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J. Biol. Chem., Vol. 280, Issue 31, 28221-28229, August 5, 2005

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Adenylate Kinase of Escherichia coli, a Component of the Phage T4 dNTP Synthetase Complex^*

JuHyun Kim, Rongkun Shen, Michael C. Olcott, Indira Rajagopal, and Christopher K. Mathews

From the Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305

Received for publication, February 25, 2005 , and in revised form, May 17, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Adenylate kinase, which catalyzes the reversible ATP-dependent phosphorylation of AMP to ADP and dAMP to dADP, can also catalyze the conversion of nucleoside diphosphates to the corresponding triphosphates. Lu and Inouye (Lu, Q., and Inouye, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5720–5725) showed that an Escherichia coli ndk mutant, lacking nucleoside diphosphate kinase, can use adenylate kinase as an alternative source of nucleoside triphosphates. Bacteriophage T4 can reproduce in an Escherichia coli ndk mutant, implying that adenylate kinase can meet a demand for deoxyribonucleoside triphosphates that increases by up to 10-fold as a result of T4 infection. In terms of kinetic linkage and specific protein-protein associations, NDP kinase is an integral component of T4 dNTP synthetase, a multienzyme complex containing phage-coded enzymes, which facilitates the synthesis of dNTPs and their flow into DNA. Here we asked whether, by similar criteria, adenylate kinase of the host cell is also a specific component of the complex. Experiments involving protein affinity chromatography, immunoprecipitation, optical biosensor measurements, and glutathione S-transferase pulldowns demonstrated direct interactions between adenylate kinase and several phage-coded enzymes, as well as E. coli nucleoside diphosphate kinase. These results identify adenylate kinase as a specific component of the complex. The rate of DNA synthesis after infection of an ndk mutant was found to be about 40% of the rate seen in wild-type infection, implying that complementation of the missing NDP kinase function by adenylate kinase is fairly efficient, but that adenylate kinase becomes rate-limiting for DNA synthesis when it is the sole source of dNTPs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Adenylate kinase catalyzes the reversible ATP-dependent phosphorylation of AMP to ADP. The reaction is involved in the de novo biosynthesis of adenine nucleotides, and it is also thought to participate in adjusting adenine nucleotide levels to meet the energy demands of a cell (1). In addition, there is evidence that the enzyme in Escherichia coli participates in phospholipid biosynthesis, although the specific involvement has not been defined (2). Temperature-sensitive mutations in adk, the structural gene for adenylate kinase, cause defective phospholipid synthesis when bacteria are grown at a nonpermissive temperature (3).

A novel capability for E. coli adenylate kinase was described when Lu and Inouye (4) found that the wild-type adk gene could complement a site-specific disruption of ndk, the structural gene for nucleoside diphosphate kinase. Lu and Inouye (5) showed that adenylate kinase could catalyze the conversion of nucleoside diphosphates to triphosphates. Experiments in our laboratory (5) showed that the phosphate donor for at least some of these reactions was ADP. So, instead of catalyzing the well known reaction, 2ADP AMP + ATP, the enzyme was evidently substituting a different nucleoside diphosphate for one of the two ADPs: (d)NDP + ADP AMP + (d)NTP.

Both NDP¹ kinase and adenylate kinase were shown, some years ago, to participate in the synthesis of dNTPs after T4 infection of E. coli (6). Most reactions in T4 dNTP and DNA synthesis are catalyzed by phage-coded enzymes, consistent with the large increase in DNA accumulation rate per cell that occurs as a result of infection. An important exception is NDP kinase, an active enzyme of low specificity, which was found capable of phosphorylating the phage-modified nucleotide 5-hydroxymethyl-dCDP (7). There is no phage-specific form of NDP kinase, and the bacterial enzyme carries out synthesis of all four dNTPs from the respective deoxyribonucleoside diphosphates. At the monophosphate level, however, a phage-coded enzyme is required, because no E. coli enzyme can phosphorylate 5-hydroxymethyl-dCMP to the diphosphate. This reaction is carried out by an unusual dNMP kinase, the product of gene 1, which also acts upon dGMP and dTMP (7, 8). This trifunctional enzyme, however, does not act upon dAMP, and it was assumed that the conversion of dAMP to dADP after infection is catalyzed by adenylate kinase; dAMP, like the other dNMPs, is derived from the breakdown of E. coli DNA, a process catalyzed by phage-coded nucleases. This is a quantitatively minor pathway, however, because nucleotides derived from host-cell DNA degradation represent only about 10% of the total nucleotides used for phage DNA synthesis (9).

The enzymes of dNTP synthesis in T4 infection interact to form a multienzyme complex, which we call the T4 dNTP synthetase complex (10), and which facilitates the flow of metabolites en route to dNTPs, and their subsequent flow into DNA. E. coli NDP kinase interacts directly or indirectly with several T4 enzymes of dNTP synthesis and DNA replication, and we consider it an integral component of the dNTP synthetase complex (11). In an early study (12) our laboratory found about 5% of the total adenylate kinase activity in the cell to cosediment with enzymes of the dNTP synthetase complex; NDP kinase behaved similarly. However, we did not inquire at the time whether the association of adenylate kinase with the T4 dNTP synthetase complex was functional, i.e. whether adenylate kinase is a specific component of the complex. This paper deals primarily with that issue.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plasmids, Phage, and Bacterial Strains—Bacteriophage T4D, a wild-type strain, has been maintained in this laboratory for many years. E. coli strains used included KA796 (ara, thi, prolac, ndk wild-type) and NR11814 (KA796ndk::cm), an ndk null mutant; both of these strains were obtained from Dr. Roel M. Schaaper, NIEHS National Institutes of Health. E. coli CV2 (fhuA22, phoA8, adk-2(ts), ompF627(T ^R₂), relA1, glpR2(glp^C), glpD3, pit-10, spoT1, rrnB-2, mcrB1, creC510), used because of its ts mutation in the adk gene (13), was obtained from the E. coli Genetic Stock Center, Yale University. E. coli Origami(DE3) was purchased from Novagen (Madison, WI).

E. coli strains overexpressing T4 enzymes as recombinant proteins were as follows. E. coli HB101/pBK5, expressing gene 1 (dNMP kinase), was obtained from Dr. Maurice J. Bessman, Johns Hopkins University (14). E. coli RR1/pSP104, expressing gene frd (dihydrofolate reductase), and E. coli BL-21(DE3)/pT7-42, expressing dCMP hydroxymethylase, were developed in this laboratory (15, 16). Plasmid pnrdAB, expressing aerobic ribonucleotide reductase, was obtained from Dr. G. Robert Greenberg, University of Michigan (17), and used to transform E. coli Origami(DE3). E. coli MB901, expressing T4 thymidylate synthase, was obtained from Dr. Marlene Belfort, New York State Department of Health (18), and E. coli BL21(DE3)/pET3c-CD5, expressing T4 dCMP deaminase, was obtained from Dr. Frank Maley, also of the New York State Department of Health (19).

Radioisotopic Compounds—[2,8-³H]Deoxyadenosine (18.4 Ci/mmol), [8-³H]deoxyguanosine (5.9 Ci/mmol), and [methyl-³H]thymidine (63 Ci/mmol) were purchased from Moravek Biochemicals and Radiochemicals, Inc. (Brea, CA).

Cloning and Expression of Adenylate Kinase—The adk gene, encoding E. coli adenylate kinase, was amplified from E. coli B genomic DNA by PCR using primers containing NdeI and BamHI restriction sites. Following ligation of the NdeI-BamHI fragment into the corresponding sites of pET-9a (Novagen), the pET9a-adk construct was transformed into competent DH5 cells. After PCR analysis of the transformants, the plasmid from one was sequenced and subsequently transformed into BL21(DE3)pLysS cells. For expression, 1-liter cultures of BL21(DE3)pLysS cells carrying the pET9a-adk plasmid were incubated in LB medium supplemented with 50 µg/ml kanamycin and 34 µg/ml chloramphenicol at 37 °C. When the cells reached an optical density (600 nm) of 0.7, the overexpression of adenylate kinase was induced by addition of 500 µM IPTG. After 2–3 h of induction at 37 °C, the cells were harvested by centrifugation, and the pellet was frozen in liquid N₂.

Purification of Recombinant E. coli Adenylate Kinase—The pellet was resuspended in 40 mM Tris-HCl (pH 8.0), 20 mM -mercaptoethanol containing leupeptin, pepstatin A, benzamidine, and aprotinin (11). Following sonication, the lysate was centrifuged at 92,000 x g for 60 min. The supernatant was transferred to another tube to which streptomycin sulfate was slowly added to a final concentration of 1%. After pelleting the nucleic acid, the resulting supernatant was desalted on an Econo-Pac 10DG column (Bio-Rad) equilibrated with 40 mM Tris-HCl (pH 7.0), and then applied to a POROS 10 HQ anion exchange column (4.6 x 100 mm) using a BioCad 700E Perfusion Chromatography Workstation (Applied Biosystems). Following a 2-min wash step in 40 mM Tris-HCl (pH 7.0), adenylate kinase was eluted with a 10-min linear gradient of NaCl (0–1.0 M in 40 mM Tris-HCl, pH 7.0). The flow rate for the entire run was 5.0 ml/min. The major peak was concentrated in a Centriprep-10 (Millipore) at 4 °C and further purified on a Superdex 200 HR 10/30 size exclusion column (Amersham Biosciences) in degassed 40 mM Tris-HCl (pH 7.5), 100 mM KCl. The purified adenylate kinase was stored on ice in 40 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM DTT.

For some early experiments the adk gene was cloned with a His₆ tag. After PCR of the adk gene and treatment of the PCR product with NdeI and XhoI, the fragment was gel purified and ligated into plasmid pET-15b. After verification of the clone by sequence analysis, induction of the tagged adenylate kinase was carried out by addition of IPTG to a log-phase culture in LB broth plus 100 µg/ml ampicillin. After induction and centrifugation, the bacterial pellet was suspended in 10 volumes of buffer A (50 mM NaH₂PO₄, 5 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 1 mM -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride) plus 1 mg/ml egg white lysozyme. After 20 min on ice, the resuspended cells were briefly sonicated, and the cell lysate was cleared by centrifugation (4 °C, 20,000 x g for 30 min), followed by filtration through a 0.45-µm syringe filter. The supernatant was applied to a HIS-select^TM HC Nickel Affinity column (Sigma). After washing off unbound proteins with buffer A, bound proteins were eluted with buffer A plus 250 mM imidazole.

Glutathione S-Transferase Pulldown Assays—GST fusion proteins were prepared, and GST pulldown experiments carried out, as described in previous publications from this laboratory (11, 20). Fusion proteins made for this study included gp1 (deoxyribonucleoside monophosphate kinase), gp42 (dCMP hydroxymethylase), gpfrd (dihydrofolate reductase), and E. coli nucleoside diphosphate kinase. Each fusion protein gene, cloned in pGEX and expressed in E. coli Origami(DE3), was induced with 0.5 mM IPTG (1 mM IPTG for GST-gpfrd). For some experiments the GST fusion proteins were partially purified as follows. After induction, bacteria were centrifuged and resuspended in 10 volumes of buffer containing 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ (pH 7.4), 2.7 mM KCl, 140 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 5 mM DTT, plus 0.1 volume of egg white lysozyme, 10 mg/ml in 10 mM Tris-HCl, pH 8.0. The resuspended cells were incubated on ice for 30 min, then briefly sonicated. DNase I was added (10 µg/ml), and incubation on ice was continued for 30 min more. Lysates were cleared by centrifugation at 4 °C at 15,000 rpm for 30 min, then filtered through a 0.45-µm syringe filter and applied to a glutathione-Sepharose 4B affinity column. Unbound proteins were removed by extensive elution with 10 mM Na₂HPO₄, 2.7 mM KCl, 1.8 mM KH₂PO₄ (pH 7.4), 140 mM NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 5 mM DTT. Bound proteins were eluted with 100 mM Tris-HCl (pH 8.0), 120 mM NaCl, 10 mM DTT, and 20 mM glutathione, and then dialyzed in 1000 volumes of 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ (pH 7.4), 2.7 mM KCl, 140 mM NaCl, and 1 mM -mercaptoethanol.

Antisera and Coimmunoprecipitation Experiments—Purity of the histidine-tagged adenylate kinase was assured by SDS-polyacrylamide gel electrophoresis. Approximately 500 µg of purified enzyme, 1 mg/ml, was mixed with TiterMax gold adjuvant (Sigma) in a 1:1 ratio, and the solution was injected subcutaneously into two New Zealand White rabbits from which preimmune sera had been collected before injection. After 1 month, the rabbits were boosted intramuscularly with 250 µg of His-tagged ADK mixed 1:1 with TiterMax gold adjuvant. Subsequent boosts were done similarly. After antisera had been collected, their quality was tested by immunoblotting for endogenous ADK in a bacterial extract, and purified His-ADK was separated by 12% SDS-PAGE. To purify E. coli ADK antibodies, 3 mg of purified His-ADK was immobilized on Affi-Gel 10 (Bio-Rad) as described previously (21, 22). Antibodies in the antiserum were precipitated by adding ammonium sulfate to 40% saturation, and the precipitate was dissolved in 10 mM Na₂HPO₄ (pH 6.8), 500 mM NaCl, and dialyzed overnight at 4 °C against 1000 volumes of the same buffer. The dialyzed solution was applied to the column containing immobilized His-tagged ADK. Unbound proteins were washed off with the same buffer. Bound proteins were then eluted with 100 mM glycine (pH 2.5), followed by elution with 100 mM triethylamine (pH 11.5). These collections were immediately neutralized with 0.1 volume of 1.5 M Tris-HCl (pH 8.0), followed by dialysis against 1000 volumes of phosphate-buffered saline. Antibodies in the preimmune serum were purified similarly, on a column containing, not His-ADK, but Protein A-Sepharose CL-4B (Sigma).

Coimmunoprecipitation experiments were carried out by procedures similar to those described previously (20). Thirty µl of purified antibodies to E. coli ADK or preimmune IgG (0.4 mg/ml) were incubated for 2 h on ice with 20 µl of radiolabeled cell extract in 500 µl of Nonidet P-40 buffer (0.1 M potassium glutamate (pH 7.5), 200 mM NaCl, 1 mM -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P-40). Each mixture was treated with Protein A-Sepharose beads, and the beads were washed and centrifuged as described (20). Each pellet was suspended in 50 µl of 2x SDS sample buffer, and the suspension was placed in a boiling water bath for 5 min, then subjected to one-dimensional SDS-PAGE. Alternatively, pellets were suspended in nonequilibrium pH gradient buffer at room temperature prior to analysis by two-dimensional gel electrophoresis (20).

Immobilized Protein Affinity Chromatography—Adenylate kinase (10 mg) and bovine serum albumin (12 mg) were immobilized on Affi-Gel 10 as previously described (21, 22). A [³⁵S]methionine-labeled extract of early T4 proteins (labeled from 3 to 8 min after infection at 37 °C) was applied to the ADK and BSA control columns, and elution was carried out with step increases of NaCl as described previously (21, 22). Proteins in each eluted fraction were subjected to two-dimensional gel electrophoresis and autoradiography by a modification (20) of our previous technique (21, 22). Protein spots on the two-dimensional gels were identified as described in our previous studies (21, 22).

Optical Biosensor Measurements—Direct measurements of protein-protein associations, involving the resonant mirror technique, used an IAsys optical biosensor (Affinity Sensors, Cambridge, UK), as described in previous publications (11, 20). Protein was immobilized in a carboxylate cuvette via 1-ethyl-3(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide, as specified by the instrument manufacturer. The cuvette containing immobilized protein was equilibrated with running buffer (0.15 M potassium glutamate, 4 mM magnesium chloride, 20 mM Tris-HCl, pH 7.4) until a buffer baseline was established. The test protein was added to the specified concentration, and association was monitored as shown. Real-time refractive index data (in arc seconds) were recorded as specified by the manufacturer, for further analysis. After each experiment, immobilized protein in the cuvette was regenerated as specified by the manufacturer.

View larger version (17K): [in this window] [in a new window]   FIG. 1. DNA synthesis rates, as measured by incorporation of [methyl-3H]thymidine in T4-infected E. coli cells. In the presence of 100 µM thymidine, T4 DNA synthesis was measured in the ndk wild-type, KA796 (), and the ndk null mutant, NR11814 (), infected with T4D at 30 °C.

DNA synthesis was measured also in infected bacteria permeabilized by sucrose plasmolysis. Bacteria were grown, infected, subjected to plasmolysis, and sampled for measurement of DNA synthesis as previously described (24, 25). Each reaction mixture contained in 0.5 ml of 1 x 10¹⁰ infected bacteria, 32 mM Tris-HCl (pH 8.4), 64 mM KCl, 12 mM MgCl₂, and 1 mM DTT. In one set of experiments (dNMP-dependent system), each incubation mixture contained 83 µM each of dAMP, dTMP, dCMP, and dGMP, 0.83 mM ATP, 0.4 mM ADP, and 100 µM [2,8-³H]deoxyadenosine at 0.09 mCi/µmol. In another series of experiments (dNDP-dependent system), each incubation mixture contained 83 µM each of dADP, dTDP, dCDP, and dGDP, plus ADP at 0.4 mM, ATP at 0.83 mM, and 42 µM each of CTP, UTP, and GTP, plus 100 µM [8-³H]deoxyguanosine at 0.03 mCi/µmol.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Effect of ndk Mutation on Rate of T4 DNA Synthesis—Although adenylate kinase complements an ndk mutation for growth of E. coli (4) and T4 can productively infect an ndk mutant host strain (26), the quantitative effect of an ndk mutation on T4 reproduction has not been reported. Therefore, we determined rates of radiolabeled thymidine incorporation into DNA after T4 infection at 30 °C of an isogenic pair of hosts, under conditions where thymidine incorporation gives a quantitative measure of the rate of DNA synthesis. As seen in Fig. 1, the rate of DNA synthesis was reduced by about 60%, relative to wild-type, in infection of an ndk mutant host. The yield of infectious phage was reduced proportionately, from 74 plaque-forming units per infected cell at 40 min after wild-type infection, to 36 for the mutant. Thus, complementation of the ndk function was not complete. Similar findings have been made in our laboratory in infection of a different pair of host strains (27).

Although complementation by adk was incomplete, its apparent efficiency was surprising. Lu and Inouye (4) reported the NDP kinase specific activity of adenylate kinase to be only 1–5% that of NDP kinase itself, depending upon the specific substrates used. This observation, to which we return to under "Discussion," raises the question whether adenylate kinase is specifically associated with the dNTP synthetase complex.

Affinity Chromatography with Adenylate Kinase as the Ligand—Each of the known enzymes in the dNTP synthetase complex, when immobilized on Affi-Gel^TM, has been shown to retain a specific ensemble of 6–12 early T4 proteins of dNTP synthesis and DNA metabolism (21, 22). If adenylate kinase is a specific component of the complex, we expect it to behave similarly. Accordingly, an extract of T4 proteins, labeled with [³⁵S]methionine from 3 to 8 min after infection, was applied to an ADK column, and the moderately strongly bound proteins (retained at 0.2 M NaCl, eluted at 0.6 M NaCl) were displayed by two-dimensional gel electrophoresis and autoradiography. Results of this experiment are shown in Fig. 2, panel A. Panel B shows a control experiment carried out with immobilized bovine serum albumin. Proteins retained by the ADK column, but not by BSA, are assumed to be specifically associated, either directly or indirectly, with adenylate kinase. Prominent among these were gpfrd (dihydrofolate reductase), gp42 (dCMP hydroxymethylase), and several proteins of DNA replication and recombination, gp32 (single-strand DNA-binding protein), gpu-vsY (recombination and recombination-dependent DNA initiation), and gppseT (DNA 3'-phosphatase, 5'-kinase). An earlier analysis, using immobilized His-tagged ADK, showed the same proteins bound (data not shown), indicating that with this protein at least, the histidine tag does not interfere with protein-protein associations.

Coimmunoprecipitation—Associations involving adenylate kinase were also investigated by an immunoprecipitation experiment. Antiserum was generated to purified His-tagged adenylate kinase. Fig. 3, panel A, shows that this antiserum strongly and specifically recognizes adenylate kinase in an extract of total T4 proteins. Purified antibodies from both ADK antiserum and preimmune serum were used to precipitate proteins from a [³⁵S]methionine-labeled extract of T4-infected bacteria. Two-dimensional gel electrophoresis and autoradiography (Fig. 3, panel B) showed several phage proteins precipitated by anti-ADK, but not by preimmune serum, the most prominent of which we identified as dihydrofolate reductase. Smaller amounts of proteins identified as thymidine kinase, gpuvsX (T4 RecA counterpart), and gp60 (topoisomerase subunit) were also seen. An immunoblotting experiment (bottom of panel B) showed that the ADK antibodies specifically precipitated nucleoside diphosphate kinase from this extract, suggesting that the two bacterial enzymes, NDP kinase and adenylate kinase, interact with each other.

Direct Protein-Protein Interactions, as Shown by GST Pulldown—Because the analyses involving affinity chromatography and immunoprecipitation cannot distinguish between direct protein-protein interactions and indirect associations mediated by a common ligand, it was of interest to determine whether E. coli adenylate kinase can interact directly with T4 enzymes in the dNTP synthetase complex. GST pulldown experiments can generate this kind of information provided one analyzes either purified proteins or extracts when only limited numbers of phage proteins are present. The latter condition is readily met by using extracts of E. coli in which only one T4 enzyme is expressed, namely, as a recombinant protein.

View larger version (59K): [in this window] [in a new window]   FIG. 2. Analysis of radiolabeled T4 proteins in E. coli infected with T4D bound to immobilized adenylate kinase (A) or bovine serum albumin (B). Columns of both immobilized proteins were prepared as described under "Experimental Procedures," and equivalent amounts of radiolabeled T4 protein extract were applied to each column. The proteins bound to each column after washing with 50 mM NaCl-containing buffer were eluted by successive increases of NaCl concentration (0.2, 0.6, and 2 M NaCl). Proteins shown here, which were bound to the column at 0.2 M NaCl and eluted at 0.6 M NaCl, were analyzed by two-dimensional gel electrophoresis, and the gel was exposed in a PhosphorImager. The nonspecifically bound protein, ipIII, serves as an internal control; similar intensities of this spot in experimental and control gels shows that equal amounts of total protein were analyzed on each column.

View larger version (60K): [in this window] [in a new window]   FIG. 3. Analysis of T4 proteins coimmunoprecipitated by ADK antibodies in E. coli infected with T4D. A, Western blotting for endogenous ADK in E. coli cell lysate (left lane) and purified His-tagged ADK (right lane), using E. coli ADK antiserum generated from a rabbit as described under "Experimental Procedures." B, autoradiogram of one-dimensional gel electrophoresis for T4 proteins coimmunoprecipitated with E. coli ADK antibodies. T4 proteins were radiolabeled with [35S]methionine from 3 to 8 min after infection of E. coli with T4D. Coimmunoprecipitation of T4 proteins was performed in the reaction mixture with purified E. coli ADK antibodies from the immunized antiserum (A) or the purified immunoglobulin G (IgG) from preimmune serum (P). Coimmunoprecipitates (IP) were separated by 12.5% SDS-PAGE and visualized by exposure in a PhosphorImager. Analysis of total proteins in each extract is shown in the two left-hand lanes. E. coli NDP kinase in coimmunoprecipitates was identified by immunoblotting with E. coli NDP kinase antibodies, following 12.5% SDS-PAGE (panel B, bottom). C, autoradiogram of two-dimensional gel electrophoresis of T4 proteins coimmunoprecipitated with E. coli ADK antibodies, the same extract as that shown in panel B. The proteins identified as ipIII and 61 appear to be the two major proteins precipitated by the preimmune serum (panel B). Protein spots not marked have not been identified.

View larger version (62K): [in this window] [in a new window]   FIG. 4. Interactions involving T4 dNMP kinase, as shown by analysis of GST pulldowns. A, three E. coli strains, Origami(DE3) (lane 1), Origami(DE3)/pGEX (lane 2), and Origami(DE3)/pGEX-gp1 (lane 3), were cultured in superbroth at 37 °C, and expression of GST-gp1 was induced with 1 mM IPTG. Cell lysates were prepared and were mixed with a cell lysate of E. coli strain Origami(DE3)/pnrdAB, which expresses T4 aerobic ribonucleotide reductase (RNR, gpnrdAB). GST pulldown was carried out with pre-equilibrated glutathione-Sepharose 4B, and proteins pulled down were separated in 12% SDS-PAGE, followed by staining with Coomassie Blue (top panel) or by immunoblotting with antisera to the respective enzymes. B, an identical analysis, except that the pulldown involved an extract of E. coli Origami(DE3), which expressed no phage proteins. Top panels in both A and B represent Coomassie Blue-stained 12% SDS-PAGE gels. All of the other gels involved immunoblotting with antisera to the respective enzymes. Total protein in each reaction (Input) is shown below each pulldown reaction. This experiment used the same rNDP reductase R1 and R2 proteins, and show the same input panels, as those used in a contemporaneous experiment that was previously reported (11).

View larger version (63K): [in this window] [in a new window]   FIG. 5. Analysis of GST pulldown of E. coli ADK and NDP kinase using purified GST fusion proteins. A, interactions involving adenylate kinase. Each partially purified GST fusion protein, GST-NDPK, GST-gp42, GST-gp1, or GST-gpfrd, at 10 µM was mixed with an amount of E. coli ADK designated, respectively, as – (0 nM), + (16 nM), ++ (80 nM), or +++ (400 nM), in 50 µl of reaction buffer containing pre-equilibrated glutathione-Sepharose 4B. The pulldown products were resolved by SDS-PAGE and analyzed by Coomassie Blue staining for the respective GST fusion protein (above, in each pair of panels) and by immunostaining with ADK antibody (below, in each pair of panels) for the ADK pulled down by the respective fusion protein. Total ADK input (top panel) was analyzed by immunostaining. B, interactions involving NDP kinase. This experiment was carried out essentially as described for the ADK analysis in A, except for the concentrations of input NDP kinase: – (0), + (0.5 µM), ++ (2 µM), +++ (10 µM).

Fig. 5, panel B, shows a comparable experiment in which purified NDP kinase in varying amounts was incubated with GST fusion proteins. At the highest NDP kinase concentration used (+++, corresponding to 10 µM), the results are unreliable, because GST itself, with no fusion partner, pulled down NDP kinase. However, the results of experiments at the two lower NDP kinase concentrations are reliable, and they confirm our finding (Fig. 4B) that NDP kinase does not interact directly with dNMP kinase (gp1). Also this experiment supports our previously reported finding (10) of direct interactions between E. coli NDP kinase and T4 dihydrofolate reductase, and in addition it shows that NDP kinase does not interact with dCMP hydroxymethylase (gp42).

Fig. 6 depicts another pulldown experiment with purified proteins, this one carried out to assess the effects of nucleotides upon binding affinity among the interacting pairs we are exploring, because we have found certain nucleotides, principally ATP, to have strong effects upon protein-protein interactions involving NDP kinase (11) and ribonucleotide reductase (20). In the experiment of Fig. 6A, purified NDP kinase was incubated with purified GST fusion proteins involving dCMP hydroxymethylase (gp42) and dihydrofolate reductase (gpfrd). NDP kinase was seen to interact with both dCMP hydroxymethylase, as we have reported previously (11), and dihydrofolate reductase. What was striking in this experiment was the pronounced and specific effect of dADP, among the nucleotides tested, upon the affinity of NDP kinase for dihydrofolate reductase, and to a lesser extent, for dCMP hydroxymethylase. The direct interaction between NDP kinase and dCMP hydroxymethylase was almost completely dependent upon substrate nucleotides for NDP kinase (dADP in this experiment). Panel B shows a similar experiment involving purified adenylate kinase and several purified GST fusion proteins. This experiment suggests a much smaller effect of the nucleotides tested on these interactions, although dADP showed a small effect in stimulating the binding of ADK to dNMP kinase (gp1).

View larger version (37K): [in this window] [in a new window]   FIG. 6. Effects of nucleotides on the interactions of E. coli NDP kinase or adenylate kinase with proteins. A, interactions involving NDP kinase. 2 µM E. coli NDP kinase was mixed with the same amount of each GST fusion protein, GST-gp42 or GST-gpfrd, in reaction buffer containing no nucleotide (lane 1) or 1 mM designated nucleotide (lane 2, dTMP; lane 3, dADP; lane 4, ATP; lane 5, AMP-PNP), 2 mM MgCl2, and 5 µl of pre-equilibrated glutathione beads. GST pulldown was carried out, and NDP kinase pulled down was identified by immunoblotting with antiserum to E. coli NDP kinase. B, interactions involving adenylate kinase. 80 nM E. coli ADK was mixed with the same amount of each GST fusion protein, GST-NDPK, GST-gp1, or GST-gp42, in the reaction buffer containing 1 mM designated nucleotide (lane 1, no nucleotide; lane 2, dAMP; lane 3, dTMP; lane 4, dADP; lane 5, ATP; lane 6, AMP-PNP), 2 mM MgCl2, and 5 µl of pre-equilibrated glutathione-Sepharose 4B. Each pulldown product was analyzed by immunoblotting with adenylate kinase antiserum.

View larger version (16K): [in this window] [in a new window]   FIG. 7. Enhancement by ATP of the association of T4 dCMP deaminase with immobilized E. coli adenylate kinase. To ADK (2.5 ng) immobilized in a carboxylate IAsys cuvette was added 311 nM T4 dCMP deaminase (CD) at 0 min, followed 6 min later by addition of 1 mM ATP (final concentration). The graph was generated by using FASTplot (Affinity Sensors, Cambridge, UK).

DNA Synthesis in Permeabilized Infected Bacteria—The experiment of Fig. 1 shows the effect upon T4 DNA synthesis of knocking out the expression of the host cell ndk gene. It would be desirable to explore the function of adenylate kinase by carrying out a similar knockdown of adk gene expression. However, even though adk expression can be ablated with a temperature-sensitive adk mutation, the results would be difficult to interpret because of the known functions of adenylate kinase in adenine ribonucleotide metabolism. Accordingly, we designed experiments using bacteria that had been permeabilized by sucrose plasmolysis (24), so that we could bypass these latter functions by providing ribonucleotides, including both ADP and ATP. In the experiment of Fig. 9, panel A, we asked whether adenylate kinase plays an essential role in T4 DNA synthesis when the precursors are supplied as deoxyribonucleoside monophosphates. E. coli CV2, which contains a ts adk mutation, was infected, and the infected cells were permeabilized and incubated with the indicated nucleotides at either 25 or 42 °C. Incorporation of radiolabeled deoxyadenosine was inhibited at 42 °C, confirming the necessity of adenylate kinase for converting dAMP to dADP under these conditions. Comparable inhibition was not seen when bacteria with a wild-type adk gene were treated similarly. Panel B shows a similar experiment, in which DNA synthesis was made dependent upon added deoxyribonucleoside diphosphates. Under these conditions, DNA synthesis was not dependent upon a functional ADK protein. Both in the ts adk mutant and the wild-type host, incorporation of radiolabeled deoxyguanosine into DNA was actually stimulated at 42 °C relative to what was seen at 25 °C. These experiments confirm the role of adenylate kinase in converting dAMP to dADP, but they suggest that the enzyme does not play an additional essential function, so long as nucleoside diphosphate kinase is available and active.

View larger version (9K): [in this window] [in a new window]   FIG. 8. Quantitative analysis of protein-protein interactions. A, binding of adenylate kinase in solution to 7.2 ng of immobilized E. coli nucleoside diphosphate kinase. Binding reactions were followed in 0.15 M potassium glutamate, 4 mM magnesium acetate, 20 mM Tris-HCl (pH 7.4). B, binding of T4 thymidylate synthase (TS) in solution to 2.5 ng of immobilized adenylate kinase. For this experiment, binding reactions were followed in 10 mM phosphate-buffered saline (pH 7.4), 0.05% Tween 20. For both experiments, initial rate constants for binding (kon) were plotted against the concentration of the protein in solution.

View larger version (16K): [in this window] [in a new window]   FIG. 9. Measurement of T4 DNA synthesis in situ. A, sucrose-plasmolyzed E. coli cells, adk wild-type (KA796), and thermosensitive adk mutant (CV2), infected with T4D, were prepared as described under "Experimental Procedures." The incorporation rate of radiolabeled deoxyadenosine into T4 DNA was measured in the sucrose-plasmolyzed cells at 25 or 42 °C with dNMPs and other nucleotides provided as described previously. B, the incorporation rate of radiolabeled deoxyguanosine into T4 DNA was measured in the sucroseplasmolyzed cells at 25 or 42 °C with dNDPs and other nucleotides provided as described previously. , T4-infected adk wild-type (KA796) at 25 °C; , T4-infected adk mutant (CV2) at 25 °C; •, T4-infected adk wild-type (KA796) at 42 °C; , T4-infected adk mutant (CV2) at 42 °C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The conversion of deoxyribonucleoside diphosphates to triphosphates is a transition point, between small-molecule metabolism and DNA replication. Nucleoside diphosphate kinase appears to be positioned to link these two processes; for example, we have demonstrated direct associations of E. coli NDP kinase with both T4 DNA polymerase and T4 gp32, the single-strand DNA-binding protein (5). What about adenylate kinase? It seems improbable that a process as central as linking dNTP synthesis to dNTP polymerization would be designed to function with interchangeable parts. However, our data indicate that adenylate kinase is a specific component of the T4 dNTP synthetase complex, just as is NDP kinase, and adenylate kinase is definitely capable of synthesizing dNTPs from dNDPs, at least, in the absence of NDP kinase. The GST pulldown experiments show direct contacts between adenylate kinase and T4 dNMP kinase (gene 1), dCMP hydroxymethylase (gene 42), and dihydrofolate reductase (gene frd), as well as E. coli NDP kinase. The immunoprecipitation and affinity chromatography experiments show associations, either direct or indirect, with T4 dihydrofolate reductase and thymidine kinase. The biosensor experiments show associations with T4 thymidylate synthase and dCMP deaminase. Thus, we conclude that adenylate kinase is a specific, possibly integral, component of the complex, held in place by multiple protein-protein interactions. Fig. 10 is a schematic diagram, showing all of those direct protein-protein interactions identified in our work to date. Note that a significant number of those interactions do not involve enzymes that act sequentially. However, all of the enzymes are functionally related, and the interactions with the gene 32 single-strand DNA-binding protein, as we have discussed elsewhere (20), suggest a functional relationship with the replisome as well.

Our finding that the two known host-cell enzymes in the complex, NDP kinase and adenylate kinase, interact with each other may be significant with respect to the physiology of the uninfected bacterium, because both enzymes participate in the redistribution of high-energy phosphate that arises initially as ATP. More to the point of this paper, the interaction may be significant to the operation of a "replication hyperstructure" in uninfected E. coli (29, 30), a supramolecular complex linking dNTP synthesis and DNA replication, comparable with what we see in the T4 system.

View larger version (33K): [in this window] [in a new window]   FIG. 10. Protein-protein interactions in the T4 dNTP synthetase complex. In this schematic diagram, adapted from Ref. 20, each solid line depicts a direct interaction shown either in this article or in previous papers from this laboratory (5, 10, 11, 20, and 22). Interactions involving adenylate kinase, which have not previously been reported, are depicted with double-headed arrows. 1, gene 32 single-stranded DNA-binding protein; 2, E. coli NDP kinase; 3, aerobic ribonucleotide reductase; 4, thymidylate synthase; 5, dihydrofolate reductase; 6, dCMP hydroxymethylase; 7, dCMP deaminase; 8, dNMP kinase; 9, dCTPase/dUTPase; 10, E. coli adenylate kinase.

Under these "unnatural" conditions, most of the dNTPs used by the replisome would not have been synthesized in situ at replication sites, and the replisome would be drawing from non-compartmentalized dNTP pools, distributed throughout the cell. Even though all of the ADK molecules might be contributing to dNTP synthesis under these conditions, its low catalytic efficiency as an NDP kinase would limit the total rate of DNA synthesis.

The other issue pertains to the effects of nucleotides. Numerous observations in our laboratory (11, 22, 31) point to the effects of nucleotides, often, but not always, ATP, in stabilizing associations within the dNTP synthetase complex. The associations involving NDP kinase may be particularly significant (Ref. 11 and Fig. 6A). Bacterial NDP kinases are tetrameric proteins. Using nondenaturing gel electrophoresis, we have found that ATP promotes tetramerization of the enzyme in vitro,² although we do not yet know whether this effect stimulates other activities of the complex. Comparable observations on promotion of oligomerization of NDP kinase have been made with the hexameric Dictyostelium enzyme (32), where ADP has been found to stabilize the enzyme, evidently by stimulating subunit interactions within the enzyme. More recently, Shen et al. (33) have described effects of nucleotides upon interactions between Arabidopsis phytochrome and one of the plant isoforms of NDP kinase. dCDP was found to stimulate the binding of hexameric NDPK2 to the Pfr form of phytochrome, and phytochrome was found to stimulate binding of substrate nucleoside diphosphates by NDPK2, evidently by accelerating formation of the phosphorylated enzyme intermediate. All of these observations with other NDP kinases, plus our own findings in the T4/E. coli system suggest that the effects of nucleotides upon the dNTP synthetase complex may involve oligomerization of NDP kinase, as well as its interaction with other enzymes. Some of the results shown in Fig. 6 show elements of specificity that suggest physiological significance to the effects of nucleotides. The pronounced stimulation by dADP of the binding of NDP kinase to both dCMP hydroxymethylase and dihydrofolate reductase is particularly intriguing. Also of interest is the significant stimulatory effect of the nonhydrolyzable ATP analog, AMP-PNP, whereas ATP has no such effect. This result may indicate that the phosphorylation state of the enzyme influences both its homotypic and heterotypic interactions in a way that might affect activities of the complex. In any event, these preliminary observations raise the intriguing possibility that intracellular flux rates for dNTP synthesis are controlled in part by the composition of the small-molecule pool. The pronounced effects of nucleotides, shown both here and in our previous articles (11, 22, 31), suggest that we can use this information to stabilize the dNTP synthetase complex in vitro, perhaps allowing systematic analysis of the effects of small molecules upon activities and substrate specificity of the complex.

	FOOTNOTES

 
^* This work was supported by National Science Foundation Research Grant MCB 01 03760 and NIEHS National Institutes of Health Grant ES 00210. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 541-737-1865; Fax: 541-737-0481; E-mail mathewsc{at}onid.orst.edu.

¹ The abbreviations used are: NDP, nucleoside diphosphate; IPTG, isopropyl--D-thiogalactoside; DTT, dithiothreitol; ADK, adenylate kinase; BSA, bovine serum albumin; GST, glutathione S-transferase; dNMP, dNDP, and dNTP, deoxyribonucleoside mono-, di-, and triphosphate, respectively; AMP-PNP, 5'-adenylyl-,-imidodiphosphate.

² J.-H. Kim, unpublished observations.

	ACKNOWLEDGMENTS

 
We thank Linda Wheeler for outstanding technical assistance.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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