Deoxynucleotide Kinase Research Articles

Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP.

NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP. The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement. The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop. The second domain, consisting of five alpha-helices, forms the NMP binding pocket. A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding. Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination.

Crystallization and preliminary X-ray analysis of bacteriophage T4 deoxynucleotide kinase.

T4 deoxynucleotide kinase catalyzes the phosphorylation of 5-hydroxymethyldeoxycytidylate, dTMP and dGMP while excluding dCMP and dAMP. In order to understand the mechanism of this remarkable specificity, the enzyme was over-expressed in Escherichia coli, purified and crystallized for X-ray diffraction analysis. The crystals belong to the monoclinic space group C2 with cell dimensions a = 155.2, b = 58.5, c = 75.7 A, beta = 108.1 degrees. There are two protein monomers in the asymmetric unit related by a twofold axis. Diffraction data to 2.0 A resolution have been collected.

Chemical modification of bacteriophage T4 deoxynucleotide kinase. Evidence of a single catalytic region.

The mechanism underlying the unusual specificity of bacteriophage T4 deoxynucleotide kinase, which catalyzes the phosphorylation of 5-hydroxymethyldeoxycytidylate, dTMP, and dGMP, has been investigated by chemical modification of the protein. Pyridoxal 5'-phosphate inactivates deoxynucleotide kinase by modifying a single lysine out of the 17 per monomer. Lysine 10 has been tentatively identified as the site of modification, although the possibility of mutually exclusive reactive residues has not been eliminated. Diethylpyrocarbonate also inactivates the enzyme, suggesting that histidine plays a role in catalytic function. With either reagent, the three activities are lost at equal rates, supporting the contention that one active site is responsible for the exclusive phosphorylation of three dissimilar deoxynucleotides. These studies also identify two distant regions of the primary sequence that are likely to be closely associated in the active region of the folded protein.

Bacteriophage T4 deoxynucleotide kinase: gene cloning and enzyme purification

Gene 1 of bacteriophage T4 has been cloned into a lambda pL expression vector, resulting in the overproduction of deoxynucleotide kinase. A procedure that includes affinity chromatography on Cibacron Blue F3GA-agarose has been used to purify milligram quantities of enzymes from a 2-liter culture. The enzyme has been partially characterized in vitro and in vivo, and it appears to be identical to the deoxynucleotide kinase isolated from T4-infected Escherichia coli. These results prove the earlier contention that the phosphorylation of three dissimilar deoxynucleotides (5-hydroxymethyldeoxycytidylate, dTMP, and dGMP), to the exclusion of most others, is catalyzed by a single protein.

Antineoplastic Agents III: Effects of Dibromoethyl and Vinyl Esters of N-Benzyloxycarbonyl-l-phenylalanine on Ehrlich Ascites Tumor Cell Metabolism

Evidence is presented that N-benzyloxycarbonyl-l-phenylalanine vinyl ester and 1,2-dibromoethyl ester are inhibitors of Walker 256 carcinosarcoma and Ehrlich ascites carcinoma tumor growth. The major effects of these two agents on Ehrlich ascites cell metabolism were the inhibition of deoxyribonucleic acid and protein synthesis and the alteration of cellular regulatory processes controlling cytokinetics. Deoxynucleotide (purine) kinase enzymes appeared to be the focal site for inhibition of deoxyribonucleic acid synthesis with marginal inhibition of thymidylate synthetase activity. Cyclic adenosine monophosphate levels were elevated by drug treatment whereas chromatin protein phosphorylation, cell respiration, and lysosomal activities were inhibited. N-Benzyloxycarbonyl-l-phenylalanine 1,2-dibromoethyl ester was a latent in vitro chymotrypsin inhibitor. Some preliminary evidence suggests that these activated esters may inhibit cellular enzymatic activity by alkylating imidazole and lysine residues of proteins.

Identification of the transcribing DNA strand for the deoxynucleotide kinase gene of bacteriophage T2

A modified procedure was developed which allows RNA--DNA hybridization reactions to be performed without the loss in translational capacity of mRNA which accompanies hybridization at elevated temperatures or in the presence of the denaturing agent formamide. Separated l and r strands of bacteriophage T2 DNA were hybridized in the presence of 4 M sodium perchlorate at 37 degrees C with total RNA from infected cells. After passage of the hybridization mixture through a nitrocellulose column to remove single-strand DNA and DNA--RNA hybrids, the eluent was measured for its capacity to promote deoxynucleotide kinase (gene 1) synthesis in an in vitro protein-synthesizing system derived from uninfected Escherichia coli. With this procedure, which should be of general use for any message whose product can be measured either enzymatically, immunologically, or by location in an acrylamide gel, it was demonstrated that deoxynucleotide kinase mRNA is transcribed from the l strand of bacteriophage T2 DNA. By titrating with l strand DNA, the number of deoxynucleotide kinase transcripts present 9 min after T2 phage infection at 30 degrees C was estimated to be about 38 copies per cell.

Regulation of ribosome function following bacteriophage T4 infection

The rate of translation in bacteriophage T4-infected Escherichia coli has been studied. It was observed that at about ten minutes after infection at 37 °C the rate of protein synthesis declines to 40 to 50% of the rate observed during the first ten minutes, yet all cells remain intact for at least 60 minutes. This drop in the rate of general protein synthesis is correlated with a change in the ability of initiation factor-free ribosomes to translate both global T4 messenger RNAs and a specific T4 messenger, deoxynucleotide kinase (EC 2.7.4.4) mRNA. The alteration in ribosome function begins between five and ten minutes after infection and minimum ribosome activity is reached at approximately 20 minutes after infection. A late T4 gene is involved, as shown by the fact that the alteration in ribosome function is not observed in amB1292-infected cells (i.e. cells which synthesize early but not late T4 mRNAs).

The role of ribosomes in stabilizing bacteriophage T4 deoxynucleotide kinase mRNA [formula omitted

In the present investigation, an approach toward defining the role of ribosomes in stabilizing functional messenger RNA in cell-free extracts is described. The data presented show that initiation of protein synthesis is necessary for maximal functional stability of bacteriophage T4 deoxynucleotide kinase mRNA in vitro and suggest that much of the stability is attained by interaction of the deoxynucleotide kinase mRNA initiation site with a 30S ribosomal subunit. Data is also presented which suggest that any of several E . coli ribonucleases could serve as a messenger ribonuclease in vivo .

Role of potassium in the synthesis and decay of two specific bacteriophage T4 messages

The role of K+ in the in vivo metabolism of specific phage T4 messengers was studied. By using a mutant of Escherichia coli defective in its ability to accumulate K+ from the growth medium, it was possible to rapidly deplete cells of their intracellular K+ and in this way determine K+-dependent reactions in vivo. The rate constants for accumulation, synthesis, and decay of the early enzymes deoxynucleotide kinase and alpha-glucosyl transferase were determined. It was shown that there is a very close association between mRNA synthesis and its decay, indicating that a mechanism may be present in the cell that can regulate the concentration of these RNAs. Since the mRNA's for these enzymes are very stable in cells depleted of K+, K+ depletion may be a useful method for the isolation of functional T4 mRNA.

Use of antibiotics to determine ribosome-messenger RNA interactions necessary for in vivo stability of specific messengers

With the use of inhibitors of individual reactions in protein synthesis, a method has been developed for (a) determining the role of ribosome messenger RNA interactions in specific bacteriophage T4 mRNA stability and (b) localizing the primary site of interaction of messenger ribonuclease (mRNase) on messengers. Antibiotics that freeze ribosomes in or near the initiation site stabilize T4 deoxynucleotide kinase mRNA. In contrast, T4 α-glucosyltransferase mRNA is stable only when the polysome configuration is kept intact. These results indicate the deoxynucleotide kinase mRNA initiation site is most susceptible to mRNase action, whereas the primary site of mRNase action on the α-glucosyltransferase mRNA is distal to the initiation site. Determining the role of ribosome-mRNA interactions in mRNA stability by the use of specific inhibitors of protein synthesis may be applicable to any procaryotic or eucaryotic mRNA that can be translated in vitro.

Initiation of synthesis of messenger RNA of deoxynucleotide kinase by oligoribonucleotides.

The effects of nucleoside triphosphates and oligoribonucleotides on the initiation of synthesis of messenger RNA of the T4 phage-specific enzyme, deoxynucleotide kinase, have been studied. The procedure involved incubation of T4 DNA, purified RNA polymerase from Escherichia coli, and selected nucleotide compounds during a brief period to permit initiation of RNA synthesis. Further initiation was arrested by the addition of ribampicin, and completion of the transcription of the newly initiated RNA was permitted to take place in the presence of the full complement of nucleoside triphosphates. After translation of the messenger RNA into phage-specific enzymes, the measured activities of the latter whe first incubation period. The effectiveness of individual nucleoside triphosphates, when present singly or in combination during the initiation period, was compared to that when all four nucleoside triphosphates were available. ATP alone was extremely effective as an initiator of the synthesis of the messenger RNA for deoxynucleotide kinase. The addition of UTP to ATP not only enhanced the magnitude of initiation but also affected the kinetics of ATP interaction with T4 DNA and RNA polymerase during the initiation period. Several oligoribonucleotides including a series ApA to ApApApA, UpU to UpUpUpU, and the heteropolymers, Ap1pU and ApApApU, were tested as initiators of kinase mRNA synthesis. A sequence of nucleotides in the promoter region of T4 DNA for the deoxynucleotide kinase gene has been proposed as a result of these experiments.

Evidence for a diffusible T4 bacteriophage protein governing the initiation of delayed early RNA synthesis

Two forms of prereplicative phage RNA can be discerned in Escherichia coli early after infection with bacteriophage T4, immediate early and delayed early RNA. The transition from immediate early to delayed early RNA synthesis is inhibited by chloramphenicol. The present work presents evidence for the existence of a phage-specific protein, which effects this transition. Delayed early RNA formation was measured by a cistron-specific enzyme-forming-capacity method, in which RNA synthesized in the presence of chloramphenicol was allowed to express itself into enzyme activity after (i) the addition of rifampin to inhibit further transcription and (ii) subsequent removal of chloramphenicol. As representatives of delayed early transcription, the two phage-specific enzymes dCTPase and deoxynucleotide kinase were chosen. Primary infection with a phage mutant defective in one of these two enzymes was found to induce a diffusible factor, which in the presence of chloramphenicol could effect the formation of delayed early RNA corresponding to the missing enzyme, upon superinfection with wild-type phage. The activity of this factor, acting in trans, was abolished by the amino acid analogue ethionine. Mutation in the suA gene of the host did not relieve phage of the apparent need for protein synthesis in the transition from immediate early to delayed early phage RNA synthesis.

The distribution of functional bacteriophage T4 mRNA on polysomes

Functional bacteriophage T4 deoxynucleotide kinase and α-glucosyl transferase mRNAs can be isolated from polysomes extracted from cells 8 min after infection. At least 55% of the 8-min deoxynucleotide kinase mRNA is associated with polysomes and is released from the cell membrane by deoxyribonuclease (DNase) treatment (soluble mRNA). Approximately 20% of the kinase mRNA remains tightly bound to membrane after DNase treatment (membrane mRNA) and 25% of the kinase mRNA is routinely lost during fractionation. The membrane-bound kinase mRNA is about three times as stable in vitro as the soluble kinase mRNA. Soluble kinase mRNA (14.5S) is found associated with as few as one ribosome and as many as 22 ribosomes; however, 14.5S α-glucosyl transferase mRNA is found predominantly in six ribosome polysomes. The size of the α-glucosyl transferase mRNA is heterogenous, ranging between 14.5 and 20S. The larger α-glucosyl transferase mRNAs are never found on small polysomes but appear only in polysomes containing at least nine ribosomes (18S α-glucosyl transferase mRNA). Maximum-size α-glucosyl transferase mRNA (approximately 20S) appears on polysomes containing at least 14 ribosomes. The relationships between decay of T4 mRNA and polysome size and the location of ribosome loading sites on the 20S α-glucosyl transferase message are also discussed.

Transcription-translation and translation-messenger RNA decay coupling: separate mechanisms for different messengers.

Antibiotics were used to inhibit protein synthesis at specific steps in the biosynthetic pathway. In this way, it was possible to study the coupling of protein synthesis to the accumulation of biologically active mRNA in T4-infected Escherichia coli. Functional mRNA for the phage enzymes deoxynucleotide kinase (EC 2.7.4.4; ATP: nucleoside monophosphate phosphotransferase or nucleosidemonophosphate kinase) and alpha-glucosyltransferase (EC 2.4.1.5; 1, 4-alpha-D-glucan: 1, 6-alpha-D-glucan 6-alpha-glucosyltransferase or dextrin dextranase) accumulated during inhibition of protein synthesis irrespective of the step in the synthesis of protein that was blocked. Under these conditions, however, the rate of mRNA synthesis for both enzymes was significantly inhibited. In contrast, the rate of degradation of these mRNAs was markedly dependent on the step in protein synthesis that was inhibited. That is, the site for mRNase action was different for each message. The most important step in protein synthesis required for the stability of deoxynucleotide kinase mRNA is the initiation step. A single ribosome bound to the 5' end of the deoxynucleotide kinase mRNA can stabilize the molecule. On the other hand, the initiation event does not seem to be important for stabilizing the alpha-glucosyltransferase mRNA. Instead, a high ribosome denisty on the alpha-glucosyltransferase messenger is required to achieve significant stability. Therefore, in studying messenger metabolism, it is important to focus on the functional stability of specific mRNAs instead of on total messenger since each mRNA can be metabolized differently.

Level of specific prereplicative mRNA's during bacteriophage T4 regA-, 43- and T4 43- infection of Escherichia coli B

The role of the T4 bacteriophage regA gene in stabilizing early mRNA was investigated by assaying the level of functional mRNA from eight prereplicative genes (56 [dCMP hydroxymethylase], cd [dCMP deaminase], 1 [deoxynucleotide kinase], rIIA, rIIB, 46 [DNA arrest], and 45) during extended infection of Escherichia coli B with T4 regA-, 43- and T4 43- bacteriophage. The above gene-specific transcripts in RNA isolated from infected cells were quantitated by translation with an E. coli B cell-free system. Conditions were chosen to insure that the amount of gene product formed in vitro, measured either as an enzyme activity or as a radioactive band in acrylamide gel, was directly proportional to the level of mRNA present. The failure of T4 regA-, 43- phage to terminate prereplicative synthesis (Wiberg et al., 1973) resulted in an enhanced production of many early gene products over those formed during T4 43- infection. This increase did not appear to be associated with an increment in mRNA levels, since in the present study gene-specific early mRNA's were found to be only marginally elevated and slightly more stable in T4 regA-, 43-- than in T4 43--infected cells. Of interest was the observation that significant quantities of all of the mRNA's studied; with the exception of those from genes 45 and 46, could be isolated from T4 43--infected cells after synthesis of the respective gene products had ceased. On termination of normal prereplicative synthesis during infection with T4 43- phage, polyribosomes were found to be dissociated completely, a finding which suggests that the residual mRNA present in these cells is free in the cytoplasm. The persistence in T4 43--infected cells of translatable mRNA for many prereplicative genes after product synthesis ceased indicates that the impairment in protein synthesis is not due solely to regA-mediated messenger degradation or modification. Rather, the results suggest that the regA gene product may act either by interfering with early mRNA polypeptide chain initiation or by promoting prereplicative polysome dissociation.

Sizing of two bacteriophage T4 specific messenger ribonucleic acids formed [formula omitted] and [formula omitted

Messenger RNA for two T4 specific enzymes, deoxynucleotide kinase and α glucosyltransferase, have been sized by sedimentation on sucrose density gradients. The sedimentation constants of transferase and kinase mRNAs formed in vitro were 21.5S and 14.5S respectively, regardless of the duration of incubation up to 20 min. Although the kinase mRNA isolated from cells infected with T4 phage for 10 min was the same size as found in vitro (14.5S), the transferase mRNA was found in a segment approximating the size of the kinase mRNA (14.5S). The experiments indicate that α glucosyltransferase mRNA is formed first as a polycistronic message and is then processed to the smaller unit.

Synthesis of functional bacteriophage T4-delayed early mRNA in the absence of protein synthesis

When Escherichia coli B207 is grown either aerobically or under limited aerobic conditions, pretreated with chloramphenicol to block protein synthesis, and then infected with bacteriophage T4, the phage RNA which accumulates, termed "immediately early" (IE), contains the transcripts of a limited number of prereplicative genes. Among the transcripts which accumulate is the mRNA which serves as a template for deoxycytidylate hydroxymethylase (HMase) synthesis. Among the prereplicative gene transcripts which do not accumulate under these conditions are deoxycytidine triphosphatase (dCTPase), alpha-glucosyl transferase (alphg-gt), and deoxynucleotide kinase (kinase); these genes have been termed "delayed early" (DE). In contrast, when protein synthesis is inhibited by depleting aerobically grown E. coli B207 of K+, both IE and DE T4 RNA accumulate, but these transcripts do not contain functional HMase, dCTPase, alpha-gt, or kinase mRNA's. However, if E. coli is grown under conditions of limited aeration and then depleted of K+ prior to T4 infection, the T4 RNA which accumulates contains both IE and DE transcripts and functional HMase, dCTPase, and alpha-gt mRNA's. Functional kinase mRNA does not accumulate under these conditions. The results of these experiments indicate that the synthesis of functional DE RNA in the absence of simultaneous protein synthesis, depends on the physiological condition of the cells and the way in which protein synthesis is inhibited. In addition, data is presented which suggests that extensive transcription of DE genes in the absence of protein synthesis results in the inhibition of transcription of certain IE genes.

The influence of potassium and rifampicin on the expression of bacteriophage T2 prereplicative genes in vitro and in vivo

The transcription of specific mRNA from T2 phage DNA by purified Escherichia coli RNA polymerase was markedly affected by the concentration of KCl. Thus the genes for dihydrofolate reductase and deoxynucleotide kinase were transcribed at 50 m m KCl, but at 200 m m KCl the synthesis of the kinase mRNA was nearly eliminated and that of the reductase was increased slightly. Rifampicin-resistant initiation complexes formed at 200 m m KCl failed to produce appreciable kinase mRNA despite reduction of the KCl concentration to 50 m m following initiation of RNA chain elongation. This result suggests that the RNA polymerase holoenzyme does not bind to deoxynucleotide kinase promoters in the presence of 200 m m KCl in a manner capable of initiating synthesis of this message. Initiation complex formation at 50 m m KCl, however, permitted the synthesis of deoxynucleotide kinase mRNA at 200 m m KCl indicating that once formed the initiation complex is capable of producing message even at high salt. The KCl effect on gene expression was reproduced in a coupled E. coli cell-free system directed by T2 DNA. However, in this case the inhibition of protein synthesis at high concentrations of KCl was due primarily to the chloride ions and could be alleviated by substituting acetate for chloride. The synthesis of mRNA in the coupled system was similar with either anion. Analysis of proteins synthesized in the coupled system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, like deoxynucleotide kinase, the synthesis of several additional gene products was influenced by the concentration of KCl. The genes coding for these products, including deoxynucleotide kinase, belong to a class whose transcription is reduced or eliminated in vivo on addition of rifampicin at 1 min postinfection and are referred to as “quasilate.” Evidence is presented to indicate that the mRNAs for deoxynucleotide kinase, dihydrofolate reductase, and deoxycytidylate deaminase are produced in excess of the levels needed for optimal synthesis of these enzymes during normal infection.

Transcriptional regulation of T4 bacteriophage-specific enzymes synthesized in vitro.

In contrast to dihydrofolate reductase and four other phage-specific enzymes, the initiation of deoxynucleotide kinase is essentially prevented if rifampin is added to a culture of Escherichia coli B cells within 1.5 min after infection with T4. Deoxynucleotide kinase thus belongs to a group of so-called delayed-early enzymes that is not initiated from an immediate-early promoter site. We prepared crude extracts from infected cells in a manner designed to maintain the integrity of the complexes of native, endogenous T4 DNA with bacterial structural and enzymatic units concerned with RNA synthesis. The initiation of the synthesis of the mRNA for dihydrofolate reductase, an example of an immediate-early enzyme, and deoxynucleotide kinase, a special type of delayed-early enzyme, was studied with these extracts prepared from cells infected in the absence or presence of chloramphenicol. Initiation of transcription of the dihydrofolate reductase gene is immediate when programmed by extracts made either from cells treated with chloramphenicol prior to infection (CM extracts) or from cells 3 min into the normal infection cycle (3-min extracts). However, initiation of transcription of the deoxynucleotide kinase gene programmed by CM extracts is delayed 2 min relative to the immediate initiation of transcription of the deoxynucleotide kinase gene programmed by 3-min extracts. These experiments duplicated in vitro effects of the antibiotics on the synthesis of phage-specific mRNA previously noted only in vivo.

Post-transcriptional regulation of T4 enzyme synthesis

The in vivo accumulation of functional messenger RNA (mRNA) specific for the bacteriophage T4-induced enzymes, lysozyme and deoxynucleotide kinase, has been studied. For each enzyme, two distinct cycles of mRNA accumulation and degradation can be observed during the first 40 min of infection at 37 °C. Lysozyme mRNA activity peaks at 15 min after infection and again at 29 min, whereas peaks of deoxynucleotide kinase mRNA are observed at 8 min and 32 min after infection. These cyclic patterns are not reflected in the kinetics of synthesis of the enzymes in vivo. Deoxynucleotide kinase mRNA extracted at 8 min or at 30 min after infection sediments in linear sucrose gradients over a broad range with a major component at 15S and minor component at 18S. Lysozyme mRNA extracted at 15 min or at 30 min after infection similarly sediments as two bands with sedimentation coefficients of 15S and 18S. These results indicate that the regulation of these two enzymes in T4-infected E. coli is mediated via post-transcriptional control mechanisms.