The pool size of deoxyguanosine 5′-triphosphate and deoxycytidine 5′-triphosphate in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

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.     

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.     

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.     

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.     

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.     

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.     

Error-prone bypass of O6-methylguanine by DNA polymerase of Pseudomonas aeruginosa phage PaP1

O⁶-Methylguanine (O⁶-MeG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, generally leads to G:C to A:T mutagenesis. To study DNA replication encountering O⁶-MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O⁶-MeG by gp90 exo⁻. O⁶-MeG partially inhibited full-length extension by gp90 exo⁻. O⁶-MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo⁻ extends beyond T:O⁶-MeG 2-fold more efficiently than C:O⁶-MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O⁶-MeG show fast burst phases. The pre-steady-state incorporation efficiency (k_pol/K_d,dNTP) is decreased in the order of dCTP:G > dTTP:O⁶-MeG > dCTP:O⁶-MeG. The presence of O⁶-MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg²⁺. Misincorporation of dTTP opposite O⁶-MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O⁶-MeG is originated from the fact that dTTP can induce a faster conformational change step and a faster chemical step than dCTP. This study reveals that gp90 bypasses O⁶-MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1.     

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.     

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.     

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.     

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.     

UV-induced imbalance of the deoxyribonucleoside triphosphate pool in E. coli

The effect of UV irradiation on the intracellular DNA precursor pool in E. coli was investigated. UV irradiation of E. coli, followed by post-incubation for 1-1.5 h, altered the relative sizes of the deoxyribonucleoside triphosphate (dNTP) pool. The total amount of dNTPs increased: both dATP and dTTP increased several-fold, dCTP about twofold, while dGTP remained almost unchanged. In recA- and umuC- strains, which are defective in UV-induced mutagenesis, the pattern of nucleotide pool alterations was similar to that of wild-type strains.     

Fidelity of the human mitochondrial DNA polymerase

We have quantified the fidelity of polymerization of DNA by human mitochondrial DNA polymerase using synthetic DNA oligonucleotides and recombinant holoenzyme and examining each of the possible 16-base pair combinations. Although the kinetics of incorporation for all correct nucleotides are similar, with an average Kd of 0.8 microM and an average k(pol) of 37 s(-1), the kinetics of misincorporation vary widely. The ground state binding Kd of incorrect bases ranges from a low of 25 microM for a dATP:A mispair to a high of 360 microM for a dCTP:T mispair. Similarly, the rates of incorporation of incorrect bases vary from 0.0031 s(-1) for a dCTP:C mispair to 1.16 s(-1) for a dGTP:T mispair. Due to the variability in the kinetic parameters for misincorporation, the estimates of fidelity range from 1 error in 3563 nucleotides for dGTP:T to 1 error in 2.3 x 10(6) nucleotides for dCTP:C. Interestingly, the discrimination against a dGTP:T mismatch is 16.5 times lower than that of a dTTP:G mismatch due to a tighter Kd for ground state binding and a faster rate of incorporation of the dGTP:T mismatch relative to the dTTP:G mismatch. We calculate an average fidelity of 1 error in 440,000 nucleotides.     

Toward understanding the mutagenicity of an environmental carcinogen: structural insights into nucleotide incorporation preferences

Bulky carcinogen-DNA adducts, including (+)-trans-anti-[BP]-N(2)-dG derived from the reaction of (+)-anti-benzo[a]pyrene diol epoxide with guanine, often block the progression of DNA polymerases. However, when rare bypass of the lesions does occur, they may be misreplicated. Experimental results have shown that nucleotides are inserted opposite the (+)-trans-anti-[BP]-N(2)-dG adduct by bacteriophage T7 DNA polymerase with the order of preference A>T>or=G>C. To gain structural insights into the effects of the bulky adduct on nucleotide incorporation within the polymerase active site, molecular modeling and molecular dynamics simulations were carried out using T7 DNA polymerase to permit the relation of function to structure. We modeled the (+)-trans-anti-[BP]-N(2)-dG adduct opposite incoming dGTP, dTTP and dCTP nucleotides, as well as unmodified guanine opposite its normal partner dCTP as a control, to compare with our previous simulation with dATP opposite the adduct. The modeling required that the (+)-trans-anti-[BP]-N(2)-dG adduct adopt the syn conformation in each case to avoid deranging essential protein-DNA interactions. While the dATP: (+)-trans-anti-[BP]-N(2)-dG pair was well accommodated within the active site of T7 DNA polymerase, dCTP fit poorly opposite the adduct, adopting an orientation perpendicular to the plane of the syn modified guanine during the simulation. Rotation about the glycosidic bond of the dCTP residue to this abnormal position was allowed because only one hydrogen bond between dCTP and the (+)-trans-anti-[BP]-N(2)-dG residue evolved during the simulation, and this hydrogen bond was directly across from the dCTP glycosidic bond. The dTTP and dGTP nucleotides, incorporated with an intermediate preference opposite (+)-trans-anti-[BP]-N(2)-dG, were accommodated reasonably well, but not as stably as the dATP nucleotide, due to a skewed primer-template alignment and more exposed BP moiety, respectively. In addition, the extent of stabilizing interactions between the nascent base-pair in each simulation was correlated positively with the incorporation preference of that particular nucleotide. The dATP nucleotide is accommodated most stably opposite the adduct, with protein-DNA hydrogen bonding interactions and an active-site pocket size that do not deviate significantly from those of the control simulation. The simulations of dTTP and dGTP opposite (+)-trans-anti-[BP]-N(2)-dG exhibited more instability in interactions between the protein and the nascent base-pair than the dATP system. However, the active-site pocket size of the dTTP and dGTP simulations remained stable. The dCTP: (+)-trans-anti-[BP]-N(2)-dG system had the least number of stabilizing interactions, and the active-site pocket of this system increased in size significantly compared to the control and other dNTPs opposite the adduct. These simulations elucidated why A is inserted opposite (+)-trans-anti-[BP]-N(2)-dG most frequently, while T and G are inserted opposite the adduct to an extent intermediate between A and C, and C is most rarely incorporated. Structural rationalization of the incorporation preference opposite (+)-trans-anti-[BP]-N(2)-dG by T7 DNA polymerase contributes to providing a molecular explanation for mutations caused by this carcinogen-DNA adduct in a model system.     

Intracellular conversions of deoxyribonucleosides by Novikoff rat hepatoma cells and effects of hydroxyurea

The incorporation of ³H-labeled deoxyadenosine and deoxyguanosine into nucleic acids by cultured Novikoff rat hepatoma cells is about 80% into RNA and 20% into DNA. The pathways of incorporation have been elucidated in studies with whole cells and cell-free extracts. Deoxyadenosine is very rapidly deaminated to deoxyinosine. Most of the deoxyinosine formed by whole cells is transported out of the cells and accumulates in the medium. A portion of the deoxyinosine, and deoxyguanosine are phosphorolyzed by purine nucleoside phosphorylase to hypoxanthine and guanine, respectively. The latter are subsequently converted by hypoxanthine-guanine phosphoribosyl transferase to IMP and GMP, respectively. Incorporation of the purine deoxyribonucleosides into DNA is mainly via this pathway and the subsequent reduction of ADP and GDP by ribonucleoside reductase, although a small proportion of the deoxyadenosine and deoxyguanosine taken up by the cells seems to be directly phosphorylated to dAMP and dGMP, respectively. Deoxyguanosine is incorporated only into guanine residues of RNA and DNA. Deoxyadenosine is also mainly incorporated into guanine residues of RNA and DNA, although the radioactivity of deoxyadenosine in the acid-soluble pool is almost exclusively associated with ATP. A similar labeling pattern is observed with labeled deoxyinosine, inosine or hypoxanthine. The pyrimidine deoxyribonucleosides, on the other hand, are specific precursors for their respective bases in DNA. Hydroxyurea inhibits the incorporation of all deoxyribonucleosides into DNA. Results from pulse-chase experiments indicate that the inhibition of DNA synthesis is prevented by the presence of high concentrations of deoxyadenosine plus deoxyguanosine in the medium. Either purine deoxyribonucleoside alone or deoxycytidine, hypoxanthine or inosine alone or in combination with deoxyadenosine or deoxyguanosine are ineffective. The results are consistent with the conclusion that the inhibition of DNA synthesis is due to a depletion of the dATP and dGTP pools as a result of the hydroxyurea treatment. On the other hand, hydroxyurea causes an increased incorporation of thymidine and deoxycytidine into the dTTP and dCTP pools, respectively. Evidence is presented to indicate that this effect of hydroxyurea is due to an increased synthesis of dTTP and dCTP rather than to an inhibition of their turnover.     

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.     

Human deoxythymidine kinase II: substrate specificity and kinetic behavior of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia.

Cytoplasmic and mitochondrial deoxythymidine kinase isozymes derived from the blast cells of acute myelocytic leukemia differ in their substrate specificity and kinetic behavior. These enzymes require divalent cations for their activity. The data suggest that the major role of idvalent cations is to chelate with ATP; the complex thus formed serves as the phosphate donor for the reaction. The activity of various triphosphate nucleosides as a phosphate donor for cytoplasmic deoxythymidine kinase is as follows: ATP = dATP greater than ara-ATP greater than GTP greater than CTP greater than dGTP = dCTP greater than dUTP, whereas for mitochondrial deoxythymidine kinase, the order of activity is ATP greater than CTP greater than UTP = dATP greater than ara-ATP greater than dGTP = dCTP greater than dUTP. Neither IdUTP nor dTTP could serve as a phosphate donor in the reaction catalyzed by either isozyme. From the many pyrimidine analogues tested for their binding affinity to each of these isozymes, I-dUrd and Br-dUrd had high good affinity which was equivalent to that of deoxythymidine. 5-Allyl-dUrd, 5-ethyl-dUrd, and 5-propyl-dUrd were only weakly bound to each isozyme. 5-I-dCyd, 5-Br-dCyd, dCyd, and 5-vinyl-dUrd were tightly bound to mitochondrial deoxythymidine kinase but not to the cytoplasmic isozyme. dTTP and I-dUTP are potent inhibitors of the reaction catalyzed by both isozymes. In contrast, dCTP and ara-CTP are potent inhibitors only of the mitochondrial isozyme, but not of the cytoplasmic isozyme. ATP-MG2+ acts as a sigmoidal substrate of the cytoplasmic isozyme with a"Km" of 0.22 mM, and as a regular substrate of the mitochondrial isozyme with a Km of 0.1 mM. Deoxythymidine acts as a regular substrate for both cytoplasmic and mitochondrial isozyme with a Km of 2.6 and 5.2 muM, respectively. Initial velocity as well as product inhibition studies suggest that the cytoplasmic isozyme catalyzes the reaction via a "sequential" mechanism. In contrast, mitochondrial deoxythymidine kinase catalyzes the reaction via a "ping-pong" mechanism.