JOURNAL OF BACTERIOLOGY, June 1990, p. 2935-2939 Vol. 172, No. 6 0021-9193/90/062935-05$02.00/0 Copyright © 1990, American Society for Microbiology Bacteriophage T4 Deoxynucleotide Kinase: Gene Cloning and Enzyme Purificationt GEORGE S. BRUSH, SATISH K. BHATNAGAR, AND MAURICE J. BESSMAN* McCollum-Pratt Institute and Department ofBiology, The Johns Hopkins University, Baltimore, Maryland 21218 Received 9 November 1989/Accepted 2 March 1990 Gene 1 of bacteriophage T4 has been cloned into a A 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. In Escherichia coli, Saccharomyces cerevisiae, and higher MATERIALS AND METHODS eucaryotes, four distinct and highly specific enzymes cata- lyze the phosphorylation of the canonical deoxynucleoside Chemicals and chromatographic media. All deoxynucle- monophosphates to the corresponding diphosphates accord- otides were purchased from P-L Biochemicals, Inc., and ing to the following equation: deoxynucleoside monophos- Sigma Chemical Co., except for dHMP, which was prepared phate + ATP = deoxynucleoside diphosphate + ADP. essentially as previously described (8, 9). ATP, phosphoe- However, after infection of E. coli with the T-even phages nolpyruvate, P-NADH, streptomycin sulfate, and ampicillin (T2, T4, and T6), enzymatic activities are induced (EC were also purchased from Sigma. The Geneclean kit for 2.7.4.12) that catalyze the phosphorylation of three deoxy- isolating DNA fragments from agarose gels was purchased nucleoside monophosphates (5-hydroxymethyldeoxycytidy- from Bio-101. Sepharose 6B was obtained from Pharmacia late [dHMP], dTMP, and dGMP); these three activities Fine Chemicals, and Affi-Gel Blue was from Bio-Rad Labo- appear to reside in one protein (2, 3, 8). The enzyme is ratories. specific for these three structurally dissimilar nucleotides (2, Enzymes. T4 DNA ligase and restriction endonucleases 8), and it would be of considerable interest to discover how BamHI, HaeII, HpaII, and SmaI were purchased from the protein recognizes these nucleotides to the exclusion of Bethesda Research Laboratories, Inc. Restriction endonu- most others. cleases AvaIl, BsmI, and MnlI were from New England Since only small quantities of the kinase can be extracted BioLabs, Inc. Calf intestinal alkaline phosphatase was from from large-scale cultures of phage-infected E. coli, the Boehringer Mannheim Biochemicals. Lactic dehydrogenase number and types of experiments that have been performed (type II) and pyruvate kinase (type II) were from Sigma. T4 to date have been limited. Sakiyama and Buchanan (21) DNA polymerase was from both Bethesda Research Labo- reported that the T4 protein exists as a homodimer with a ratories and Boehringer Mannheim. subunit molecular weight of 23,000. This value is comparable Bacteria, bacteriophages, and plasmids. E. coli B, CR63 to the molecular weight of 27,300 calculated from the DNA (22), DH5a, HB101 (4), and JM83 (24) were taken from sequence data of Broida and Abelson (5). Duckworth and stocks maintained in this laboratory at -80°C in 8.0% Bessman (8) provided supportive evidence that only one dimethyl sulfoxide. Bacteriophages T4D and T4amE957 (8) enzyme, encoded by gene 1 of T4 phage, is responsible for were from liquid stocks kept at 4°C. We are very thankful to the three activities. It has also been shown with T4 phage Donna Duckworth for providing us with the amber mutant. that dHMP, dTMP, and dGMP are competitive inhibitors The expression vector pHE6 (18) and the plasmid pTFR1501 of each other (2). This would imply the existence of only (J. Velten, Ph.D. thesis, Department of Chemistry, Univer- one active site for the three substrates, overlapping sites, sity of California, San Diego, 1981) were the generous gifts or substrate-induced conformational changes in the protein. of Lawrence Grossman and John Abelson, respectively. The resolution of this question will require substantial Growth media. All media used were prepared as described quantities of pure enzyme. In this report we describe a by Miller (17). For antibiotic selection, ampicillin was added procedure for the molecular cloning of gene 1 into an to a final concentration of 50 ,ug/ml. expression vector, the overproduction of the enzyme, and Assays. The method of Lowry et al. (15) was used to the large-scale preparation of the pure protein. We also determine protein concentrations, with bovine serum albu- demonstrate unequivocally that one protein is responsible min as the standard. To determine enzymatic activity, a for all three activities. spectrophotometric assay was used in which the formation of nucleoside diphosphates from monophosphates is coupled to the oxidation of 1-NADH (8, 12, 14). The conditions were as previously described (8), with the following exceptions. (i) * Corresponding author. Purified pyruvate kinase and lactic dehydrogenase fractions t Publication no. 1452 of the McCollum-Pratt Institute. were used rather than a crude extract. As a result, no 2935 2936 BRUSH ET AL. J. BACTERIOL. detectable nucleoside diphosphokinase was present in the column equilibrated with buffer A. The flow rate was ad- system. Ten units of each enzyme was added to each justed to 0.4 ml/min, and fractions containing approximately reaction mixture. (ii) Approximately 1.5 to 15 U of deoxy- 75% of the loaded activity were pooled. nucleotide kinase was added to the assay mixtures. As (v) Affinity chromatography. A 2.5- by 16-cm Cibacron before, 1 U of deoxynucleotide kinase is the amount of Blue F3GA-agarose (Affi-Gel Blue) column was equilibrated enzyme that catalyzes the phosphorylation' of 1 nmol of with buffer A, 'and 10 ml of the previous fraction was applied dTMP per min under the standard assay conditions. to the gel bed. The sample was washed into the column with Cloning procedures. Most of the cloning procedures were 2 column volumes of buffer A, followed by 5 column carried out by the methods of Maniatis et al. (16), except for volumes of 50 mM Tris chloride (pH 7.4)-i mM dithiothre- blunt-end ligations (6) and transformations (19). When cells itol-150 mM NaCl (buffer B). Activity was then eluted with were being transformed with recombinant pHE6 (18) plas- buffer B containing 5 mM ATP and 10 mM MgC12, and the mids, they were incubated at 30'C rather than 37°C after the active fractions were combined. Apparently, Cibacron Blue 42°C pulse. In addition, the plated cells and small-scale binds to the same site of the enzyme as ATP, the phosphoryl cultures used for plasmid preparations were incubated at donor in the reaction (2, 8). 30°C. These modifications were included so that transcrip- (vi) Pressure filtration. The pooled Affi-Gel eluent was tion from the k PL promoter of pHE6 would be repressed by concentrated by filtration through an Amicon PM10 mem- the temperature-sensitive cI857 repressor during cell recov- brane and then dialyzed extensively versus buffer A. Glyc- ery and growth. erol was added to a final concentration of 20%, and the Complementation. These studies were done by modifying purified enzyme was stored at -80°C. the single-step bacteriophage growth curve procedure de- Polyacrylamide gel electrophoresis. Denaturing gel electro- scribed by Adams (1). Bacteria were grown in H broth phoresis was carried out by the method of Laemmli (13), and containing ampicillin at 30°C to an A6. of 0.3 and then native slab gel electrophoresis was a modification of the disc infected with phage at a low multiplicity (-0.02). The method of Ornstein (20) and Davis (7). temperature was shifted to 42°C to allow for transcription Amino acid analysis. An Applied Biosystems 470A gas- from recombinant plasmids. After a 5-min incubation period phase protein sequencer coupled to a PTH analyzer (model for phage adsorption, a 10-,ul sample, was filtered onto a 120A) was used to determine the N-terminal amino acid Micropore disc (0.45-,um pore size), and the retained cells sequence of'the purified enzyme. were washed with H broth. This filter was then placed in a tube containing 10 ml of H broth (tube 1) and incubated with RESULTS aeration at 42°C. At regular intervals, samples of this culture were plated on H-agar petri plates, using H soft agar with E. Subcloning of gene 1. The expression vector pHE6, which coli CR63 as the indicator. Plaques were scored after an contains both the strong A PL promoter upstream from a overnight incubation at 37°C. multiple cloning site and the gene for the temperature- Enzyme purification. (i) Crude extract. E. coli HB101 sensitive repressor cI857, was used to overproduce T4 containing pBK5 was used for the enzyme purification. deoxynucleotide kinase. This plasmid also contains the These cells were grown in 2 liters of LB with ampicillin at 1-lactamase gene, allowing for ampicillin selection of trans- 30°C until an A6. of 0.3 was reached. At this point the formed cells. culture was shifted to 42°C to activate K PL and induce the Velten (Ph.D. thesis) cloned several fragments from the production of deoxynucleotide kinase, and the culture was T4 tRNA region into pBR322, some of which contained the incubated overnight. proximal gene 1. One of the recombinant plasmids, The cells were harvested by centrifugation at 4,200 x g pTFR1501, was the source of this gene for our subcloning (this and subsequent steps were carried out at 4°C) and then procedure.