Structural basis for recognition and catalysis by the bifunctional dCTP deaminase and dUTPase from Methanococcus jannaschii

Potentially mutagenic uracil-containing nucleotide intermediates are generated by deamination of dCTP, either spontaneously or enzymatically as the first step in the conversion of dCTP to dTTP. dUTPases convert dUTP to dUMP, thus avoiding the misincorporation of dUTP into DNA and creating the substrate for the next enzyme in the dTTP synthetic pathway, thymidylate synthase. Although dCTP deaminase and dUTPase activities are usually found in separate but homologous enzymes, the hyperthermophile Methanococcus jannaschii has an enzyme, DCD-DUT, that harbors both dCTP deaminase and dUTP pyrophosphatase activities. DCD-DUT has highest activity on dCTP, followed by dUTP, and dTTP inhibits both the deaminase and pyrophosphatase activities. To help clarify structure-function relationships for DCD-DUT, we have determined the crystal structure of the wild-type DCD-DUT protein in its apo form to 1.42A and structures of DCD-DUT in complex with dCTP and dUTP to resolutions of 1.77A and 2.10A, respectively. To gain insights into substrate interactions, we complemented analyses of the experimentally defined weak density for nucleotides with automated docking experiments using dCTP, dUTP, and dTTP. DCD-DUT is a hexamer, unlike the homologous dUTPases, and its subunits contain several insertions and substitutions different from the dUTPase beta barrel core that likely contribute to dCTP specificity and deamination. These first structures of a dCTP deaminase reveal a probable role for an unstructured C-terminal region different from that of the dUTPases and possible mechanisms for both bifunctional enzyme activity and feedback inhibition by dTTP.     

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.     

Structures of dCTP deaminase from Escherichia coli with bound substrate and product: reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.     

Regulation of dCTP deaminase from Escherichia coli by nonallosteric dTTP binding to an inactive form of the enzyme

The trimeric dCTP deaminase produces dUTP that is hydrolysed to dUMP by the structurally closely related dUTPase. This pathway provides 70-80% of the total dUMP as a precursor for dTTP. Accordingly, dCTP deaminase is regulated by dTTP, which increases the substrate concentration for half-maximal activity and the cooperativity of dCTP saturation. Likewise, increasing concentrations of dCTP increase the cooperativity of dTTP inhibition. Previous structural studies showed that the complexes of inactive mutant protein, E138A, with dUTP or dCTP bound, and wild-type enzyme with dUTP bound were all highly similar and characterized by having an ordered C-terminal. When comparing with a new structure in which dTTP is bound to the active site of E138A, the region between Val120 and His125 was found to be in a new conformation. This and the previous conformation were mutually exclusive within the trimer. Also, the dCTP complex of the inactive H121A was found to have residues 120-125 in this new conformation, indicating that it renders the enzyme inactive. The C-terminal fold was found to be disordered for both new complexes. We suggest that the cooperative kinetics are imposed by a dTTP-dependent lag of product formation observed in presteady-state kinetics. This lag may be derived from a slow equilibration between an inactive and an active conformation of dCTP deaminase represented by the dTTP complex and the dUTP/dCTP complex, respectively. The dCTP deaminase then resembles a simple concerted system subjected to effector binding, but without the use of an allosteric site.     

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.     

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.     

Direct photoaffinity-labelling of human deoxycytidine kinase with the feedback inhibitor dCTP.

Deoxycytidine kinase (dCyd kinase, EC 2.7.1.74) is a key enzyme in the salvage pathway of deoxyribonucleosides, and the human enzyme is a dimer of two 30 kDa polypeptides with a broad substrate specificity, phosphorylating both purine and pyrimidine nucleosides and using various nucleoside triphosphates as phosphate donors. The enzyme is efficiently feedback-inhibited by dCTP, which presumably is the main regulator of its activity in vivo. Submicromolar concentrations of [32P]dCTP could be used for direct photoaffinity labelling of pure dCyd kinase isolated from leukaemic spleen. A clearcut saturation of photoincorporation occurred with half-maximal incorporation at 0.07 microM-dCTP. However, the total molar incorporation of dCTP was very low (approx. 0.1%), in part due to a substantial u.v. inactivation of the enzyme. Proteinase digestion of labelled enzyme showed that dCTP was incorporated predominantly into a single peptide. Addition of equimolar concentrations of dCyd or dCMP as compared with dCTP inhibited photoincorporation approx. 50%. The presence of other nucleoside substrates, as well as phosphate donors, also inhibited photolabelling of the enzyme. Thus photoincorporation of dCTP seems to occur at a site which can bind both the phosphate donors and acceptors of dCyd kinase, which strongly support the hypothesis that dCTP functions as a multi-substrate analogue, binding and bridging both substrate sites of the enzyme.     

The Methanococcus jannaschii dCTP deaminase is a bifunctional deaminase and diphosphatase

Most bacteria produce the dUMP precursor for thymine nucleotide biosynthesis using two enzymes: a dCTP deaminase catalyzes the formation of dUTP and a dUTP diphosphatase catalyzes pyrophosphate release. Although these two hydrolytic enzymes appear to catalyze very different reactions, they are encoded by homologous genes. The hyperthermophilic archaeon Methanococcus jannaschii has two members of this gene family. One gene, at locus MJ1102, encodes a dUTP diphosphatase, which can scavenge deoxyuridine nucleotides that inhibit archaeal DNA polymerases. The second gene, at locus MJ0430, encodes a novel dCTP deaminase that releases dUMP, ammonia, and pyrophosphate. Therefore this enzyme can singly catalyze both steps in dUMP biosynthesis, precluding the formation of free, mutagenic dUTP. Besides differing from the previously characterized Salmonella typhimurium dCTP deaminase in its reaction products, this archaeal enzyme has a higher affinity for dCTP and its steady-state turnover is faster than the bacterial enzyme. Kinetic studies suggest: 1) the archaeal enzyme specifically recognizes dCTP; 2) dCTP deamination and dUTP diphosphatase activities occur independently at the same active site, and 3) both activities depend on Mg(2+). The bifunctional activity of this M. jannaschii enzyme illustrates the evolution of a suprafamily of related enzymes that catalyze mechanistically distinct reactions.     

Bromodeoxyuridine mutagenesis in mammalian cells is related to deoxyribonucleotide pool imbalance.

The relationship between bromodeoxyuridine (BrdUrd) mutagenesis in mammalian cells and the effects of BrdUrd on deoxyribonucleoside triphosphate pools was analyzed. It was found that the exposure of Syrian hamster melanoma cells to mutagenic concentrations of BrdUrd resulted in the formation of a large bromodeoxyuridine triphosphate (BrdUTP) pool, which remained at a high level for several days. In contrast, the size of the deoxycytidine triphosphate (dCTP) pool dropped rapidly after the addition of BrdUrd, reached a minimum at about 6 h, and then expanded gradually to nearly its original level over the next 3 days. The addition of lower concentrations of BrdUrd, which had less of a mutagenic effect, resulted in the formation of a smaller BrdUTP pool and a slightly smaller drop in the dCTP pool. When a high concentration of deoxycytidine was added at the same time as a normally mutagenic concentration of BrdUrd, the drop in the dCTP pool was prevented, as was BrdUrd mutagenesis. In all of these experiments, mutagenesis was related to the ratio of BrdUTP to dCTP in the cells. In addition, it was shown that mutagenesis occurred primarily during the first 24 h of BrdUrd exposure, when the BrdUTP/dCTP ratio was at its highest level. It appears that there is a critical ratio of BrdUTP to dCTP that must be attained for high levels of mutagenesis to occur and that the extent of mutagenesis is related to the ratio of the BrdUrd and dCTP pools.     

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.     

Compartmentation of dCTP pools disappears after hydroxyurea or araC treatment in lymphocytes.

The calculated rate of DNA synthesis using [5-3H]TdR was about 4 times higher than in the case of [5-3H]CdR labeling, even after correction for the specific radioactivities of the intracellular pools. These data show a compartmentation of dCTP pools in lymphocytes. Hydroxyurea increased the specific activities of both dTTP and dCTP pools so that the calculated rate of DNA synthesis became equal. The same effect was found for araC treatment, but not for fluorodeoxyuridine. dCTP was supplied from CTP which is the lowest ribonucleotide pool in lymphocytes. Different functions of the two dCTP pools are proposed: one serving DNA replication; the other one supplies phospholipid precursors and DNA repair.     

Chloroviruses encode a bifunctional dCMP-dCTP deaminase that produces two key intermediates in dTTP formation

The chlorovirus PBCV-1, like many large double-stranded DNA-containing viruses, contains several genes that encode putative proteins involved in nucleotide biosynthesis. This report describes the characterization of the PBCV-1 dCMP deaminase, which produces dUMP, a key intermediate in the synthesis of dTTP. As predicted, the recombinant protein has dCMP deaminase activity that is activated by dCTP and inhibited by dTTP. Unexpectedly, however, the viral enzyme also has dCTP deaminase activity, producing dUTP. Typically, these two reactions are catalyzed by proteins in separate enzyme classes; to our knowledge, this is the first example of a protein having both deaminase activities. Kinetic experiments established that (i) the PBCV-1 enzyme has a higher affinity for dCTP than for dCMP, (ii) dCTP serves as a positive heterotropic effector for the dCMP deaminase activity and a positive homotropic effector for the dCTP deaminase activity, and (iii) the enzymatic efficiency of the dCMP deaminase activity is about four times higher than that of the dCTP deaminase activity. Inhibitor studies suggest that the same active site is involved in both dCMP and dCTP deaminations. The discovery that the PBCV-1 dCMP deaminase has two activities, together with a previous report that the virus also encodes a functional dUTP triphosphatase (Y. Zhang, H. Moriyama, K. Homma, and J. L. Van Etten, J. Virol. 79:9945-9953, 2005), means that PBCV-1 is the first virus to encode enzymes involved in all three known pathways to form dUMP.     

Formation of deoxycytidine diphosphate-diglyceride by nuclei isolated from HeLa cells.

Isolated nuclei from HeLa cells synthesize dCDP-diglyceride from dCTP at the rapid rate of 5–10 nmol/20 min/10⁸ nuclei. The incorporation of dCTP into this phospholipid precursor is thus 10 to 20 times faster than the incorporation of dCTP into DNA, in vitro, under the same conditions. ATP, phosphatidic acid, and MgCl₂ are required for optimal synthesis of dCDP-diglyceride. The reaction is completely inhibited by the presence of 0.04% Triton N-101. Liponucleotide formation occurs equally well with dCTP or CTP in this system and competition studies suggest that a single enzyme catalyzes the formation of dCDP- and CDP-diglyceride.     

Efficient and high fidelity incorporation of dCTP opposite 7,8-dihydro-8-oxodeoxyguanosine by Sulfolobus solfataricus DNA polymerase Dpo4

DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed >/=95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured K(d)(,dCTP) was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The E(a) for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca(2+). The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5' C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.     

Studies on the mechanism of Escherichia coli DNA polymerase I large fragment. Effect of template sequence and substrate variation on termination of synthesis

Termination of Escherichia coli DNA polymerase I large fragment after processive synthesis on natural and other well-defined template.primer systems has been examined. We found that after any given deoxynucleoside monophosphate incorporation termination occurs in a nonrandom manner with phi X174 DNA as template: Termination is much more likely at some nucleotide residues along the template than at others. Analysis of these stronger termination sites indicates that the template base:incoming nucleotide combination influences termination. Introduction of a double-stranded region along the phi X174 template induces termination, and reducing dNTP concentrations or substituting 2'-deoxynucleoside 5'-O-(1-thio)triphosphate substrates also increases termination. Observations with the phi X174 DNA template system were extended with a defined template containing 1 inosine residue in an otherwise d(T)n homopolymer. Termination at the I residue is modulated by dCTP and decreases as dCTP concentration increases. A similar relationship is seen with the dCTP (1-thio) derivative, but termination is higher at given concentrations of this derivative than with dCTP. Pyrophosphate decreases general processivity in this system, but does not counteract the effect of increasing dCTP. Hill plot analysis of the dCTP effect in the inosine-containing template system gave a linear plot with Hill coefficient of 0.34, suggesting that dCTP influences termination at several steps in the polymerase reaction scheme. Substituting a methylated template base for I also increased termination, producing very strong blocks to processive synthesis. The results are consistent with a model in which termination occurs with several enzyme forms that are in equilibrium in an ordered catalytic mechanism.     

Probing DNA polymerase alpha with monoclonal antibodies

DNA polymerase alpha was purified from human KB cells by immunoaffinity chromatography. Enzyme activity was inhibited by three different monoclonal antibodies (SJK-132, SJK-211, SJK-287). Kinetic analysis showed that each antibody neutralized polymerase activity by a different mechanism. SJK-132 was competitive with DNA indicating it interacts with the DNA binding domain of the polymerase. SJK-287 showed a biphasic response to dCTP suggesting two dCTP binding sites exist on polymerase alpha. SJK-211 was non-competitive with DNA, dCTP and dATP.     

Specific amino acid residues are involved in substrate discrimination and template binding of human REV1 protein

REV1 is a member of the Y-family DNA polymerases, but is atypical in utilizing only dCTP with a preference for guanine (G) as the template. Crystallography of the REV1-DNA-dCTP ternary complex has revealed a unique mechanism by which template G is evicted from the DNA helix and incoming dCTP is recognized by an arginine residue in an α-loop, termed the N-digit. To better understand functions of its individual amino acid residues, we made a series of mutant human REV1 proteins. We found that R357 and L358 play vital roles in template binding. Furthermore, extensive mutation analysis revealed a novel function of R357 for substrate discrimination, in addition to previously proposed specific interaction with incoming dCTP. We found that the binding pocket for dCTP of REV1 has also significant but latent affinity for dGTP. The results suggest that the positive charge on R357 could prevent interaction with dGTP. We propose that both direct and indirect mechanisms mediated by R357 ensure specificity for dCTP.     

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.     

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.     

Characterization of a novel dUTP-dependent activity of CTP synthetase from Saccharomyces cerevisiae

CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] from the yeast Saccharomyces cerevisiae catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In this work, we demonstrated that CTP synthetase utilized dUTP as a substrate to synthesize dCTP. The dUTP-dependent activity was linear with time and with enzyme concentration. Maximum dUTP-dependent activity was dependent on MgCl(2) (4 mM) and GTP (K(a) = 14 microM) at a pH optimum of 8.0. The apparent K(m) values for dUTP, ATP, and glutamine were 0.18, 0.25, and 0.41 mM, respectively. dUTP promoted the tetramerization of CTP synthetase, and the extent of enzyme tetramerization correlated with dUTP-dependent activity. dCTP was a poor inhibitor of dUTP-dependent activity, whereas CTP was a potent inhibitor of this activity. The enzyme catalyzed the synthesis of dCTP and CTP when dUTP and UTP were used as substrates together. CTP was the major product synthesized when dUTP and UTP were present at saturating concentrations. When dUTP and UTP were present at concentrations near their K(m) values, the synthesis of dCTP increased relative to that of CTP. The synthesis of dCTP was favored over the synthesis of CTP when UTP was present at a concentration near its K(m) value and dUTP was varied from subsaturating to saturating concentrations. These data suggested that the dUTP-dependent synthesis of dCTP by CTP synthetase activity may be physiologically relevant.