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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Although SARS-CoV-2 was reported to alter several cellular pathways, its impact on DNA integrity and the mechanisms involved remain unknown. Here we show that SARS-CoV-2 causes DNA damage and elicits an altered DNA damage response. Mechanistically, SARS-CoV-2 proteins ORF6 and NSP13 cause degradation of the DNA damage response kinase CHK1 through proteasome and autophagy, respectively. CHK1 loss leads to deoxynucleoside triphosphate (dNTP) shortage, causing impaired S-phase progression, DNA damage, pro-inflammatory pathways activation and cellular senescence. Supplementation of deoxynucleosides reduces that. Furthermore, SARS-CoV-2 N-protein impairs 53BP1 focal recruitment by interfering with damage-induced long non-coding RNAs, thus reducing DNA repair. Key observations are recapitulated in SARS-CoV-2-infected mice and patients with COVID-19. We propose that SARS-CoV-2, by boosting ribonucleoside triphosphate levels to promote its replication at the expense of dNTPs and by hijacking damage-induced long non-coding RNAs’ biology, threatens genome integrity and causes altered DNA damage response activation, induction of inflammation and cellular senescence.

Traumas and chronic damages can hamper the regenerative power of nervous, muscle, and connective tissues. Tissue engineering approaches are promising therapeutic tools, aiming to develop reliable, reproducible, and economically affordable synthetic scaffolds which could provide sufficient biomimetic cues to promote the desired cell behaviour without triggering graft rejection and transplant failure. Here, we used 3D-printing to develop 3D-printed scaffolds based on either PLA or graphene@PLA with a defined pattern. Multiple regeneration strategies require a specific orientation of implanted and recruited cells to perform their function correctly. We tested our scaffolds with induced pluripotent stem cells (iPSC), neuronal-like cells, immortalised fibroblasts and myoblasts. Our results demonstrated that the specific “lines and ridges” 100 µm-scaffold topography is sufficient to promote myoblast and fibroblast cell alignment and orient neurites along with the scaffolds line pattern. Conversely, graphene is critical to promote cells differentiation, as seen by the iPSC commitment to neuroectoderm, and myoblast fusions into multinuclear myotubes achieved by the 100 µm scaffolds containing graphene. This work shows the development of a reliable and economical 3D-printed scaffold with the potential of being used in multiple tissue engineering applications and elucidates how scaffold micro-topography and graphene properties synergistically control cell differentiation.

Mitochondrial diseases impair oxidative phosphorylation and ATP production, while effective treatment is still lacking. Defective complex III is associated with a highly variable clinical spectrum. We show that pyocyanin, a bacterial redox cycler, can replace the redox functions of complex III, acting as an electron shunt. Sub-μM pyocyanin was harmless, restored respiration and increased ATP production in fibroblasts from five patients harboring pathogenic mutations in TTC19, BCS1L or LYRM7, involved in assembly/stabilization of complex III. Pyocyanin normalized the mitochondrial membrane potential, and mildly increased ROS production and biogenesis. These in vitro effects were confirmed in both DrosophilaTTC19KO and in Danio rerioTTC19KD, as administration of low concentrations of pyocyanin significantly ameliorated movement proficiency. Importantly, daily administration of pyocyanin for two months was not toxic in control mice. Our results point to utilization of redox cyclers for therapy of complex III disorders., Mitochondrial diseases, including those caused by defects in complex III of the respiratory chain, lack curative treatments. Here the authors report that the small molecule pyocyanin has beneficial effects in cells derived from patients with complex III-deficiency as well as in fly and zebrafish genetic models with reduced complex III activity.

The Euganean Thermal District (Italy) represents the oldest and largest thermal center in Europe, and its therapeutic mud is considered a unique product whose beneficial effects have been documented since Ancient Roman times. Mud properties depend on the heat and electrolytes of the thermal water, as well as on the bioactive molecules produced by its biotic component, mainly represented by cyanobacteria. The investigation of the healing effects of compounds produced by the Euganean cyanobacteria represents an important goal for scientific validation of Euganean mud therapies and for the discovering of new health beneficial biomolecules. In this work, we evaluated the therapeutic potential of exopolysaccharides (EPS) produced by Phormidium sp. ETS05, the most abundant cyanobacterium of the Euganean mud. Specifically, Phormidium EPS resulted in exerting anti-inflammatory and pro-resolution activities in chemical and injury-induced zebrafish inflammation models as demonstrated using specific transgenic zebrafish lines and morphometric and expression analyses. Moreover, in vivo and in vitro tests showed no toxicity at all for the EPS concentrations tested. The results suggest that these EPS, with their combined anti-inflammatory and pro-resolution activities, could be one of the most important therapeutic molecules present in the Euganean mud and confirm the potential of these treatments for chronic inflammatory disease recovery.

Terminally differentiated cells are defined by their inability to proliferate. When forced to re-enter the cell cycle, they generally cannot undergo long-term replication. Our previous work with myotubes has shown that these cells fail to proliferate because of their intrinsic inability to complete DNA replication. Moreover, we have reported pronounced modifications of deoxynucleotide metabolism during myogenesis. Here we investigate the causes of incomplete DNA duplication in cell cycle-reactivated myotubes (rMt). We find that rMt possess extremely low levels of thymidine triphosphate (dTTP), resulting in very slow replication fork rates. Exogenous administration of thymidine or forced expression of thymidine kinase increases deoxynucleotide availability, allowing extended and faster DNA replication. Inadequate dTTP levels are caused by selective, differentiation-dependent, cell cycle-resistant suppression of genes encoding critical synthetic enzymes, chief among which is thymidine kinase 1. We conclude that lack of dTTP is at least partially responsible for the inability of myotubes to proliferate and speculate that it constitutes an emergency barrier against unwarranted DNA replication in terminally differentiated cells.

YAP/TAZ, downstream transducers of the Hippo pathway, are powerful regulators of cancer growth. How these factors control proliferation remains poorly defined. Here, we found that YAP/TAZ directly regulate expression of key enzymes involved in deoxynucleotide biosynthesis and maintain dNTP precursor pools in human cancer cells. Regulation of deoxynucleotide metabolism is required for YAP-induced cell growth and underlies the resistance of YAP-addicted cells to chemotherapeutics targeting dNTP synthesis. During RAS-induced senescence, YAP/TAZ bypass RAS-mediated inhibition of nucleotide metabolism and control senescence. Endogenous YAP/TAZ targets and signatures are inhibited by RAS/MEK1 during senescence, and depletion of YAP/TAZ is sufficient to cause senescence-associated phenotypes, suggesting a role for YAP/TAZ in suppression of senescence. Finally, mechanical cues, such as ECM stiffness and cell geometry, regulate senescence in a YAP-dependent manner. This study indicates that YAP/TAZ couples cell proliferation with a metabolism suited for DNA replication and facilitates escape from oncogene-induced senescence. We speculate that this activity might be relevant during the initial phases of tumour progression or during experimental stem cell reprogramming induced by YAP.

Background: Genetic loss of SAMHD1, a catabolic enzyme preventing dNTP accumulation outside S phase, leads to disease in humans but not in mice. Results: Allosteric regulation by dNTPs works similarly in pure recombinant human and mouse SAMHD1. Conclusion: Human SAMHD1 is more efficiently regulated at low dNTP concentrations. Significance: Inside cells dATP and dTTP are the main regulators of both human and mouse SAMHD1., 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 μm for mouse and 1 μm for human SAMHD1, for dTTP the corresponding values are 50 and 2 μm. 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.

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 [3H]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 non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level.

Background: Deoxyguanosine kinase (dGK) deficiency causes mtDNA depletion in quiescent cells where SAMHD1 restricts dGTP concentration. Results: siRNA silencing of SAMHD1 corrects mt dNTP imbalance boosting mtDNA content in quiescent dGK-mutated human fibroblasts. Conclusion: In noncycling cells SAMHD1 activity worsens the phenotype of dGK genetic deficiency, limiting dGTP supply. Significance: Nuclear SAMHD1 and mitochondrial dGK functionally interact in the regulation of mt dGTP for mtDNA maintenance., The dNTP triphosphohydrolase SAMHD1 is a nuclear antiviral host restriction factor limiting HIV-1 infection in macrophages and a major regulator of dNTP concentrations in human cells. In normal human fibroblasts its expression increases during quiescence, contributing to the small dNTP pool sizes of these cells. Down-regulation of SAMHD1 by siRNA expands all four dNTP pools, with dGTP undergoing the largest relative increase. The deoxyguanosine released by SAMHD1 from dGTP can be phosphorylated inside mitochondria by deoxyguanosine kinase (dGK) or degraded in the cytosol by purine nucleoside phosphorylase. Genetic mutations of dGK cause mitochondrial (mt) DNA depletion in noncycling cells and hepato-cerebral mtDNA depletion syndrome in humans. We studied if SAMHD1 and dGK interact in the regulation of the dGTP pool during quiescence employing dGK-mutated skin fibroblasts derived from three unrelated patients. In the presence of SAMHD1 quiescent mutant fibroblasts manifested mt dNTP pool imbalance and mtDNA depletion. When SAMHD1 was silenced by siRNA transfection the composition of the mt dNTP pool approached that of the controls, and mtDNA copy number increased, compensating the depletion to various degrees in the different mutant fibroblasts. Chemical inhibition of purine nucleoside phosphorylase did not improve deoxyguanosine recycling by dGK in WT cells. We conclude that the activity of SAMHD1 contributes to the pathological phenotype of dGK deficiency. Our results prove the importance of SAMHD1 in the regulation of all dNTP pools and suggest that dGK inside mitochondria has the function of recycling the deoxyguanosine derived from endogenous dGTP degraded by SAMHD1 in the nucleus.

Deoxynucleoside triphosphates (dNTPs) used for mitochondrial DNA replication are mainly formed by phosphorylation of deoxynucleosides imported into mitochondria from the cytosol. We earlier obtained evidence for a mitochondrial 5′-nucleotidase (dNT2) with a pronounced specificity for dUMP and dTMP and suggested that the enzyme protects mitochondrial DNA replication from excess dTTP. In humans, accumulation of dTTP causes a mitochondrial genetic disease. We now establish that dNT2 in vivo indeed is located in mitochondria. The native enzyme shows the same substrate specificity and affinity for inhibitors as the recombinant dNT2. We constructed ponasterone-inducible cell lines overproducing dNT2 with and without the green fluorescent protein (GFP) linked to its C terminus. The fusion protein occurred in mitochondria mostly in an inactive truncated form, with only a short C-terminal fragment of dNT2 linked to GFP. No truncation occurred when dNT2 and GFP were not linked. The cell mitochondria then contained a large excess of active dNT2 with or without the mitochondrial presequence. After removal of ponasterone overproduced dNT2 disappeared only slowly from the cells, whereas dNT2-mRNA was lost rapidly. Overproduction of dNT2 did not lead to an increased excretion of pyrimidine deoxyribonucleosides, in contrast to overproduction of the corresponding cytosolic deoxynucleotidase, suggesting that the mitochondrial enzyme does not affect overall cellular deoxynucleotide turnover.

Two of the five known mammalian 5 0 -nucleotidases show a preference for the dephosphorylation of deoxynucleoside-5 0 -phosphates. One is a cytoplasmic enzyme (dNT-1), the other occurs in mitochondria (dNT-2). The human mitochondrial enzyme, recently discovered and cloned by us, is encoded by a nuclear gene located on chromosome 17 p11.2 in the critical region deleted in the Smith‐Magenis syndrome (SMS), a genetic disease of unknown etiology. Looking for a model system to study the possible involvement of dNT-2 in the disease, we have cloned the cDNA of the mouse ortholog. The deduced protein sequence is 84% identical to the human ortholog, has a very basic NH2terminus, a very high calculated probability of being imported into mitochondria and contains the DXDXT/V motif conserved among nucleotidases. Expression in Escherichia coli of the predicted processed form of the protein produced an active deoxyribonucleotidase. We also identified in genomic sequences present in the data base the structures of the murine genes for the cytosolic and mitochondrial deoxyribonucleotidases (Nt5c and Nt5m). PAC clones for the two loci were isolated from a library and used for chromosomal localization by fluorescent in situ hybridization. Both genes map on chromosome 11: Nt5c at 11E and Nt5m at 11B, demonstrating the presence of the dNT-2 locus in the mouse shaker-2 critical region, the murine counterpart of the human SMS region. We performed pair-wise dot-plot and PIP (percent identity plot) analyses of mouse and human deoxyribonucleotidase genes, and found a strong conservation that extends also to some intronic sequences of possible regulatory significance. q 2002 Elsevier Science B.V. All rights reserved.

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.

Background: In developing muscle, stimulation of mitochondrial biogenesis and mtDNA expansion occur with down-regulation of deoxynucleotide synthesis. Results: siRNA silencing of mitochondrial thymidine or deoxyguanosine kinase impacts myotube differentiation causing depletion of mtDNA and of all four deoxynucleotides. Conclusion: Shortage of even a single deoxynucleotide may upset the regulation of all DNA precursors. Significance: Deoxynucleotide analysis in myotubes unveils unexpected outcomes of synthetic enzyme deficiencies., 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.

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.

We 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.

Three cytosolic and one plasma membrane-bound 5′-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5′-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5′- and 2′(3′)-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. Duringin vitroimport into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith–Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.

5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whose physiological function is not known. It was earlier purified to homogeneity from human placenta. We determined the amino acid sequences of several internal peptides and with their aid found an expressed sequence tag clone with the complete cDNA for a murine enzyme of 23.9 kDa. The DNA was cloned into appropriate plasmids and introduced into Escherichia coli and ecdyson-inducible 293 and V79 cells. The recombinant enzyme was purified to homogeneity from transformed E. coli and was found to be identical with the native enzyme. After induction with ponasterone, the transfected mammalian cells showed a gradual increase of enzyme activity. A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked the 5'-end corresponding to 51 amino acids of the murine enzyme. Several polymerase chain reaction-based approaches to find this sequence met with no success. A mouse/human hybrid cDNA that had substituted the missing human 5'-end with the corresponding mouse sequence coded for a fully active enzyme.

5'-Nucleotidases participate, together with nucleoside kinases, in substrate cycles involved in the regulation of deoxyribonucleotide metabolism. Three major classes of nucleotidases are known, one on the plasma membrane and two in the cytosol. The two cytosolic classes have been named high-Km nucleotidases and 5'(3')-nucleotidases. Starting from two plasmids with partial sequences (Oka, J., Matsumoto, A., Hosokawa, Y. & Inoue, S. (1994) Biochem. Biophys. Res. Commun. 205, 917-922) we cloned the complete cDNA of the human high-Km nucleotidase into vectors suitable for transfection of Escherichia coli or mammalian cells. After transfection, E. coli overproduced large amounts of the enzyme. Most of the enzyme was present in inclusion bodies that also contained many partially degraded products of the protein. Part of the enzyme, corresponding to approximately 2% of the soluble proteins, was in a soluble active form. Stably transfected human 293 cells were obtained with a vector where the 3'-end of the nucleotidase coding sequence is linked to the 5'-end of the green fluorescent protein coding sequence. Several green clones overproduced both mRNA and fusion protein. Two clones with 10-fold higher enzyme activity were analyzed further. The nucleotidase activity of cell extracts showed the same substrate specificity and allosteric regulation as the high-Km enzyme. The growth rate of the two clones did not differ from the controls. The cells were not resistant to deoxyguanosine or deoxyadenosine, and did not show an increased ability to phosphorylate dideoxyinosine. Both ribonucleoside and deoxyribonucleoside triphosphate pools were decreased slightly, suggesting participation of the enzyme in their regulation.

We incorporated 3H-labeled thymidine, deoxycytidine, or cytidine into dNTPs and DNA of exponentially growing CEM cells. G1 and S phase cells were separated by centrifugal elutriation, and the size and specific activity of dNTP pools were determined to study the cell cycle-dependent regulation of specific dNTP synthesizing enzymes in their metabolic context. With [3H]thymidine, we confirm the earlier demonstrated S phase specificity of thymidine kinase. Incorporation of radioactivity from [5-3H]deoxycytidine into dCTP occurred almost exclusively in G1 cells. During S phase, de novo synthesis by ribonucleotide reductase was switched on, resulting in a 70-fold dilution of [3H]dCTP, confirming that ribonucleotide reductase is an S phase-specific enzyme, whereas deoxycytidine kinase is not. [5-3H]Cytidine appeared in dCTP almost to the same extent in G1 as in S phase, despite the S phase specificity of ribonucleotide reductase. During S phase, DNA replication greatly increased the turnover of dCTP, requiring a corresponding increase in ribonucleotide reductase activity. During G1, the enzyme maintained activity to provide dNTPs for DNA repair and mitochondrial DNA synthesis. The poor incorporation of isotope from deoxycytidine into DNA earlier led to the suggestion that the nucleoside is used only for DNA repair (Xu, Y-Z., Peng, H., and Plunkett, W. (1995) J. Biol. Chem. 270, 631-637). The poor phosphorylation of deoxycytidine in S phase provides a better explanation.

Ribonucleotide reductase inhibitors reduce the cellular supply of DNA precursors(dNTP) by interfering with their de novo synthesis. A secondary effect is the stimulation of the uptake and phosphorylation of extracellular deoxynucleosides, including their analogs, e.g., 3'-azidothymidine (AZT). Both effects are relevant to HIV replication, which requires dNTP and is impaired by the triphosphate of AZT. Earlier we demonstrated that ribonucleotide reductase inhibitors--hydroxyurea, and two deoxycytidine analogs specifically active in lymphoid cells--increased the phosphorylation of AZT in CEM cells by prolonging the S phase of the cell cycle. Here we tested the effects of long-term treatments on HIV proliferation in CEM cells and stimulated human lymphocytes infected with HIV-1IIIB. Treatment with low doses of AZT (0.05-0.1 microM) and either hydroxyurea (25-100 microM) or 2'-azido-2'-deoxycytidine (0.25-4 microM) lasted 2 weeks, during which p24 in the culture medium was monitored. Noninfected CEM cells were treated in parallel to measure the inhibition of cell growth, distribution along the cell cycle, dNTP pool size, and level of tritiated AZT phosphorylation. A clear synergism between AZT and ribonucleotide reductase inhibitors was observed at nontoxic doses that induced only minor changes in the cellular parameters measured. The reductase inhibitors by themselves interfered with replication only at doses that inhibited cell proliferation.