Cloning of deoxynucleoside monophosphate kinase genes and biosynthesis of deoxynucleoside diphosphates

The genes encoding four deoxynucleoside monophosphate kinase (dNMP kinase) enzymes, including ADK1 for deoxyadenylate monophosphate kinase (AK), GUK1 for deoxyguanylate monophosphate kinase (GK), URA6 for deoxycytidylate monophosphate kinase (CK), and CDC8 for deoxythymidylate monophosphate kinase (TK), were isolated from the genome of Saccharomyces cerevisiae ATCC 2610 strain and cloned into E. coli strain BL21(DE3). Four recombinant plasmids, pET17b-JB1 containing ADK1, pET17b-JB2 containing GUK1, pET17b-JB3 containing URA6, and pET17b-JB4 containing CDC8, were constructed and transformed into E. coli strain for over-expression of AK, GK, CK, and TK. The amino acid sequences of these enzymes were analyzed and a putative conserved peptide sequence for the ATP active site was proposed. The four deoxynucleoside diphosphates (dNDP) including deoxyadenosine diphosphate (dADP), deoxyguanosine diphosphate (dGDP), deoxycytidine diphosphate (dCDP), and deoxythymidine diphosphate (dTDP), were synthesized from the corresponding deoxynucleoside monophosphates (dNMP) using the purified AK, GK, CK, and TK, respectively. The effects of pH and magnesium ion concentration on the dNDP biosynthesis were found to be important. A kinetic model for the synthetic reactions of dNDP was developed based on the Bi-Bi random rapid equilibrium mechanism. The kinetic parameters including the maximum reaction velocity and Michaelis-Menten constants were experimentally determined. The study on dNDP biosynthesis reported in this article are important to the proposed bioprocess for production of deoxynucleoside triphosphates (dNTP) that are used as precursors for in vitro DNA synthesis. There is a significant advantage of using enzymatic biosyntheses of dNDP as compared to the chemical method that has been in commercial use.     

Saccharomyces cerevisiae nucleoside-diphosphate kinase: purification, characterization, and substrate specificity.

Nucleoside-diphosphate kinase is an enzyme which catalyzes the phosphorylation of nucleoside diphosphates into the corresponding triphosphates for nucleic acid biosynthesis. In this communication, we describe the purification and characterization of nucleoside-diphosphate kinase from yeast. The purified protein appears to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis, with a molecular weight of about 17,000-18,000. An estimate from the fast protein liquid chromatography Superose 12 gel filtration shows a native molecular weight of about 68,000 to 70,000. The results suggest that yeast nucleoside-diphosphate kinase is composed of four subunits. Substrate specificity studies show that the relative activity of nucleoside diphosphates (NDP) as phosphate acceptors is in the order of dTDP greater than CDP greater than UDP greater than dUDP greater than GDP greater than or equal to dGDP greater than dCDP greater than dADP greater than ADP; and the relative activity of triphosphate donors is in the order of UTP greater than dTTP greater than CTP greater than dCTP greater than dATP greater than ATP greater than or equal to dGTP greater than GTP. The Km and Vm of dTDP, dGDP, dCDP, dUDP, CDP, and UDP have been determined. The rate constant studies indicate that the purified NDP kinase prefers using, to a slight extent, dTDP (approximately 800 min-1) as the substrate rather than other tested deoxyribo- and ribonucleotides (350-450 min-1). The broad substrate specificity and kinetic data suggest that the enzyme is involved in both DNA and RNA metabolism.     

Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP

The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.     

Deoxynucleotide-interconverting enzymes and the quantification of deoxynucleoside triphosphates in mammalian cells

We have demonstrated that methanol extracts of human cells are heterogeneous with regard to content of dNDP (deoxynucleoside diphosphate) and dNMP (deoxynucleoside monophosphate) kinases. The presence of these enzymes can affect the reliability of techniques used to measure intracellular pools of deoxynucleotides. An optimized extraction procedure and enzymic assay for dNTP species in haematopoietic cells are described which provide sensitivity to measure 0.1-40pmol of dATP, dTTP and dGTP, and 1.0-40pmol of dCTP. The extraction and assay give linear results with (2.5-15)x10(6) nucleated cells and (0.1-1.5)x10(9) red blood cells. Under these conditions, extracts equivalent to ~0.5x10(6) nucleated haematopoietic cells catalyse the phosphorylation of 0-8% of dNDP and dNMP standards to dNTP and incorporate them into deoxynucleotide polymer under circumstances where 100% of an equimolar dNTP standard would be incorporated. By contrast, extracts of 0.4x10(6) HeLa cells totally converted dADP, dTDP and dGDP into dNTP with subsequent polymerization. Conversion of dCDP was somewhat less efficient. The results demonstrate conclusively that the activities of deoxynucleotide interconverting enzymes differ in different types of human cells. They can interfere with assay of nucleotides, but may not do so in many types of cell extracts. In particular, dNTP concentrations can be measured in human haematopoietic cells after extraction with 60% (v/v) methanol and are not artificially elevated by deoxynucleotide interconversions. It is apparent that extraction and assay procedures for measurement of dNTP species should be analysed for each cell type in order to minimize contaminating enzyme activities and ensure accuracy of dNTP quantification.     

Nucleoside 5'-diphosphates as effectors of mammalian ribonucleotide reductase

It was found that nucleoside 5'-diphosphates could serve as effectors of ribonucleotide reductase. ADP was an activator of CDP reduction; ADP reduction was activated by dGDP; GDP reduction was activated by dTDP. Conversely, dADP inhibited the reduction of CDP, UDP, GDP, and ADP; dGDP inhibited UDP and GDP reductions; and dTDP inhibited UDP reduction. The inhibition of UDP reduction by dADP, dTDP, and dGDP was at least equal to that observed for dATP, dTTP, and dGTP, respectively. In these experiments with the nucleoside diphosphates as effectors, high-pressure liquid chromatography analysis of the reaction mixtures showed that no nucleoside 5'-triphosphates were found during the reaction period which could account for the effects seen with the nucleoside diphosphates as effectors. Further experiments were carried out in which adenyl-5'-yl imidodiphosphate was used as the positive effector of CDP and UDP reductions in place of ATP. Under these conditions, CDP and UDP reductions were inhibited by dADP, dTDP, and dGDP to the same extent observed in the presence of ATP. ADP served not only as a substrate for ribonucleotide reductase but also as an activator of CDP and UDP reductions. The direct products (dNDPs) also served as positive and negative effectors. Dixon plots indicated that the dNDPs were acting as noncompetitive inhibitors with respect to the substrate. ADP increased the sedimentation velocity of the ribonucleotide reductase in a manner similar to ATP. These data are consistent with the allosteric effects seen with the nucleoside 5'-triphosphates. Additionally, from the thorough study of the role of effectors on UDP reduction, it is clear that UDP reduction was most sensitive to the negative effectors dATP, dADP, dTTP, dTDP, dGTP, and dGDP.     

Characterization of a nucleotide kinase encoded by bacteriophage t7

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

Enzymatic synthesis of oligodeoxyribonucleotides of defined sequence. Polynucleotide phosphorylase catalysed addition of deoxyribonucleotides to primers which are good or poor acceptors.

Analytical high-pressure anion-exchange chromatography on RPC-5 has been used to study the behaviour of a good primer, d(pT-T-A-G), and a poor primer, d(pT-T-T-T-T-T) in the E. coli polynucleotide phosphorylase-catalysed reactions of dADP, dCDP, dGDP and dTDP where the primer is extended, predominantly, by one or two nucleotides. The experiments provide some generalizations for obtaining optimal yields in preparative reactions. In the course of the experiments, examples of anomalous behaviour of oligonucleotides on RPC-5 were encountered and these are discussed.     

Shrimp oncoprotein nm23 is a functional nucleoside diphosphate kinase

Biosynthesis of nucleoside triphosphates is critical for bioenergetics and nucleic acid replication, and this is achieved by nucleoside diphosphate kinase (NDK). As an emerging biological model and the global importance of shrimp culture, we have addressed the study of the Pacific whiteleg shrimp (Litopenaeus vannamei) NDK. We demonstrated its activity and affinity towards deoxynucleoside diphosphates. Also, the quaternary structure obtained by gel filtration chromatography showed that shrimp NDK is a trimer. Affinity was in the micro-molar range for dADP, dGDP, dTDP and except for dCDP, which presented no detectable interaction by isothermal titration calorimetry, as described previously for Plasmodium falciparum NDK. This information is particularly important, as this enzyme could be used to test nucleotide analogs that can block white spot syndrome virus (WSSV) viral replication and to study its bioenergetics role during hypoxia and fasting.     

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.     

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.     

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.     

An enzymatic method for distinguishing deoxyuridine and deoxythymidine nucleotide pools and its application for determining ribonucleotide reductase activity.

A method is described for distinguishing deoxyuridine and deoxythymidine di- and triphosphate pools. The method utilizes a DNA polymerase assay for triphosphate determination and a coupled assay in which the disphosphate is converted to its corresponding triphosphate by nucleoside-diphosphate kinase and the triphosphate is measured by the DNA polymerase assay. By including deoxyruidine-triphosphate nucleotidohydrolase in the reaction mixture, dUTP is removed as a substrate for the polymerase. By determining differences in labelled acid-insoluble product formed in the reaction it is possible to determine dUTP, dUDP, dTDP and dTTP pools. Ribonucleotide reductase activity was determined by converting either CDP or ADP to its corresponding deoxyribonucleoside disphosphate and then using the diphosphate assay described for deoxyribonucleoside pools.     

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.     

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.     

Sulfonylpiperidines as novel,antibacterial inhibitors of Gram-positive thymidylate kinase (TMK)

Thymidylate kinase (TMK) is an essential enzyme for DNA synthesis in bacteria, phosphorylating deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP), and thus is a potential new antibacterial drug target. Previously, we have described the first potent and selective inhibitors of Gram-positive TMK, leading to in vivo validation of the target. Here, a structure-guided design approach based on the initial series led to the discovery of novel sulfonylpiperidine inhibitors of TMK. Formation of hydrogen bonds with Arg48 in Staphylococcus aureus TMK was key to obtaining excellent enzyme affinity, as verified by protein crystallography. Replacement of a methylene linker in the series by a sulfonamide was accomplished with retention of binding conformation. Further optimization of log D yielded phenol derivative 11, a potent inhibitor of TMK showing excellent MICs against a broad spectrum of Gram-positive bacteria and >10⁵ selectivity versus the human TMK homologue.     

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.     

Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity.

A real-time, simple and sensitive method for detection of nucleoside diphosphate (NDP) kinase activity has been developed. The assay is based on detection of ATP, generated in the NDP kinase reaction between a nucleoside triphosphate and adenosine diphosphate (ADP), by the firefly luciferase system. In the presence of 0.3 mM dGTP, the Km for ADP was found to be approximately 30 microM for the NDP kinase from Baker's yeast. In the presence of 250 microM ADP, the Km for dATP alpha S, dTTP alpha S, dGTP, dTTP, dCTP and GTP was found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respectively. The assay is sensitive and yields linear responses between 0.05-50 mU. The detection limit was found to be 0.05 mU of NDP kinase. The method was used to detect NDP kinase contamination in commercial enzyme preparations.     

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.     

Development of a sensitive and selective LC/MS/MS method for the simultaneous determination of intracellular 1-beta-d-arabinofuranosylcytosine triphosphate (araCTP), cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in a human follicular lymphoma cell line

A method was developed for the quantification of araCTP, CTP and dCTP in a human follicular lymphoma cell line. This method involves solid phase extraction (SPE) using a weak anion-exchanger (WAX) cartridge, a porous graphitic carbon high-performance liquid chromatography (HPLC) column separation, and tandem mass spectrometry (MS/MS) detection. By using a triple quadrupole mass spectrometer operating in negative ion multiple reaction monitoring (MRM) mode, the method was able to achieve a lower limit of quantification (LLOQ) of 0.1 μg mL^?1 for araCTP and of 0.01 μg mL^?1 for both CTP and dCTP. The method was validated and used to determine the amount of araCTP, CTP and dCTP formed after incubation of araC and an araCMP prodrug in the human follicular lymphoma cell line RL.     

Inhibition of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity of the antimutagenic human MTH1 protein by nucleoside 5'-diphosphates

The hMTH1 protein, a human homologue of E. coli MutT protein, is an enzyme converting 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) to 8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) and inorganic pyrophosphate. It is thought to play an antimutagenic role by preventing the incorporation of promutagenic 8-oxo-dGTP into DNA. As found in our previous investigations, 8-oxo-2'-deoxyguanosine 5'-diphosphate (8-oxo-dGDP) strongly inhibited 8-oxo-dGTPase activity of MTH1. Following this finding, in the present study we have tested the canonical ribo- and deoxyribonucleoside 5'-diphosphates (NDPs and dNDPs) for possible inhibition of 8-oxo-dGTP hydrolysis by hMTH1 extracted from CCRF-CEM cells (a human leukemia cell line). Among them, the strongest inhibitors appeared to be dGDP (Ki=74 microM), dADP (Ki=147 microM), and GDP (Ki=502 microM). Other dNDPs and NDPs, such as dCDP, dTDP, ADP, CDP, and UDP were much weaker inhibitors, with Ki in the millimolar range. Based on the present results and published data, we estimate that the strongest inhibitors, dGDP and dADP, at physiological concentrations not exceeding 5 microM and GDP at mean concentration of 30 microM, taken together, can decrease the cellular hMTH1 enzymatic activity vs. 8-oxo-dGTP (expected to remain below 500 pM) by up to 15%. The other five NDPs and dNDPs tested cannot markedly affect this activity.