The mechanism of inhibition and "reversal" of mitogen-induced lymphocyte activation in a model of adenosine deaminase deficiency

The biochemical mechanism of lymphocyte dysfunction with adenosine deaminase deficiency has been investigated using cultured phytohemagglutinin stimulated normal peripheral blood lymphocytes and the adenosine deaminase (ADA) inhibitor 2'-deoxycoformycin. The addition of deoxyadenosine to ADA-inhibited (but not to uninhibited) cells generated increased dATP pools (up to 50-fold greater than controls) and depressed the mitogen response. dATP Accumulation was accompanied by depletion of the other three deoxynucleoside triphosphate (dNTP) pools (dTTP, dCTP, and dGTP). Suppression of the mitogen response could be prevented ("reversed") to 90% of control levels by the addition of deoxynucleoside precursors for the depleted dNTPs at the initiation of mitogen stimulation. "Reversal" restored the dTTP and possibly the dGTP pools. Thus the mechanism of toxicity in this model appears to be inhibition of ribonucleotide reductase by massive accumulation of dATP, resulting in starvation for the other three deoxyribonucleoside triphosphates. "Reversibility" of this toxicity by providing sources for the missing three deoxynucleoside triphosphates argues for ribonucleotide reductase inhibition rather than other mechanisms of deoxyadenosine toxicity in this model.     

Deoxynucleoside Kinases of Bacillus megaterium KM

Dialyzed extracts of Bacillus megaterium KM contain thymidine, deoxyadenosine, and deoxyguanosine kinase activities. Thymidine kinase activity is best with deoxyadenosine triphosphate or deoxyguanosine triphosphate (dGTP) as the phosphoryl donor, whereas the best deoxyadenosine kinase activity is obtained with dGTP or adenosine triphosphate. Deoxyguanosine kinase activity functions optimally with deoxycytidine triphosphate as the donor. Although the thymidine kinase activity of crude extracts does not have a demonstrable divalent cation requirement, the addition of Mg(2+) or Mn(2+) is necessary for the formation of thymidine di- and triphosphates. The synthesis of thymidine kinase appears to be partially derepressed by thymine starvation. Incubation of extracts with deoxyadenosine and dGTP results in the substantial accumulation of deoxyadenosine di- and triphosphates. Extracts deaminate deoxycytidine to deoxyuridine, presumably as a consequence of the action of deoxycytidine deaminase, and then convert deoxyuridine to deoxyuridylic acid. B. megaterium extracts do not contain any detectable deoxycytidine kinase activity.     

IUPAC-IUPAB-IUB Intercommission on Biothermodynamics. Recommendations for the presentation of thermodynamic and related data in biology (1985)

High levels of deoxyadenosine and deoxyguanosine in patients with inherited deficiency of either adenosine deaminase or purine-nucleoside phosphorylase, respectively, are considered to be responsible for the associated immunological disorder. The mechanism involves phosphorylation to the corresponding deoxyribonucleoside triphosphates which subsequently inhibit the CDP-reducing activity of ribonucleotide reductase. Addition of deoxycytidine protects cells from the cytotoxic effects of deoxyadenosine and deoxyguanosine by competition for phosphorylation and by replenishing dCTP, the apparent limiting DNA precursor. Addition of cytidine, but not uridine, led to a reversal of deoxyguanosine and thymidine growth inhibition, comparable to that obtained with deoxycytidine. Analysis of the intracellular nucleotide pools showed that increased levels of cytidine ribonucleotides were sufficient to overcome the inhibitory effects of dGTP and dTTP on CDP reduction, thereby circumventing a depletion of the dCTP pool. A partial reversal of deoxyadenosine toxicity was also obtained with addition of cytidine. In this case little change in the dCTP level was observed, but a decreased dGTP pool appeared to be correlated with growth inhibition. High cytidine ribonucleotide levels partially prevented this effect. The present results may encourage the use of cytidine in combination with deoxycytidine as a pharmacological regime in treatment of immunodeficiency disease associated with increased deoxyribonucleotide levels.     

Deoxyadenosine reverses hydroxyurea inhibition of vaccinia virus growth.

Hydroxyurea, an inhibitor of ribonucleotide reductase, blocks replication of vaccinia virus. However, when medium containing hydroxyurea and dialyzed serum was supplemented with deoxyadenosine, the block to viral reproduction was circumvented, provided that an inhibitor of adenosine deaminase was also present. Deoxyguanosine, deoxycytidine, and deoxythymidine were ineffective alone and did not augment the deoxyadenosine effect. In fact, increasing concentrations of deoxyguanosine and deoxythymidine, but not deoxycytidine, eliminated the deoxyadenosine rescue, an effect that was reversed by the addition of low concentrations of deoxycytidine. These results suggested that the inhibition of viral replication by hydroxyurea was primarily due to a deficiency of dATP. Deoxyribonucleoside triphosphate pools in vaccinia virus-infected cells were measured at the height of viral DNA synthesis after a synchronous infection. With 0.5 mM hydroxyurea, the dATP pool was greater than 90% depleted, the dCTP and dGTP pools were 40 to 50% reduced, and the dTTP pool was increased. Assay of ribonucleotide reductase activity in intact virus-infected cells suggested that hydroxyurea may differentially affect reduction of the various substrates of the enzyme.     

The effect of deoxyguanosine on human lymphocyte function. II. Analysis of the interference with B lymphocyte differentiation in vitro

The differentiation of normal human peripheral blood B lymphocytes into plasma cells in vitro, studied in mononuclear cells stimulated with PWM or in purified B cells stimulated with a T cell-replacing factor (TRF), can be inhibited by both deoxyguanosine (dGuo) and guanosine. The mechanism underlying this effect, which differs from the in vivo findings in PNP deficiency, was analyzed. dGuo toxicity can be antagonized by hypoxanthine but not by deoxycytidine. PNP-deficient and HGPRT-deficient B lymphocytes are not sensitive to the intoxicating properties of (deoxy)guanosine. Inhibition of PNP activity in normal B lymphocytes by 8-aminoguanosine decreases the sensitivity for dGuo intoxication. Incubation of purified B cells (stimulated with TRF) with dGuo leads to increased intracellular levels of guanosine di- and triphosphate (GDP and GTP), whereas deoxyguanosine triphosphate (dGTP) levels remain low. These observations lead to the conclusion that inhibition of B lymphocyte differentiation by dGuo is brought about by one of the end products of the pathway starting with degradation of dGuo by PNP, followed by guanine salvage by HGPRT, and possibly further phosphorylation of GMP into GDP and GTP. According to this mechanism, B lymphocyte differentiation in PNP deficiency is not sensitive to (deoxy)guanosine; because of the absence of PNP activity, these cells cannot accumulate GMP, GDP, and GTP, and therefore escape dGuo intoxication.     

Lack of specific correlation of the deoxycytidine triphosphate pool level with rate of DNA synthesis.

The levels of the four deoxyribonucleoside triphosphate pools and the distribution of cells in the various phases of the cell cycle have been examined in Chinese hamster cells as thymidine, present as a regular constituent in the growth medium, was removed in stages. The results indicate that: 1. Duration of the DNA synthetic phase was lengthened when thymidine was removed from the growth medium. 2.Temporally correlated with lengthening of the DNA synthetic phase upon thymidine removal was a 7-fold increase in level of the dCTP pool, reduction in the dGTP pools, and little or no change in dATP pool. 3.Radioactive labeling procedures indicated that expansion of the dCTP pool could be completely accounted for by increased ribonucleotide reductase activity and that the dTTP pool switched from a largely exogenous thymidine source to endogenous dTTP synthesis as the extracellular thymidine concentration was reduced. 4.Deoxyuridine and thymidine were apparently transported by the same system in Chinese hamster cells, while deoxycytidine was transported by a different system. Although deoxycytidine transport was unaffected by thymidine, phosphorylation of intracellular deoxycytidine compounds to the triphosphate level was stimulated by thymidine. Cytidine transport was not significantly affected by thymidine.     

Mechanisms of deoxyguanosine lymphotoxicity. Human thymocytes, but not peripheral blood lymphocytes accumulate deoxy-GTP in conditions simulating purine nucleoside phosphorylase deficiency

This study was designed to simulate purine nucleoside phosphorylase (PNP) deficiency by preincubating with guanosine (Guo) to minimize PNP activity while investigating the metabolism of [14C] deoxyguanosine (dGuo) at physiologic concentrations (10 microM) by unstimulated thymocytes, tonsil-derived T and B lymphocytes, and peripheral blood cells over short time periods. GTP was the principal metabolite formed from dGuo by all cell types with functional PNP and hypoxanthine-guanine phosphoribosyltransferase, confirming formation via degradation to guanine with subsequent salvage by hypoxanthine-guanine phosphoribosyltransferase. Thymocytes also formed a small amount of deoxyguanosine triphosphate (dGTP), presumably through direct phosphorylation by deoxycytidine kinase. Incorporation of dGuo into GTP was effectively inhibited in all instances under PNP deficiency conditions and dGTP levels increased up to 10-fold in thymocytes, but tonsil-derived B or T lymphocytes and unfractionated PBL still accumulated no detectable dGTP. E and platelets formed low amounts of dGTP under these conditions. Preincubation with adenine (50 microM) to reverse any Guo-induced toxicity reduced the incorporation of dGuo into GTP without inhibitor in all cell types with intact adenine phosphoribosyltransferase, but had no effect on dGTP accumulation in thymocytes, with or without inhibitor, thus excluding any indirect formation of dGTP via the de novo route. The rapid metabolism of dGuo to GTP, in the absence of PNP inhibition and subsequent effects of the altered GTP concentrations on cellular metabolism, may account for the differing responses reported by investigators with the use of low dGuo concentrations (enhancing), compared with high (inhibitory), concentrations in mitogen-stimulated lymphocyte studies. The exclusive ability of thymocytes to accumulate significant amounts of dGTP, and inability of B cells to do so, provides a logical explanation for the selective T cell immunodeficiency in PNP deficiency.     

Deoxyadenosine toxicity and cell cycle arrest in hydroxyurea-resistant S49 T-lymphoma cells

Hydroxyurea-resistant S49 T-lymphoma cells have increased ribonucleotide reductase activity and deoxyribonucleoside triphosphate pools when compared with wild-type cultures. If ribonucleotide reductase inhibition is the mechanism by which deoxyadenosine is cytotoxic, then hydroxyurea (HU)-resistant S49 cells might be more resistant to deoxyadenosine toxicity when adenosine deaminase is inhibited than wild-type cells. Five S49 cell lines resistant to varying concentrations of HU were compared with wild-type cells by measuring CDP reductase activity, deoxyribonucleoside triphosphate pools, and deoxyadenosine toxicity. All five cell lines resistant to increasing concentrations of HU exhibited a twofold increase in resistance to deoxyadenosine toxicity when compared to wild type, and the resistance was proportional to the twofold increased pools of dNTPs in these cell lines but was less than the six- to eight fold increase in ribonucleotide reductase activity. In both wild-type and mutant cell lines, deoxyadenosine toxicity was accompanied by the accumulation of deoxyadenosine triphosphate and reduction of the other dNTPs; however, only dGTP greatly diminished. Exogenous addition of deoxycytidine decreased the dATP accumulation by about 20%, but also resulted in increases in the dCTP, dTTP, and dGTP pools. The S49 cells arrested in G1 phase when exposed to dAdo, although hydroxyurea-resistant cells required higher dAdo concentrations to elicit G1-phase arrest than wild-type cells. Deoxycytidine prevented dAdo-induced G1 arrest in all cell types. In summary, these data support the hypothesis that deoxyadenosine-induced dATP accumulation results in inhibition of ribonucleotide reductase and that this may be the mechanism for both cell cycle arrest and cytotoxicity in S49 T-lymphoma cells.     

Purine and pyrimidine metabolism in human T lymphocytes. Regulation of deoxyribonucleotide metabolism

Purine and pyrimidine deoxyribonucleoside metabolism was studied in G1 and S phase human thymocytes and compared with that of the more mature T lymphocytes from peripheral blood. Both thymocyte populations have much higher intracellular deoxyribonucleoside triphosphate (dNTP) pools than peripheral blood T lymphocytes. The smallest dNTP pool in S phase thymocytes is dCTP (5.7 pmol/10(6) cells) and the largest is dTTP (48 pmol/10(6) cells), whereas in G1 thymocytes, dATP and dGTP comprise the smallest pools. While both G1 and S phase thymocytes have active deoxyribonucleoside salvage pathways, only S phase thymocytes have significant ribonucleotide reduction activity. We have studied ribonucleotide reduction and deoxyribonucleoside salvage in S phase thymocytes in the presence of extracellular deoxyribonucleosides. Based on these studies, we propose a model for the interaction of deoxyribonucleoside salvage and ribonucleotide reduction in S phase thymocytes. According to this model, extracellular deoxycytidine at micromolar concentrations is efficiently salvaged by deoxycytidine kinase. However, due to feedback inhibition of deoxycytidine kinase by dCTP, the maximal level of dCTP which can be achieved is limited. The salvage of both deoxyadenosine and deoxyguanosine (up to 10(-4) M) is completely inhibited in the presence of micromolar concentrations of deoxycytidine, whereas the salvage of thymidine is unregulated resulting in large increases in dTTP levels. Moreover, significant amounts of the salvaged deoxycytidine is used for dTTP synthesis resulting in further increase of dTTP pools. The accumulated dTTP inhibits the reduction of UDP and CDP while stimulating GDP reduction and subsequently also ADP reduction. The end result of the proposed model is that S phase thymocytes in the presence of a wide range of extracellular deoxyribonucleoside concentrations synthesize their pyrimidine dNTP by the salvage pathway, whereas purine dNTPs are synthesized primarily by ribonucleotide reduction. Using the proposed model, it is possible to predict the relative intracellular dNTP pools found in fresh S phase thymocytes.     

Production of 2-Methylisocitric Acid from n-Paraffins by Mutants of Candida lipolytica

Vitamin B₁₂-dependent ribonucleotide reductase purified from Rhizobium meliloti catalyzes the reduction of 5′-diphosphates of guanosine, adenosine, cytidine and uridine (GDP, ADP, CDP and UDP). The enzyme activities were regulated by Mg²⁺ and deoxyribonucleoside triphosphate effectors as follows: in the presence of Mg²⁺, allosteric effector deoxyguanosine triphosphate (dGTP) had the most stimulatory effect on reduction of ADP and UDP; deoxyadenosine triphosphate (dATP) on reduction of CDP; and thymidine triphosphate (dTTP) on reduction of GDP. These stimulatory effectors were active at a low concentration of 10 μm. Other deoxyribonucleotides may be negative or weakly positive effectors. Without effectors, the rate profile of ADP and GDP reduction showed a sigmoidal curve. In the absence of Mg²⁺, the activities of the reductase showed nearly maximal levels, and the addition of effectors rather decreased the activities, except in the case of UDP reduction which was most strongly stimulated by dGTP. The effect of Mg²⁺ can be replaced by Ca²⁺. Monovalent cations such as Na⁺ and K⁺ had a negligible effect on the activities of ribonucleotide reductase.     

Deoxyguanosine toxicity on lymphoid cells as a cause for immunosuppression in purine nucleoside phosphorylase deficiency.

To delineate the pathogenesis of the immunodeficiency disease associated with purine nucleoside phosphorylase deficiency, the effects of guanosine, inosine, deoxyguanosine and deoxyinosine on the growth of a mouse T cell lymphoma line in culture were studied. Of these four purine nucleosides, deoxyguanosine was the most toxic. At 5 x 10^?6 to 10^?5 M, deoxyguanosine inhibits growth of the lymphoma cells; higher concentrations result in complete killing. The cytotoxic effects of this deoxynucleoside can be prevented by simultaneous addition to culture medium of deoxycytidine and hypoxanthine. Determination of nucleotide pools in deoxyguanosine-treated cells shows a marked reduction of the deoxycytidine triphosphate and the adenine ribonucleotide pools, accompanied by a sharp rise in the guanosine deoxyribonucleotide and a smaller increase in the corresponding ribonucleotide pools.Deoxyguanosine as well as guanosine, inosine and deoxyinosine were known to accumulate to relatively high levels in the plasma of a patient with T cell immunodeficiency disease associated with purine nucleoside phosphorylase deficiency. The other three purine nucleosides are much less toxic than deoxyguanosine. Thus it is very probable that the patient's clinical manifestations of T lymphocytopenia are the consequence of deoxyguanosine inhibition of lymphoid cell proliferation, resulting from depletion of deoxycytidine triphosphate and adenine nucleotides.     

Deoxyguanosine toxicity in a mouse T lymphoma: relationship to purine nucleoside phosphorylase-associated immune dysfunction.

The absence of either of the enzymes adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with an immunodeficiency disease. Because all four nucleoside substrates of the enzyme purine nucleoside phosphorylase accumulate in the urine of patients who lack this enzyme (Cohen et al., 1976), we examined the toxicity of each of the four substrates using a mouse T cell lymphoma (S49) in continuous culture. Of the four substrates (inosine, deoxyinosine, guanosine and deoxyguanosine), only deoxyguanosine is cytotoxic at concentrations lower than 100 μM; furthermore, only deoxyguanosine is directly phosphorylated in S49 cells. Mutant S49 cells lacking deoxycytidine kinase (EC 2.7.1.74) are resistant to the toxic effects of deoxyguanosine, and these same mutants do not phosphorylate deoxyguanosine. Thus the cytotoxicity of exogenous deoxyguanosine correlates with the intracellular concentration of accumulated deoxyGTP.The addition of deoxyguanosine results in the depletion of deoxyCTP in S49 cells, indicating that deoxyGTP is an inhibitor of ribonucleotide reductase. Furthermore, the addition of deoxycytidine prevents the toxic effects of deoxyguanosine. Thus a therapy for purine nucleoside phosphorylase-deficient patients might include deoxycytidine to alleviate the proposed deoxyCTP starvation in those tissues capable of phosphorylating deoxyguanosine.     

Deoxyribonucleotide pools in mouse-fibroblast cell lines with altered ribonucleotide reductase.

Mutant cells lines of 3T6 mouse fibroblasts, resistant to thymidine and deoxyadenosine, have an altered allosteric regulation of the enzyme ribonucleotide reductase (Meuth, M. and Green, H., Cell, 3, 367, 1974). Compared to 3T6, these lines contain larger pools of deoxynucleoside triphosphates, in particular deoxycytidine triphosphate, but show a normal rate of DNA synthesis. Addition of thymidine or deoxyadenosine to 3T6 cells results in large accumulations of the corresponding triphosphates and a dramatic decrease in the dCTP pool, concomitant with inhibition of DNA synthesis. Addition of thymidine to the mutant cell lines also leads to an increase in the dTTP pool but does not result in a depletion of dCTP or inhibition of DNA synthesis. Addition of deoxyadenosine only leads to a small increase of the dATP pool. In general the change in the allosteric regulation of bibonucleotide reductase is reflected in the deoxynucleotide pools.     

Changes of deoxyribonucleoside triphosphate pools induced by hydroxyurea and their relation to DNA synthesis

Hydroxyurea inactivates ribonucleotide reductase from mammalian cells and thereby depletes them of the deoxynucleoside triphosphates required for DNA replication. In cultures of exponentially growing 3T6 cells, with 60-70% of the cells in S-phase, 3 mM hydroxyurea rapidly stopped ribonucleotide reduction and DNA synthesis (incorporation of labeled thymidine). The pool of deoxyadenosine triphosphate (dATP) decreased in size primarily, but also the pools of the triphosphates of deoxyguanosine and deoxycytidine (dCTP) were depleted. Paradoxically, the pool of thymidine triphosphate increased. After addition of hydroxyurea this pool was fed by a net influx and phosphorylation of deoxyuridine from the medium and by deamination of intracellular dCTP. An influx of deoxycytidine from the medium contributed to the maintenance of intracellular dCTP. 10 min after addition of hydroxyurea, DNA synthesis appeared to be completely blocked even though the dATP pool was only moderately decreased. As possible explanations for this discrepancy, we discuss compartmentation of pools and/or vulnerability of newly formed DNA strands to nuclease action and pyrophosphorolysis.     

Deoxyribonucleoside triphosphate metabolism and the mammalian cell cycle. Effects of hydroxyurea on mutant and wild-type mouse S49 T-lymphoma cells

DNA precursor synthesis can be blocked specifically by the drug hydroxyurea (HU) which has therefore been used for anticancer therapy. High concentrations of HU, however, affect other processes than DNA synthesis; nevertheless, most studies on the biological action of HU have been made with concentrations at least one order of magnitude higher than those needed for cell-growth inhibition. In this study we characterized the effects of low concentrations of HU (i.e. concentrations leading to 50% inhibition of cell growth in 72 h) on cell cycle kinetics and nucleotide pools in mouse S49 cells with various defined alterations in DNA precursor synthesis. The effect of 50 microM HU on deoxyribonucleoside triphosphate pools was a 2-3-fold decrease in the dATP and dGTP pools, with no change in the dCTP pool and a certain increase in the dTTP pool. Addition of deoxycytidine or thymidine led to a partial reversal of the growth inhibition and cell-cycle perturbation caused by HU, and was accompanied by an increased level of the deoxyribonucleoside triphosphates. Addition of purine deoxyribonucleoside gave no protection, indicating that salvage of these nucleosides could not supply precursors for DNA synthesis in T-lymphoma cells. We observed a higher sensitivity to HU of cells lacking purine nucleoside phosphorylase or with a ribonucleotide reductase with altered allosteric regulation. Cells lacking thymidine kinase or deoxycytidine kinase were just as sensitive as wild-type cells.     

Metabolic interrelations within guanine deoxynucleotide pools for mitochondrial and nuclear DNA maintenance

Mitochondrial deoxynucleoside triphosphates are formed and regulated by a network of anabolic and catabolic enzymes present both in mitochondria and the cytosol. Genetic deficiencies for enzymes of the network cause mitochondrial DNA depletion and disease. We investigate by isotope flow experiments the interrelation between mitochondrial and cytosolic deoxynucleotide pools as well as the contributions of the individual enzymes of the network to their maintenance. To study specifically the synthesis of dGTP used for the synthesis of mitochondrial and nuclear DNA, we labeled hamster CHO cells or human fibroblasts with [(3)H]deoxyguanosine during growth and quiescence and after inhibition with aphidicolin or hydroxyurea. At time intervals we determined the labeling of deoxyguanosine nucleotides and DNA and the turnover of dGTP from its specific radioactivity in the separated mitochondrial and cytosolic pools. In both cycling and quiescent cells, the import of deoxynucleotides formed by cytosolic ribonucleotide reductase accounted for most of the synthesis of mitochondrial dGTP, with minor contributions by cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. A dynamic isotopic equilibrium arose rapidly from the shuttling of deoxynucleotides between mitochondria and cytosol, incorporation of dGTP into DNA, and degradation of dGMP. Inhibition of DNA synthesis by aphidicolin marginally affected the equilibrium. Inhibition of DNA synthesis by blockage of ribonucleotide reduction with hydroxyurea instead disturbed the equilibrium and led to accumulation of labeled dGTP in the cytosol. The turnover of dGTP decreased, suggesting a close connection between ribonucleotide reduction and pool degradation.     

Deoxyribonucleoside triphosphate pools and differential thymidine sensitivities of cultured mouse lymphoma and myeloma cells

The effects of various concentrations of thymidine on DNA synthesis and deoxyribonucleoside triphosphate contents of a highly thymidine-sensitive cultured mouse lymphoma cell line (WEHI-7) and a relatively resistant mouse myeloma cell line (HPC-108) have been studied by 32P-labelling techniques. DNA synthesis in the myeloma cells was inhibited by thymidine at concentrations of 10(-3) M or greater, while DNA synthesis in the lymphoma cells was inhibited by concentrations 30-fold lower, consistent with the 25-fold difference between the two cell lines in sensitivity to growth inhibition by thymidine. Thymidine caused marked elevation of the dTTP and dGTP pools, slight elevation or no change in the dATP pool and a marked decrease in the dCTP pool in cells of both lines. The greater resistance of HPC-108 cells to thymidine inhibition was related to the finding that they normally contained a much higher concentration of dCTP than did the WEHI-7 cells. Pool size measurements on thymidine-treated (10(-4) M) cells of an additional seven sensitive lymphoma and six relatively resistant myeloma cell lines indicated that in all 15 lines studied, with one exception, a critical concentration of dCTP of about 32 nmol per ml of cell volume was required for the maintenance of normal rates of DNA synthesis. The dCTP content found normally in the lymphoma cells was only a little above this concentration. Amongst the myeloma lines, three contained similarly low levels of dCTP, but were more resistant to thymidine inhibition probably because of their inefficient production of dTTP from thymidine. Cells of the other four myeloma lines (including HPC-108) normally contained much higher dCTP concentrations. The mechanism of thymidine action was explained by reference to the known allosteric properties of ribonucleotide reductase.     

The role of deoxynucleoside triphosphate pools in the inhibition of DNA-excision repair and replication in human cells by hydroxyurea

The effects of hydroxyurea (HU) on the DNA-excision repair process in human cells has been systematically examined. It is demonstrated that HU induces DNA single-strand break accumulation in a dose-dependent fashion in ultraviolet-irradiated and MMS-treated confluent but not log-phase fibroblasts and that these breaks are clearly the consequence of the inhibition by HU of the enzyme, ribonucleotide reductase. The breaks form rapidly, are stable for at least 10 h and largely disappear by 20 h. The production of these DNA-strand breaks is antagonized by a combined treatment of 10 microM deoxyadenosine, deoxycytidine and deoxyguanosine whereas thymidine potentiates strand-break formation at low HU concentrations. It is also confirmed that HU, while inhibiting replicative synthesis has no apparent inhibitory effect on unscheduled DNA synthesis (UDS) although the increased uptake of labeled DNA precursors into HU-treated cells makes it difficult to assess the actual effects on the repair-synthetic process. Analysis of the effects of HU on deoxynucleoside triphosphate pool levels and the demonstration of the failure of the HU block to replicative synthesis to be reversed by high (1 mM) concentrations of added deoxynucleosides lend support to the notion of compartmentalized dNTP pools for repair and replication.     

Deoxyribonucleoside triphosphate pools in regenerating rat liver: effect of hydroxyurea and exogenous deoxypyrimidines

Hydroxyurea (HU) causes inhibition of DNA synthesis in regenerating rat liver due to an inhibition of the ribonucleotide reductase. We studied the consequences of a continuous HU infusion for deoxyribonucleoside triphosphate (dNTP) pools in the liver after partial hepatectomy and tried to modify imbalances by application of deoxyribonucleosides in vivo. In normal liver, an intracellular concentration of 0.16, 0.84, 0.33 and 0.27 pmol/micrograms DNA was observed for dATP, dCTP, dGTP and dTTP, respectively. In regenerating liver the dNTP pools show minor changes until 18 h after partial hepatectomy. During and after a continuous HU infusion 14--24 h after partial hepatectomy, the intracellular dNTP pools change considerably. At 19.5 h after partial hepatectomy, 5.5 h after the start of HU infusion, and at 25 h after partial hepatectomy, 1 h after termination of HU infusion, the dTTP pool was more than 10-times, and the dGTP pool about 2-times higher than in controls, while the dATP and dCTP pools remain relatively unchanged. Simultaneous infusion of HU and deoxythymidine (dThd) 14--25 h after partial hepatectomy results in a further increase of the dTTP pool during and after HU infusion. Administration of deoxycytidine (dCyd) leads to a moderate increase of the dCTP pool and a weak decrease of the dTTP pool during HU infusion. The combined application of dCyd and dThd after HU infusion had similar effects on dNTP pools as observed with dThd alone. These results show that intracellular pools of dNTPs in hepatocytes can be altered by exogenous factors in a controlled pattern. This system can be used as a model for studying the implications of induced dNTP pool dysbalances for the initiation of liver carcinogenesis by mutagenic chemicals.     

Characterization of a nucleotide kinase encoded by bacteriophage t7

Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ～12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg(2+) in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The K(m) of 4.4 × 10(-4) m obtained with dTTP for dTMP kinase is ～3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10(-4) m), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.