Altered CTP synthetase activity confers resistance to 5-bromodeoxyuridine toxicity and mutagenesis

A V79 Chinese hamster fibroblast cell line selected for resistance to the toxic effects of 5-fluorouracil (Kaufman, 1984b) was found to be cross-resistant to the toxic effects of the thymidine analog 5-bromodeoxyuridine (BrdUrd). When tested for sensitivity to BrdUrd mutagenesis, the fluorouracil-resistant cells were found to be resistant to mutagenesis induced by high concentrations of BrdUrd in the medium (INC mutagenesis) but not to mutagenesis induced by the replication of DNA containing 5-bromouracil (REP mutagenesis). Analyses of deoxyribonucleoside triphosphate pools indicated that high endogenous dCTP levels in the mutant prevented the high BrdUTP/dCTP ratio associated with INC mutagenesis. However, the mutant phenotype had no effect on the nucleotide pool imbalance associated with REP mutagenesis. This mutant provides further genetic evidence for the existence of two independent mechanisms for BrdUrd mutagenesis in mammalian cells.     

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.     

Effect of bromodeoxyuridine on the proliferation and growth of ethyl methanesulfonate-exposed P3 cells: Relationship to the induction of sister-chromatid exchanges

Although sister-chromatid exchange (SCE) analysis is recognized as an indicator of exposure to DNA-damaging agents, the results of these analyses have been confounded by the use of bromodeoxyuridine (BrdUrd) to differentially label the sister chromatids. Not only does BrdUrd itself induce SCE, it also modulates the frequency of SCE induced by certain DNA-damaging agents. In order to examine this effect of BrdUrd on SCE frequency, an indirect method which lends itself to measurements both with and without BrdUrd was employed. Human teratocarcinoma-derived (P3) cells were exposed to ethyl methanesulfonate (EMS) and cultured with increasing concentrations of BrdUrd for lengths of time corresponding to one, two, and three generations of cell growth. At each time point, the distribution of nuclei among the phases of the cell-cycle and cell growth were evaluated for each concentration and chemical. A statistical model was employed which tested both for the main effects of chemicals and culture times and for interactions between these factors. Both EMS and BrdUrd significantly affected the percentages of nuclei within the cell-cycle. Exposure to EMS resulted in decreases in the percentages of nuclei in G0 + G1 and increases in the G2 + M compartment. Exposure to BrdUrd affected the size of the G0 + G1 compartment as well as the percentage of S-phase nuclei. Cell growth was reduced as a consequence of increasing EMS concentration and as a function of BrdUrd concentration; the effects of these chemicals were more readily apparent at the later time points. Most importantly, for both the cell-cycle kinetics data and the cell growth data, no evidence of an interaction between the effects of EMS and the effects of BrdUrd was detected statistically. These results may be interpreted to mean that while both EMS and BrdUrd affect the induction of SCE, under the conditions of this experiment, the effects are additive rather than interactive.Abbreviations: EMS, ethyl methanesulfonate - BrdUrd, bromodeoxyuridine - BrdUTP, bromodeoxyuridine triphosphate - dCTP, deoxycytidine triphosphate - SCE, sister-chromatid exchange - P3, human teratocarcinoma derived - HBSS, Hank's Balanced Salt Solution - HOUR, culture time - REP, replicate     

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.     

The role of deoxyribonucleotide metabolism in 5-bromo-2′-deoxyuridine mutagenesis in mammalian cells

The effects of deoxyribonucleoside triphosphate (dNTP) pool imbalance on the induction of mutations and siste-chromatid exchanges (SCEs) by 5-bromo-2′-deoxyuridine (BrdUrd) in mammalian cells is reviewed. The INC BrdUrd mutagenesis protocol involves the incorporation of BrdUrd into DNA under conditions of specific dNTP pool imbalance, while the REP BrdUrd mutagenesis protocol involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of specific (but different) dNTP pool imbalance. Biochemical and genetic analyses of both the INC and REP mutagenesis protocols provided evidence that (1) INC mutagenesis resulted from errors of incorporation due to the mispairing of BrdUTP with a guanine residue in replicating DNA leading to GC to AT transitions and (2) REP mutagenesis resulted from errors of replication due to the mispairing of dGTP with a BrURA residue in replicating DNA leading to AT to GC transitions. Further analyses involving different cell lines has led to an hypothesis describing the role of mismatch repair in the induction of mutations and SCEs.     

Pyrimidine Nucleotide Metabolism and Pathways of Thymidine Triphosphate Biosynthesis in Salmonella typhimurium

The nucleoside triphosphate pools of two cytidine auxotrophic mutants of Salmonella typhimurium LT-2 were studied under different conditions of pyrimidine starvation. Both mutants, DP-45 and DP-55, are defective in cytidine deaminase and cytidine triphosphate (CTP) synthase. In addition, DP-55 has a requirement for uracil (uridine). Cytidine starvation of the mutants results in accumulation of high concentrations of uridine triphosphate (UTP) in the cells, while the pools of CTP and deoxy-CTP drop to undetectable levels within a few minutes. Addition of deoxycytidine to such cells does not restore the dCTP pool, indicating that S. typhimurium has no deoxycytidine kinase. From the kinetics of UTP accumulation during cytidine starvation, it is concluded that only cytidine nucleotides participate in the feedback regulation of de novo synthesis of UTP; both uridine and cytidine nucleotides participate in the regulation of UTP synthesis from exogenously supplied uracil or uridine. Uracil starvation of DP-55 in presence of cytidine results in extensive accumulation of CTP, suggesting that CTP does not regulate its own synthesis from exogenous cytidine. Analysis of the thymidine triphosphate (dTTP) pool of DP-55 labeled for several generations with (32)P-orthophosphate and (3)H-uracil in presence of (12)C-cytidine shows that only 20% of the dTTP pool is derived from uracil (via the methylation of deoxyuridine monophosphate); 80% is apparently synthesized from a cytidine nucleotide.     

Ribonucleotide reductase: A determinant of 5-bromodeoxyuridine mutagenesis in phage T4

Summary Mutagenesis by 5-bromodeoxyuridine (BrdUrd) can result from base-pairing errors either during replication of a BrdUrd-containing template or at the nucleotide incorporation step. Replication errors give rise predominantly to AT-to-GC transitions, while incorporation errors, in which 5-bromo-dUTP competes with dCTP at a template guanine site, should give rise to GC-to-AT transitions. The latter pathway should be sensitive to deoxyribonucleoside triphosphate (dNTP) pool fluctuations. Since dNTP pools are regulated through allosteric control of ribonucleotide reductase, the control of this enzyme should be a determinant of BrdUrd mutagenesis — if mutagenesis results largely from incorporation errors. Since T4 phage-encoded ribonucleotide reductase is insensitive to feedback inhibition, we established conditions under which phage DNA replication is dependent upon ribonucleotide reductase of the host, Escherichia coli. We examined BrdUrd mutagenesis of rII mutants known to revert to wild type either by AT-to-GC or GC-to-AT transition pathways. While both reversion pathways were stimulated under all conditions analyzed, the AT-to-GC pathway was stimulated more when the E. coli reductase was functioning, while the GC-to-AT pathway was more specifically enhanced when the T4 reductase was active. These results confirm that ribonucleotide reductase is a determinant of BrdUrd mutagenesis, but our observations, plus experiments showing that BrdUrd has relatively small effects upon dNTP pool sizes, indicate that the relationship between deoxyribonucleotide metabolism and BrdUrd mutagenesis is more complex than anticipated.     

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.     

Intracellular compartmentation of deoxycytidine nucleotide pools in S phase mouse 3T3 fibroblasts

We labeled mouse 3T3 fibroblasts, synchronized in G0 or S phase, from [3H]cytidine or [3H]deoxycytidine and measured the flow of isotope into and through deoxycytidine nucleotide pools, including the two deoxyliponucleotides dCDP choline and dCDP ethanolamine. Compared to G0 cells, S phase cells had much larger pools with a 20-40-fold faster turnover. The dCTP pool of S phase cells during steady state conditions attained a 6-fold higher specific activity than the pool of G0 cells when labeled from cytidine but a 10-fold lower specific activity when labeled from deoxycytidine. The dCTP pool of G0 cells showed a slow but measurable turnover indicating a limited amount of de novo synthesis also in resting cells. The labeling pattern of dCTP and deoxyliponucleotides of G0 cells was compatible with a simple precursor-product relationship. In S phase cells, however, dCDP choline had a 4-6 times higher specific activity during steady state conditions than dCTP and dCMP when the cells were labeled with [3H]deoxycytidine. We suggest that 3T3 cells contain two distinct intracellular dCTP pools, one labeled preferentially from cytidine and used for DNA replication, the other labeled from deoxycytidine and used for deoxyliponucleotide synthesis. We speculate that the latter pool during S phase may be temporarily sequestered in the cell's membrane fraction before equilibration with the much larger dCTP pool originating in S phase cells from the reduction of CDP.     

Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

Two protocols have been developed, both of which utilize the thymidine analog 5-bromodeoxyuridine (BrdUrd) to induce mutations in mammalian cells in culture (E. R. Kaufman and R. L. Davidson, Proc. Natl. Acad. Sci. USA 75:4982-4986, 1978; E. R. Kaufman, Mol. Cell. Biol. 4:2449-2454, 1984). The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP. The second protocol, termed replicational (REP) mutagenesis, entails the incorporation of BrdUrd into DNA under nonmutagenic conditions, the removal of all BrdUrd from the culture medium, and the subsequent replication of the bromouracil-containing DNA in the presence of high intracellular levels of dTTP and dGTP. Genetic studies using reversion analysis at the hypoxanthine-guanine phosphoribosyltransferase locus were used to determine whether the mechanisms of these two BrdUrd mutagenesis protocols had enough specificity to be distinguishable by their ability to revert various mutants. The results of these studies indicated that (i) mutants induced by INC mutagenesis were induced to revert only by REP mutagenesis and not by INC mutagenesis, (ii) mutants induced by REP mutagenesis were more efficiently reverted by INC mutagenesis than by REP mutagenesis, and (iii) both spontaneous mutants and mutants induced by the chemical mutagen ethyl methanesulfonate showed a high degree of specificity when tested for reversion by the BrdUrd mutagenesis protocols.     

Deoxycytidine kinase-deficient mutants of Chinese hamster ovary cells are hypersensitive to DNA alkylating agents

Chinese hamster ovary cell strains deficient in deoxycytidine kinase activity were selected by isolating mutants resistant to high concentrations of the analogue arabinosyl cytosine. Mutants isolated were deficient in the pool of dCTP, supporting earlier a suggestion that the deoxycytidine kinase may play a role in the turnover and maintenance of the dCTP pool. Consistent with earlier observations that increased intracellular levels of dTTP relative to dCTP lead to increased sensitivy to monofunctional DNA alkylating agents, deoxycytidine kinase-deficient mutants showed a 2–5-fold increase in sensitivity to the cytotoxic and mutagenic effects of one agent, ethyl methanesulfonate (EMS). The survival of the two kinase-deficient strains after mutagen treatment was clearly related to dCTP level as the strain with lowest dCTP was most sensitive to EMS. Thus hypersensitivity to this class of DNA damaging agents can result from cellular mutations decreasing the intracellular level of dCTP.     

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.     

Sensitivity of a mutator gene in Chinese hamster ovary cell to deoxynucleoside triphosphate pool alterations.

The Thy- mutants of Chinese hamster ovary cells have a 5- to 10-fold elevated pool of deoxycytidine 5'-triphosphate (dCTP) and are auxotrophic for thymidine as an apparent consequence of a single mutation. thy is also a mutator gene, elevating the spontaneous rate of mutation 5- to 200-fold for at least two genetic markers. Previous experiments suggested that this mutator activity was caused by the elevated pool of dCTP in Thy- cells. To test this, the dCTP and deoxythymidine 5'-triphosphate (dTTP) pools were manipulated by altering the external concentration of thymidine in the growth medium. The rate of mutation at one genetic locus, ouabain resistance, was directly related to cellular dCTP content. At the highest level of dCTP the rate in one Thy- strain was approximately 200 times that of wild-type cells. However, the relationship between dCTP content and the rate of mutation at the ouabain locus was different for two mutator strains and wild-type cells. The rate of mutation at a second locus, thioguanine resistance, was increased approximately 10-fold over wild type regardless of the dCTP-dTTP pools. These experiments suggest that the mutator activity of thy is clearly related to dCTP content, but the dCTP level alone does not appear to be the cause of the mutator.     

Deoxyribonucleotide pool imbalance stimulates deletions in HeLa cell mitochondrial DNA

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder associated with multiple mutations in mitochondrial DNA, both deletions and point mutations, and mutations in the nuclear gene for thymidine phosphorylase. Spinazzola et al. (Spinazzola, A., Marti, R., Nishino, I., Andreu, A., Naini, A., Tadesse, S., Pela, I., Zammarchi, E., Donati, M., Oliver, J., and Hirano, M. (2001) J. Biol. Chem. 277, 4128-4133) showed that MNGIE patients have elevated circulating thymidine levels and they hypothesized that this generates imbalanced mitochondrial deoxyribonucleoside triphosphate (dNTP) pools, which in turn are responsible for mitochondrial (mt) DNA mutagenesis. We tested this hypothesis by culturing HeLa cells in medium supplemented with 50 microM thymidine. After 8-month growth, mtDNA in the thymidine-treated culture, but not the control, showed multiple deletions, as detected both by Southern blotting and by long extension polymerase chain reaction. After 4-h growth in thymidine-supplemented medium, we found the mitochondrial dTTP and dGTP pools to expand significantly, the dCTP pool to drop significantly, and the dATP pool to drop slightly. In whole-cell extracts, dTTP and dGTP pools also expanded, but somewhat less than in mitochondria. The dCTP pool shrank by about 50%, and the dATP pool was essentially unchanged. These results are discussed in terms of the recent report by Nishigaki et al. (Nishigaki, Y., Marti, R., Copeland, W. C., and Hirano, M. (2003) J. Clin. Invest. 111, 1913-1921) that most mitochondrial point mutations in MNGIE patients involve T --> C transitions in sequences containing two As to the 5' side of a T residue. Our finding of dTTP and dGTP elevations and dATP depletion in mitochondrial dNTP pools are consistent with a mutagenic mechanism involving T-G mispairing followed by a next-nucleotide effect involving T insertion opposite A.     

Cell cycle-dependent effects on deoxyribonucleotide and DNA labeling by nucleoside precursors in mammalian cells.

dCTP pools equilibrated to equivalent specific activities in Chinese hamster ovary cells or in nuclei after incubation of cells with radiolabeled nucleosides, indicating that dCTP in nuclei does not constitute a distinct metabolic pool. In the G1 phase, [5-3H]deoxycytidine labeled dCTP to unexpectedly high specific activities. This may explain reports of replication-excluded DNA precursor pools.     

Deoxyribonucleoside triphosphate metabolism and the mammalian cell cycle: Effects of thymidine on wild-type and dCMP deaminase-deficient mouse S49 T-lymphoma cells

The size of the dCTP pool has been implicated as a possible regulator of DNA synthesis. In this investigation we correlate large intracellular variations in deoxyribonucleoside triphosphate levels to the growth rates and cell-cycle kinetics of mouse S49 T-lymphoma cells. Wild-type and a mutant line AzidoC-100-5, lacking dCMP-deaminase activity resulting in a 10-fold expanded dCTP pool were studied and compared using flow cytometry, centrifugal elutriation and nucleoside triphosphate determinations. An increase in the dCTP pool was closely correlated to the passage of cells from G1 to S phase in both cell types. Addition of thymidine to wild-type and mutant cells resulted in an accumulation of cells in early S phase, concomitant with a decreased dCTP level. Mutant cells excreted large amounts of deoxycytidine into the medium which partially protected the cells from thymidine inhibition. The doubling times for the mutant and wild-type cells were very similar but the mutant had a somewhat prolonged S phase and shortened G1 phase compared with the wild-type cells. Large changes in the DNA precursor levels were produced by addition of thymidine to mutant cultures. This gave no change in the growth rate but a somewhat shortened S phase and prolonged G1. The biochemical background for these effects is discussed.     

The mutagenic properties of BrdUTP in a random mutagenesis process

To explore the mutagenic properties of the nucleotide analogue bromodeoxyuridine triphosphate (BrdUTP), the wild type α-amylase (xamy) gene from Xanthomonas campestris pv. campestris 8004 was used as a mutational target. It was mutated using PCR techniques to partially replace deoxythymidine triphosphate (dTTP) with BrdUTP. A total of 18 mutants were selected for DNA sequencing from the mutagenesis libraries by their ability to hydrolyze the starch. The results showed that 70% of the total mutations were single base-pair substitutions; BrdUTP also induced deletion and insertion mutation types. Among single base-pair substitutions, the predominant mutation type is transition (84%), but three kinds of transversions (16%) were also detected. It thus mainly induces A:T → G:C and T:A → C:G transitions. This result indicated that when bromouracil is present as a deoxyribonucleoside triphosphate substrate it mainly paired with dAMP, and when it is present as a template base it could pair with free dGTP. Three mutational hot spots induced by BrdUTP were revealed in this work.     

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.     

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.     

Compartmentation of dCTP pools. Evidence from deoxyliponucleotide synthesis

The nucleotide fraction of cultured 3T6 and 3T3 mouse fibroblasts contains deoxy-CDP choline and deoxy-CDP ethanolamine as well as the corresponding riboliponucleotides. In permeabilized cells both deoxyliponucleotides were formed from dCTP. In intact cells they could be labeled from [5-3H] deoxycytidine or cytidine via transformation of the nucleosides to dCTP. Their turnover was slow compared to that of dCTP. When rapidly growing 3T3 cells were labeled during 90 min from deoxycytidine the specific activity of dCDP choline was 2.4 times higher than that of dCTP while after labeling from cytidine both nucleotides (and CTP) reached the same specific activity under steady state conditions. Also dCDP ethanolamine was labeled more rapidly from deoxycytidine than from cytidine. Our results suggest that the deoxyliponucleotides were synthesized from a dCTP pool that was labeled preferentially from deoxycytidine. Earlier work (Nicander, B., and Reichard, P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1347-1351) had demonstrated synthesis of DNA from a dCTP pool labeled preferentially from cytidine. Taken together our results suggest that deoxyliponucleotides and DNA are synthesized from separate dCTP pools.