Mitochondrial thymidine kinase and the enzymatic network regulating thymidine triphosphate pools in cultured human cells

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.     

Depletion of deoxyribonucleoside triphosphate pools in tumor cells by nitric oxide

Nitric oxide displays pro- and anti-tumor activities, prompting further studies to better understand its precise role. Nitric oxide inhibits ribonucleotide reductase (RnR), the limiting enzyme for de novo dNTP synthesis. We report here the first detailed analysis of dNTP variations induced in tumor cells by NO. NO prodrugs induced a depletion in dNTP pools and an activation of the pyrimidine salvage pathway, as did hydroxyurea, the prototypic RnR inhibitor. In the presence of dipyridamole, which blocked salvaged dNTP synthesis, depletion of dNTP pools was also observed in tumor cells cocultured with macrophages expressing the high-output iNOS activity. This effect was rapid, reversible, blocked by NO scavengers, and cGMP independent. It was quantitatively correlated to iNOS activity. In the absence of dipyridamole, NO still induced a decrease in dATP concentration in tumor cells cocultured with macrophages, whereas surprisingly, concentrations of dCTP and dTTP expanded considerably, resulting in a strong imbalance in dNTP pools. NO prodrugs did not cause such an increase in pyrimidine dNTP, suggesting that pyrimidine nucleosides were released by NO-injured macrophages. Altered dNTP levels have been reported to promote mutagenesis and apoptosis. It is suggested that abnormal changes in dNTP pools in tumors might contribute to NO-dependent toxicity.     

Evidence for the involvement of substrate cycles in the regulation of deoxyribonucleoside triphosphate pools in 3T6 cells

Pool sizes of deoxyribonucleoside triphosphates (dNTPs) in cultured cells are tightly regulated by i.al., the allosteric control of ribonucleotide reductase. We now determine the in situ activity of this enzyme from the turnover of the deoxycytidine triphosphate (dCTP) pool in rapidly growing 3T6 mouse fibroblasts, as well as in cells whose DNA replication was inhibited by aphidicolin or amethopterin, by following under steady state conditions the path of isotope from [5-3H]cytidine into nucleotides, DNA, and deoxynucleosides excreted into the medium. In normal cells as much as 28% of the dCDP synthesized was excreted as deoxynucleoside (mostly deoxyuridine), leading to an accumulation of deoxyuridine in the medium. Inhibition with amethopterin slightly increased ribonucleotide reductase activity, while aphidicolin halved the activity of this enzyme (and thymidylate synthase). In both instances all dCDP synthesized was degraded and excreted as nucleosides. This continued synthesis and turnover in the absence of DNA synthesis is in contrast to the earlier found inhibition of dCTP (and dTTP) turnover when hydroxyurea, an inhibitor of ribonucleotide reductase, was used to block DNA synthesis. To explain our results, we propose that substrate cycles between deoxyribonucleosides and their monophosphates, involving the activities of kinases and phosphatases, participate in the regulation of pool sizes. Within the cycles, a block of the reductase activates net phosphorylation, while inhibition of DNA polymerase stimulates degradation.     

Mitochondrial purine and pyrimidine metabolism and beyond

ABSTRACT

Carefully balanced deoxynucleoside triphosphate (dNTP) pools are essential for both nuclear and mitochondrial genome replication and repair. Two synthetic pathways operate in cells to produce dNTPs, e.g., the de novo and the salvage pathways. The key regulatory enzymes for de novo synthesis are ribonucleotide reductase (RNR) and thymidylate synthase (TS), and this process is considered to be cytosolic. The salvage pathway operates both in the cytosol (TK1 and dCK) and the mitochondria (TK2 and dGK). Mitochondrial dNTP pools are separated from the cytosolic ones owing to the double membrane structure of the mitochondria, and are formed by the salvage enzymes TK2 and dGK together with NMPKs and NDPK in postmitotic tissues, while in proliferating cells the mitochondrial dNTPs are mainly imported from the cytosol produced by the cytosolic pathways. Imbalanced mitochondrial dNTP pools lead to mtDNA depletion and/or deletions resulting in serious mitochondrial diseases. The mtDNA depletion syndrome is caused by deficiencies not only in enzymes in dNTP synthesis (TK2, dGK, p53R2, and TP) and mtDNA replication (mtDNA polymerase and twinkle helicase), but also in enzymes in other metabolic pathways such as SUCLA2 and SUCLG1, ABAT and MPV17. Basic questions are why defects in these enzymes affect dNTP synthesis and how important is mitochondrial nucleotide synthesis in the whole cell/organism perspective? This review will focus on recent studies on purine and pyrimidine metabolism, which have revealed several important links that connect mitochondrial nucleotide metabolism with amino acids, glucose, and fatty acid metabolism.     

Mitochondrial deoxynucleotide pools in quiescent fibroblasts: a possible model for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

Mitochondrial (mt) DNA depletion syndromes can arise from genetic deficiencies for enzymes of dNTP metabolism, operating either inside or outside mitochondria. MNGIE is caused by the deficiency of cytosolic thymidine phosphorylase that degrades thymidine and deoxyuridine. The extracellular fluid of the patients contains 10-20 microM deoxynucleosides leading to changes in dTTP that may disturb mtDNA replication. In earlier work, we suggested that mt dTTP originates from two distinct pathways: (i) the reduction of ribonucleotides in the cytosol (in cycling cells) and (ii) intra-mt salvage of thymidine (in quiescent cells). In MNGIE and most other mtDNA depletion syndromes, quiescent cells are affected. Here, we demonstrate in quiescent fibroblasts (i) the existence of small mt dNTP pools, each usually 3-4% of the corresponding cytosolic pool; (ii) the rapid metabolic equilibrium between mt and cytosolic pools; and (iii) the intra-mt synthesis and rapid turnover of dTTP in the absence of DNA replication. Between 0.1 and 10 microM extracellular thymidine, intracellular thymidine rapidly approaches the extracellular concentration. We mimic the conditions of MNGIE by maintaining quiescent fibroblasts in 10-40 microM thymidine and/or deoxyuridine. Despite a large increase in intracellular thymidine concentration, cytosolic and mt dTTP increase at most 4-fold, maintaining their concentration for 41 days. Other dNTPs are marginally affected. Deoxyuridine does not increase the normal dNTP pools but gives rise to a small dUTP and a large dUMP pool, both turning over rapidly. We discuss these results in relation to MNGIE.     

Azidocytidine is a specific inhibitor of deoxyribonucleotide synthesis in 3T6 cells

2'-Azidocytidine is a specific inhibitor of DNA synthesis in cultured 3T6 mouse fibroblasts. Earlier work (Akerblom, L., Pontis, E., and Reichard, P. (1982) 257, 6776-6782) indicated that the nucleoside, after phosphorylation, acted by inhibiting both ribonucleotide reduction and DNA strand elongation. We now demonstrate that the effect on strand elongation was due to a contamination of azidocytidine with less than 0.3% of arabinosyl cytosine. Pure azidocytidine inhibits specifically ribonucleotide reductase and its effects on DNA synthesis are secondary to this inhibition. The results with azidocytidine concerning the size and turnover of deoxyribonucleoside triphosphate pools parallel those of hydroxyurea and are distinct from those of arabinosyl cytosine. Together with hydroxyurea, azidocytidine is a useful compound in studies aiming at a specific block of the production of deoxynucleoside triphosphates in intact cells. Comparisons of the effects of azidocytidine and arabinosyl cytosine complement earlier studies with hydroxyurea and aphidicolin concerning inter-relations between dNTP synthesis and DNA replication.     

ATP:AMP phosphotransferase from baker''s yeast. Purification and properties

Synchronous cells of the green alga, Scenedesmus obliquus, cultured in a 14-h/10-h light/dark regime, contain a peak of ribonucleoside-diphosphate reductase activity and maximum deoxyribonucleoside 5'-triphosphate concentrations at the 12th hour of the cell cycle, coinciding with DNA synthesis and preceding the formation of eight daughter cells. The intracellular dTTP pool reaches 4.5 pmol and the other pools 2-3 pmol/10(6) cells. Algal reductase activity is sensitive to cycloheximide, but not to lincomycin. These correlations demonstrate the functioning of the NDP leads to dNDP leads to dNTP pathway of DNA precursor biosynthesis in plant cells. In the presence of 20 micrograms 5-fluorodeoxyuridine/ml, an inhibitor of thymidylate synthesis, the dTTP pool is rapidly depleted and DNA synthesis ceases. 5-Fluorouracil and methotrexate produce similar effects. At the same time the ribonucleotide reductase activity and also the dATP pool are greatly increased, especially when fluorodeoxyuridine treatment is combined with continued illumination of the algae. In contrast, arabinosylcytosine, an inhibitor of DNA replication, has no effect on ribonucleotide reduction. The control of de novo enzyme synthesis in the eucaryotic algae therefore appears to depend on the presence of dTTP (or a related nucleotide), but not directly coupled to DNA synthesis. This interdependence resembles the situation observed in HeLa cells, while it may differ in detail from control mechanisms of ribonucleotide reductase studied in bacteria.     

Studies of the identity of the unknown in protein hydrolysates

DNA synthesis in the adenovirus DNA replication complex, containing host DNA polymerases-α and -γ, was inhibited completely by aphidicolin and by 2′,3′-dideoxythymidine triphosphate (ddTTP). Double reciprocal plots of DNA polymerase activity in the replication complex against each dNTP gave a straight line although the complex contained two species of DNA polymerase. Inhibition by aphidicolin of DNA polymerase activity was competitive with dTTP but that of purified DNA polymerase-α isolated from adenovirus infected KB cells was competitive with dCTP. The above results suggest that DNA polymerases-α and -γ are integrated in the replication complex to behave as a single enzyme.     

Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools

The relationship between dNTP levels and DNA synthesis was investigated using alpha factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G1/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37 degrees C before being released to 25 degrees C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G1 levels. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25 degrees C were able to replicate when subsequently switched to 37 degrees C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold.     

The sources of thymidine nucleotides for virus DNA synthesis in herpes simplex virus type 2-infected cells

The thymidine nucleotide sources present during herpes simplex virus type 2 (HSV-2) infection were examined. It was concluded that the source of dTTP in HSV-2-infected cells is not only derived from the ribonucleotide reductase-catalyzed de novo pathway, but also from host DNA. When the de novo pathway was inhibited by the addition of hydroxyurea, an inhibitor of ribonucleotide reductase, the dTTP levels were maintained by a compensatory increase in dTTP derived from host DNA. The utilization of host DNA-derived dTTP for viral DNA synthesis was demonstrated. In spite of an increased contribution of dTTP from host DNA in the presence of hydroxyurea, the level of utilization of host DNA-derived dTTP appeared to remain constant. More than one dTTP pool in virus-infected cells is implicated.     

Alterations in deoxynucleoside triphosphate metabolism in DNA damaged cells: identification and consequences of poly(ADP-ribose) polymerase dependent and independent processes

Treatment of L1210 cells with increasing concentrations of MNNG produces heterogeneous perturbations of cellular deoxynucleoside triphosphate pools, with the magnitude and direction of the shift depending on the deoxynucleotide and on the concentration and time of exposure of the DNA damaging agent. 5 microM MNNG stimulated an increase in dATP, dCTP and dTTP but dGTP pools remained constant. These increases were not affected by 3-aminobenzamide, indicating that the pool size increases were produced by poly(ADP-ribose) polymerase independent reactions. 30 microM MNNG caused a time dependent decrease in dATP, dGTP, dTTP and dCTP. The dGTP pool was most drastically affected, becoming totally depleted within 3 hours. The fall in all 4 dNTP pools was substantially prevented by 3-aminobenzamide, suggesting that the decrease in dNTPs following DNA damage is mediated by a poly(ADP-ribose) polymerase dependent reaction. Severe depression of dGTP pools consequent to NAD and ATP depletion may provide a metabolic pathway for rapidly stopping DNA synthesis as a consequence of DNA damage and the activation of poly(ADP-ribose) polymerase.     

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.     

Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells

Ribonucleotide reductase (RNR) provides the cell with a balanced supply of deoxyribonucleoside triphosphates (dNTP) for DNA synthesis. In budding yeast DNA damage leads to an up-regulation of RNR activity and an increase in dNTP pools, which are essential for survival. Mammalian cells contain three non-identical subunits of RNR; that is, one homodimeric large subunit, R1, carrying the catalytic site and two variants of the homodimeric small subunit, R2 and the p53-inducible p53R2, each containing a tyrosyl free radical essential for catalysis. S-phase-specific DNA replication is supported by an RNR consisting of the R1 and R2 subunits. In contrast, DNA damage induces expression of the R1 and the p53R2 subunits. We now show that neither logarithmically growing nor G(o)/G1-synchronized mammalian cells show any major increase in their dNTP pools after DNA damage. However, non-dividing fibroblasts expressing the p53R2 protein, but not the R2 protein, have reduced dNTP levels if exposed to the RNR-specific inhibitor hydroxyurea, strongly indicating that there is ribonucleotide reduction in resting cells. The slow, 4-fold increase in p53R2 protein expression after DNA damage results in a less than 2-fold increase in the dNTP pools in G(o)/G1 cells, where the pools are about 5% that of the size of the pools in S-phase cells. Our results emphasize the importance of the low constitutive levels of p53R2 in mammalian cells, which together with low levels of R1 protein may be essential for the supply of dNTPs for basal levels of DNA repair and mitochondrial DNA synthesis in G(o)/G1 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.     

Allosteric Regulation of the Human and Mouse Deoxyribonucleotide Triphosphohydrolase Sterile α-Motif/Histidine-Aspartate Domain-containing Protein 1 (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 K_m 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 K_m 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.     

Deoxyribonucleoside triphosphate pools in mutagen sensitive mutants of Neurospora crassa

Deoxyribonucleoside triphosphate (dNTP) levels were measured in wild type Neurospora and nine mutagen-sensitive mutants, at nine different genes. Eight of these mutants are sensitive to hydroxyurea and histidine and show chromosomal instability, a phenotype which could result from altered levels of dNTPs. Two patterns were seen. Five of the mutants had altered ratios of dNTPs, with relatively high levels of dATP and dGTP and low levels of dCTP, but changes in the dTTP/dCTP ratio did not correlate with changes in spontaneous mutation levels. During exponential growth all but two of the mutants had small but consistent increases in dNTP pools compared to wild type. DNA content per microgram dry hyphae was altered in several mutants but these changes showed no correlation with the dNTP pool alterations.     

Dynamcis of the thymidine triphosphate pool during the cell cycle of synchronized 3T3 mouse fibroblasts

To investigate whether resting cells of 3T3 mouse fibroblasts carry out de novo synthesis of deoxyribonucleoside triphosphates, we determined the turnover of the thymidine triphosphate pool of G₀ cells obtained by starvation of cultures for platelet-derived growth factor. These cells were contaminated by less than 1% S-phase cells. In the absence of deoxyribonucleosides in the medium one million G₀ cells contained 5 pmole of dTTP with a turnover of 0.09 pmole/min. S-phase cells in comparison contained a 20 times larger dTTP pool with a more than 200-fold faster turnover. Our results suggest that G₀ cells carry out a slow but finite de novo synthesis of deoxyribonucleoside triphosphates to satisfy the cells' requirement for DNA repair and mitochondrial DNA synthesis.     

Quantitation of cellular deoxynucleoside triphosphates

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.     

Limited dCTP availability accounts for mitochondrial DNA depletion in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e.g., imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored.     

Deoxyribonucleoside triphosphate pools of herpes simplex virus infected cells: the influence of selective antiherpes agents and the role of the deaminase pathway.

The effect of 5-methoxymethyl-2'-deoxycytidine (MMdCyd), in combination with tetrahydrodeoxyuridine (H4dUrd) and 5-methoxymethyl-2'-deoxyuridine (MMdUrd) on deoxyribonucleoside triphosphate pools was assessed. The dNTP pool content was almost 5 times as high in herpes simplex virus (HSV) infected VERO cells compared with mock-infected cells. Significant differences in dNTP pool sizes were observed with the different treatments. Treatment of HSV-infected cells with MMdCyd and MMdUrd resulted in a massive expansion of the dTTP pool, whereas pools of dCTP and dGTP were not affected substantially. MMdUrd and MMdCyd produced dATP pools that were 4 and 2.5 times that of the controls, respectively. Treatment with H4dUrd resulted in the dCTP pool increasing 12 times and barely detectable levels of dTTP. MMdCyd in combination with H4dUrd resulted in a marked reduction of the total deoxyribonucleoside triphosphate level. These results indicate that during viral replication the bulk of the thymidine nucleotides are derived from the dCyd/dCMP deaminase de novo pathway.