK2 activity.
In cycling cells cytosolic de novo synthesis of deoxynucleotides is the main source of precursors for mitochondrial (mt) DNA synthesis. The transfer of deoxynucleotides across the inner mt membrane requires protein carriers. PNC1, a SLC25 family member, exchanges pyrimidine nucleoside triphosphates in liposomes and its downregulation decreases mtUTP concentration in cultured cells. By an isotope-flow protocol we confirmed transport of uridine nucleotides by PNC1 in intact cultured cells and investigated PNC1 involvement in the mt trafficking of thymidine phosphates. Key features of our approach were the manipulation of PNC1 expression by RNA interference or inducible overexpression, the employment of cells proficient or deficient for cytosolic thymidine kinase (TK1) to distinguish the direction of flow of thymidine nucleotides across the mt membrane during short pulses with [(3)H]-thymidine, the determination of mtdTTP specific radioactivity to quantitate the rate of mtdTTP export to the cytoplasm. Downregulation of PNC1 in TK1(-) cells increased labeled dTTP in mitochondria due to a reduced rate of export. Overexpression of PNC1 in TK1(+) cells increased mtdTTP pool size and radioactivity, suggesting an involvement in the import of thymidine phosphates. Thus PNC1 is a component of the network regulating the mtdTTP pool in human cells.
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