Elevation and hardening of the fertilization membrane in sea urchin eggs : Role of the soluble fertilization product

Requirements and optimal conditions have been studied for measurements of dGTP and dCTP in cellular extracts using the copolymer [d(1 ? C)] as primer in a reaction catalysed by the large fragment of DNA polymerase from E. coli. The pool size of dGTP and dCTP in the human lymphocytes in the absence of PHA was found to be about 0.1 and 0.15 pmoles/10⁶ cells, respectively. After treatment with PHA the pool size of both deoxynucleotides increased. The pool size of dCTP reached a maximum after 67 h simultaneously with the peak value of labelled deoxythymidine incorporation into DNA and the variation in these two parameters was very similar. The variation in the dGTP pool, however, was not so distinctly related to deoxythymidine incorporation as in the dCTP pool, since the increase in the dGTP pool was very small from 52–67 h. During transformation the dGTP pool was found to be the smallest pool. The relative cellular content of mono-, di- and triphosphate esters of deoxyadenosine, deoxyguanosine and deoxycytidine was studied.     

The deoxyribonucleoside 5′-triphosphate (dATP and dTTP) pool in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

The pool size of dATP and dTTP in human lymphocytes was studied in untreated and PHA-treated cells. Different methods of extracting the cellular content of dATP and dTTP have been investigated and extraction with 60% methanol was preferred. The pool size of dATP and dTTP in non-stimulated lymphocytes was about 0.2 and 0.05 pmoles/10⁶ cells, respectively. After treatment with PHA for about 50 h the dATP and dTTP pools reached peak values representing increases in the pools of 20 and 170 fold, respectively. The variation in the pool sizes during transformation was paralleled by the variation of the rate of incorporation of labeled deoxy-thymidine into cellular DNA.     

Two simple high-performance liquid chromatographic methods for simultaneous determination of 2′-deoxycytidine 5′-triphosphate and cytosine arabinoside 5′-triphosphate concentrations in biological samples

Cytosine arabinoside (ara-C) has been used in the treatment of leukemia, but its exact mechanism of cytotoxicity is not yet known. One of the proposed mechanisms for the effectiveness of this drug in treating leukemias suggests that a metabolite of ara-C, i.e., 2′-deoxycytidine 5′-triphosphate (araCTP), competes with cytosine arabinoside 5′-triphosphate (dCTP) for binding to DNA polymerase. The ratio of the drug metabolite to the endogenous nucleotide (araCTP/dCTP) may, therefore, be important in determining the effectiveness of ara-C therapy. This ratio may also play a role in drug resistance. Previously published methods have focused on either araCTP or dCTP, along with metabolites and analogues of one of these compounds. The methods presented here provide two simple, sensitive ways to measure dCTP and araCTP in the same biological sample.     

Incorporation of 8-hydroxyguanosine (8-oxo-7,8-dihydroguanosine) 5′-triphosphate by bacterial and human RNA polymerases

Oxidized RNA precursors formed in the nucleotide pool may be incorporated into RNA. In this study, the incorporation of 8-hydroxyguanosine 5′-triphosphate (8-OH-GTP; 8-oxo-7,8-dihydroguanosine 5′-triphosphate) into RNA by Escherichia coli RNA polymerase was examined in vitro, using a primer RNA and a template DNA with defined sequences. 8-OH-GTP was incorporated opposite C and A in the template DNA. Surprisingly, 8-OH-GTP was quite efficiently incorporated by the bacterial RNA polymerase, in contrast to the incorporation of the 2′-deoxyribo counterpart by DNA polymerases, as indicated by the kinetic parameters. The primer was further extended by the addition of a ribonucleotide complementary to the nucleobase adjacent to C or A (the nucleobase opposite which 8-OH-GTP was inserted). Thus, the incorporation of 8-OH-GTP did not completely inhibit further RNA chain elongation. 8-OH-GTP was also incorporated opposite C and A by human RNA polymerase II. These results suggest that 8-OH-GTP in the nucleotide pool can cause the formation of oxidized RNA and disturb the transmittance of genetic information.     

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.     

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.     

Biosynthesis of deoxynucleoside triphosphates,dCTP and dTTP: reaction mechanism and kinetics

The enzyme reaction mechanism and kinetics for biosyntheses of deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) from the corresponding deoxycytidine diphosphate (dCDP) and deoxythymidine diphosphate (dTDP) catalyzed by pyruvate kinase were studied. The kinetic model for the two synthetic reactions was found to follow the Bi–Bi random rapid equilibrium mechanism similar to that of the biosynthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP). Kinetic constants involved in the reactions including the maximum reaction velocity, the Michaelis–Menten constants, and the inhibition constants for dCTP and dTTP biosyntheses were experimentally determined. This enzyme reaction requires Mg²⁺ ion and the optimal Mg²⁺ concentration was also determined. The experimental results showed a good agreement with the simulation results obtained from the kinetic model developed. The kinetics of the four biosynthetic reactions for deoxynucleoside triphosphates (dNTP) including dATP, dGTP, dCTP, and dTTP from the corresponding deoxynucleoside diphosphates (dNDP) including dADP, dGDP, dCDP, and dTDP were analyzed. The results suggest that the binding kinetics of phosphoenolpyruvate (PEP) and pyruvate are similar for all four biosynthetic reactions. The affinity of the dNDP substrates to enzyme is of the same order of magnitude as the corresponding dNTP as inhibitors. The order of reactivity and substrate specificity for dNDP is dADP > dGDP > dCDP > dTDP in the pyruvate kinase (PK) reactions. The results obtained from this study can be applied to bioreactor design and production of dCTP and dTTP for biosynthesis of DNA at a significantly lower cost compared to the currently available chemical method.     

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.     

Effect of cycloheximide on pools of deoxyribonucleoside triphosphates

Inhibition of protein synthesis by cycloheximide blocks DNA replication in many eukaryotic cells. To test whether this effect was mediated through enzymes furnishing DNA precursors, pool sizes of deoxyribonucleoside triphosphates were measured following cycloheximide treatment in the synchronous mitotic cycle of Physarum. It was found that cycloheximide either did not affect the pool size of DNA precursors (dATP and dGTP) or it led to a pool expansion (dCTP and dTTP). It is concluded that the arrest of DNA replication by inhibitors of protein synthesis is not due to a lack of precursors.     

Potentiation of the anti-HIV activity of zalcitabine and lamivudine by a CTP synthase inhibitor, 3-deazauridine

Low levels of the CTP synthase inhibitor 3-deazauridine (3-DU) strongly potentiated the anti-HIV-1 activity of the 5'-triphosphates of the cytidine-based analogues [-]2'-deoxy-3'-thiacytidine (3TC; lamivudine) and 2',3'-dideoxycytidine (ddC). The potentiation was associated with a 3-DU-induced decrease in dCTP pool size; no changes were seen in cellular pool sizes of dATP, dGTP or dTTP.     

DNA polymerase activity in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

Requirements and optimal conditions have been studied for the activity of DNA polymerase from phytohemagglutinin-stimulated and non-stimulated human lymphocytes. Differences were found in thermal stability and inhibitory effect of KC1 and p-chloromercuribenzoate. The relationship was determined between DNA polymerase activity, cellular pools of dATP, dTTP and incorporation of deoxythymidine into DNA during transformation. The increase in polymerase activity was paralleled by a similar increase in the pools of dATP and dTTP. The enzyme activity and the pool sizes of both nucleotides reached a maximum simultaneously with the peak of deoxythymidine incorporation into DNA. Studies in which protein synthesis was limited by cycloheximide showed that both the DNA polymerase activity and the rise in the pool sizes of both nucleotides were abolished. This implies that the de novo synthesis is required for the enzymes involved.     

Phospholipid metabolism and lysosomal enzyme secretion by leukocytes Effects of dibutyryl cyclic adenosine 3′ : 5′-monophosphate and ATP

The effects of dibutyryl cyclic adenosine 3′ : 5′-monophosphate and ATP on isotope incorporation into phospholipids and the release of β-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3′ : 5′-monophosphate (0.1–1.0 mM) reduced β-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited ³²P_i incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-³H]inositol into the phosphoinositides. Added ATP (0.1–1.0 mM), but not other nucleotides, was found to potentiate β-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-³H]inositol. The mechanism of the inhibition of the isotope incorporation into these acidic phospholipids by the two nucleotides has not been defined. Dibutyryl cyclic adenosine 3′ : 5′-monophosphate at 2–4 mM concentration was not found to appreciably alter the incorporation of [γ-³²P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.     

DNA synthesis in cultured human fibroblasts: Regulation by 3′ : 5′-cyclic amp

The addition of serum to density-inhibited human fibroblast cultures induced a wave of DNA synthesis, measured as [³H] thymidine incorporation into acid-precipitable material, beginning after 8–12 hr and reaching maximum levels at 16–24 hr. Addition of dibutyryl-3′ : 5′-cyclic AMP (DBcAMP) together with serum inhibited [³H] thymidine incorporation by 75–95%. When DBcAMP was added for the first 4 hr of serum stimulation and then removed, the wave of DNA synthesis was not delayed. This suggested that serum could induce DNA synthesis even though cyclic AMP concentrations were maintained at high levels by DBcAMP during this initial period. These results are inconsistent with the hypothesis that it is the immediate transient reduction in 3′ : 5′-cyclic AMP concentration following the addition of serum that triggers DNA synthesis. By contrast, DBcAMP added 8 hr after serum inhibited [³H] thymidine incorporation to the same extent as DBcAMP added at the same time as serum. This indicated that a step essential for DNA synthesis and occurring late in G₁ was inhibited by high concentrations of 3′ : 5′-cyclic AMP.     

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.     

Early effects of phytohemagglutinin on induction of DNA polymerase, thymidine kinase, deoxyribonucleoside triphosphate pools and DNA synthesis in human lymphocytes.

In phytohemagglutinin stimulated human lymphocytes the time relationship was determined between induction of the parameters mentioned. The results indicate that the induction occurred in a specific sequence. Thus, a simultaneous increase in the activity of DNA polymerase and thymidinekinase occurred after 15 h of incubation with Phytohemagglutinin. Furthermore, this enhancement occurred 2 h before the expansion of the TTP and dCTP pools and 4 h before the expansion of the dATP and dGTP pools. The rate of [3H] deoxyguanosine incorporation into DNA increased simultaneously with the expansion of the TTP and dCTP pools.     

A bacteriophage-induced 5-methyldeoxycytidine 5′-monophosphate kinase

Bacteriophage XP-12-infected Xanthomonas oryzae have been found to be a source of a kinase preparation which converts m⁵dCMP to m⁵dCDP and then to m⁵dCTP using ATP as the phosphate donor. Optimal formation of the triphosphate required the presence of creatine phosphate and creatine kinase. In the presence of dGTP, dTTP and dATP, Escherichia coli DNA polymerase I and T4 DNA polymerase catalyzed the incorporation of m⁵dCTP into DNA just as efficiently as that of dCTP. Neither dTMP nor dCMP served as substrate for the m⁵dCMP monophosphate kinase. Analogous preparations from uninfected X. oryzae were unable to phosphorylate m⁵dCMP.     

Accuracy of DNA polymerase-alpha in copying natural DNA

The fidelity of DNA polymerase-alpha from calf thymus (9S enzyme) in copying bacteriophage phi174am16 DNA in vitro has been determined from the frequency of production of different revertants. In the self-priming reaction we were able to measure the frequencies of base pairing mismatches during the course of replication on biasing the ratios of deoxynucleoside triphosphates. The frequency of dGTP:T, dGTP:G and dATP:G mismatches were 7.6 x 10(-5), 4.4 x 10(-5) and 2.8 x 10(-5), respectively, at equal concentrations of the deoxynucleoside triphosphates. dCTP:A, dGTP:A, dCTP:T and dTTP:T mismatches were below the limit of detection (<5 x 10(-6)). A synthetic dodecamer primer with a 3' end covering the first two bases of the amber codon was used to determine the misinsertion frequency of the first nucleotide incorporated. This gave a misinsertion frequency of 1.5 x 10(-4) for the dGTP:T mismatch, which is slightly higher than that observed from the pool bias studies. Further, it showed no sensitivity to biasing the nucleotide pool, suggesting a different mechanism for the incorporation of the first nucleotide. These data do not support 'energy-relay'-like models for achieving high accuracy in eukaryotes. The observed misinsertion frequencies were corrected for mismatch repair of the heteroduplexes during the transfection experiments by parallel experiments using a mismatched primer. This was synthesized to have the same G:T mismatch as produced in the preceding experiment.     

Deoxyribonucleotide metabolism in Herpes simplex virus infected HeLa cells.

The effect of Rolly No. 11 strain herpes simplex virus infection of HeLa cells in culture on deoxynucleotide metabolism and the level of various enzymes concerned with the biosynthesis of DNA has been investigated. Of 18 enzyme activities studied, thymidine kinase, DNA polymerase and deoxyribonuclease were markedly augmented, a finding in agreement with previous reports. Deoxycytidine kinase, ribonucleotide reductase, thymidylate kinase and deoxycytidylate deaminase activities, in contrast with previous reports, did not increase; the activities of the other enzymes studied, also did not increase. Whereas most of the radioactivity derived from [14-C] thymidine in the acid-soluble fraction of the uninfected cells was present as deoxythymidine triphosphate, that present in the infected cells was primarily in the form of deoxythymidine monophosphate. Thus, in the infected cell deoxythymidylate kinase is a rate-limiting enzyme in the biosynthesis of deoxythymidine triphosphate. A marked increase in the pools of the four naturally occurring deoxynucleoside triphosphates (dTTP, dCTP, dATP, dGTP) was found. The rate of formation of the virus-induced enzymes was determined, as were the various nucleoside triphosphate pools and the other phosphorylated derivatives of thymidine; a maximum was reached for all these csmponents between 6 to 8 h post infection. Although an apparent greater synthesis of DNA occurred in the uninefected cells, when the specific activity of the radioactive deoxythymidine triphosphate was taken into account, there was actually a greater rate of DNA synthesis in the infected cells, with the peak at 8 h post infection.     

Aphidicolin inhibits DNA synthesis by DNA polymerase alpha and isolated nuclei by a similar mechanism.

Aphidicolin is a selective inhibitor of DNA polymerase alpha. In contrast to earlier reports, the drug was found to inhibit DNA synthesis catalyzed by DNA polymerase alpha and isolated HeLa cell nuclei by a similar mechanism. For both systems aphidicolin primarily competed with dCTP incorporation. However, the apparent Vmax for dCTP incorporation was reduced by 50-60% at relatively low concentrations of aphidicolin, thus the mechanism of inhibition is complex. Furthermore, a 2-5 fold increase in apparent Km for dTTP was observed in the presence of aphidicolin, but the apparent Km values for dATP and dGTP were essentially unaltered. This, together with additional evidence, suggested that the mechanism of action of aphidicolin involves a strong competition with dCMP incorporation, a weaker competition with dTMP incorporation and very little, if any, competition with dGMP and dAMP incorporation.