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'I Martin, R. G., and B. N. Ames, J. Biol. Chem., 236, 1372 (1961).15 For references to individual enzymes, see Snell, E. E., in The Mechanism of Action of Water

Soluble Vitamins, Ciba Foundation Study Group No. 11, ed. A. V. E8. de Reuck and A. O'Connor(London: Churchill, 1961), pp. 18-37.

16 Under the same conditions PLP was observed to interact with serum albumin with appear-ance of absorption maxima at 332 and 415 m/A, as described by W. B. Dempsey and H. N. Chris-tensen [J. Biol. Chem., 237, 1113 (1962)].

17 The activating effects of PLP and Fe+++ on these earlier preparations9 remain unexplainedbut were partially due to stabilizing effects on the enzyme under the assay conditions then in use.They could be duplicated by Tweens, and hence provide no evidence for a role of PLP in actionof the enzyme. Neither crude nor pure preparations of enzyme prepared and assayed by thepresent procedure show these effects.

18 Rabinowitz, J. C., and E. E. Snell, J. Biol. Chem., 176, 1157 (1948).19 Shore, P. A., A. Burkhalter, and V. H. Cohn, Jr., J. Pharm. Exptl. Therap., 127, 182 (1959).20 Du Vigneaud, V., and 0. K. Behrens, J. Biol. Chem., 117, 27 (1937).21 Wallach, O., Ber., 15, 644 (1882).22 Mardashev, C. D., and L. A. Seminha, Dokl. Akad. Nauk USSR, 156, 465 (1964).23 Dr. Mardashev (private communication, 1964) indicated they had not yet found a PLP

requirement for the micrococcal histidine decarboxylase.24 Blethen, S., and E. E. Snell, unpublished data.

PHOSPHORYLATION OF NUCLEIC ACID BY AN ENZYME FROMT4 BACTERIOPHAGE-INFECTED ESCHERICHIA COLI*

BY CHARLES C. RICHARDSON

DEPARTMENT OF BIOLOGICAL CHEMISTRY, HARVARD MEDICAL SCHOOL

Communicated by Eugene P. Kennedy, May 10, 1965

In recent years several enzymatic reactions have been identified which result inthe modification of nucleic acids at the polynucleotide level. These include theglucosylation of DNA,1 the methylation of both DNA and RNA,2, 3 and the addi-tion of single nucleotides to the 3'-hydroxyl termini of RNA4 and DNA5' I mole-cules. This paper describes the purification and some properties of an enzyme, poly-nucleotide kinase, identified in extracts of Escherichia coli infected with T4 phage,which catalyzes the transfer of orthophosphate from ATP to the 5'-hydroxyltermini of polynucleotides (Fig. 1). With the purified enzyme the 5'-hydroxyltermini of high-molecular-weight DNA and RNA molecules can be labeled selec-tively. The ability of this kinase to phosphorylate the 5'-terminus provides a use-ful reagent for identifying end groups in nucleic acids and for studying the effect ofthese groups on enzymes involved in nucleic acid metabolism. A preliminary re-port by Novogrodsky and Hurwitz7 describes an enzyme with similar propertiesfrom T2 phage-infected cells.

Bz By Bx Bz By Bx

APPP + HO J P`3p.pI.P - APP + P bPJP kP5I 5'

FIG. 1.-The transfer of Pi from ATP to the 5'-hydroxyl terminus of a polynuieleotide by poly-nucleotide kinase.

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Experimental Procedure.-Materials: Unlabeled nucleosides, nucleotides, and salmon spermDNA were purchased from the California Corporation for Biochemical Research, and 8-C14-ATPfrom Schwarz Biochemical Co. -y-P32-ATP was prepared by the procedure of Glynn and Chappell.8The 2'- and 3'-isomers of AMP were separated chromatographically.9 Dinucleotides bearing 5'-hydroxyl groups were obtained as described by Lehman.10 E. coli DNA and H3-labeled T7 phageDNA were prepared as before.11 Partially purified, dephosphorylated sRNA was a gift from Dr.H. Paulus.

Phosphodiesterase (Worthington Biochemical Corp.) from Crotalus adamanteus venom wasfurther purified as described by Keller.12 This preparation had no measurable activity on 5'-AMP (less than 0.1% of its diesterase activity). The 5'-nucleotidase from Crotalus adamanteusvenom was purified and characterized as previously described.13 Pancreatic DNase, RNase, andE. coli alkaline phosphatase were products of the Worthington Biochemical Co. Micrococcalnuclease, E. coli exonucleases I and III, and endonuclease I were the same enzyme preparationspreviously described.14

Concentrations of DNA and RNA are expressed as equivalents of nucleotide phosphorus unlessotherwise indicated.

Methods: (a) Preparation of DNA substrates: The 5'-hydroxyl-terminated DNA's were pre-pared by partial digestion (30% conversion to acid-soluble products) of the native DNA's withmicrococcal nuclease, and 5'-phosphoryl-terminated DNA by partial digestion (29% conversion toacid-soluble products) of native E. coli DNA with E. coli endonuclease I. The enzymatic incuba-tions and isolation of the acid-precipitable oligonucleotides have been previously described.14

(b) Assay of polynucleotide kinase: The standard assay for polynucleotide kinase measures theconversion of y-P32-ATP into an acid-insoluble product. The enzyme was routinely diluted intoa solution containing 0.05 M Tris (pH 7.6), bovine plasma albumin (0.5 mg per ml), and 0.01 M2-mercaptoethanol. The incubation mixture (0.3 ml) contained 20 smoles of Tris buffer (pH 7.6),3 gmoles of MgCl2, 5 ;&moles of 2-mercaptoethanol, 80 miumoles of 5'-hydroxyl-terminated salmonsperm DNA (micrococcal nuclease digest), 20 mjAmoles of _y-P32-ATP (specific activity, 5 X 106cpm per M&mole), and 0.05 to 0.5 unit of enzyme. After incubation at 370 for 30 min, 0.2 ml ofsalmon sperm DNA (2.5 mg per ml), 0.5 ml of cold 0.7 N perchloric acid, and 2 ml of cold waterwere added in succession. After centrifugation for 5 min at 10,000 X g, the supernatant fluid wasdiscarded. The precipitate was resuspended in 2 ml of 0.1 N perchloric acid, recentrifuged, andthe supernatant fluid discarded. The precipitate was dissolved in 0.2 ml of 0.2 N NaOH, 2 mlof cold water was added, and the DNA was then reprecipitated by the addition of 0.5 ml of cold0.7 N perchloric acid. Finally, the precipitate was collected on a glass filter, washed, dried, andthe radioactivity determined as previously described.15The precipitate obtained from control incubations with enzyme omitted contained 0.05-0.1%,

of the added radioactivity. One unit of enzyme is defined as the amount catalyzing the produc-tion of 1 mumole of acid-insoluble p32 in 30 min. The radioactivity made acid-insoluble was pro-portional to enzyme concentrations at levels of from 0.05 to 0.5 unit of enzyme;When dinucleotides and other acid-soluble compounds were tested as phosphate acceptors, the

reaction was terminated by chilling, and a 0.04-ml aliquot was spotted onto Whatman no. 3 MMpaper. The sample was subjected to electrophoresis for 2 hr in 0.05 M sodium citrate (pH 5)at a potential of 800 volts. Under these conditions ATP moved more rapidly than any of thecompounds tested. After electrophoresis, the paper was cut into strips and the radioactivity deter-mined by liquid scintillation counting.

(c) Large-scale preparation of P32-labeled DNA with polynucleotide kinase: For characterizationof the P32-labeled product (kinase reaction) a large amount of labeled polynucleotide was prepared.The incubation mixture (4.6 ml) contained 300 limoles of Tris buffer (pH 7.6), 30 MAmoles of MgCl2,30 jmoles of 2-mercaptoethanol, 2 /Amoles of 5'-hydroxyl-terminated E. coli DNA, 80 miumoles ofP32-ATP (specific activity, 5 X 108 cpm per Mmole), and 120 units of polynucleotide kinase (Frac-tion VI). After incubation for 60 min at 370, the mixture was extensively dialyzed to removeunreacted ATP. Assay of aliquots removed at 30 and 60 min of incubation showed that a limitof 25 mumoles of p32 had been incorporated into an acid-insoluble product.

(d) Other methods: Protein, deoxypentose, and phosphate measurements were as previouslydescribed.'4 Paper chromatographic separations were for 16 hr in the solvent system of Markhamand Smith'6 modified as follows: ammonium sulfate saturated in water, 80 parts; 1 M sodium

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acetate, 18 parts; isopropanol, 2 parts. Whatman no. 3 MM paper was used for chromatography.(e) Puriftcation of polynucleotide kinase: Unless otherwise indicated, all operations were per-

formed at 4'. All centrifugations were for 10 min at 15,000 X g. The purification procedureand the results of a typical preparation are summarized in Table 1.

(i) Preparation of extracts: A 50-liter culture of E. coli B was grown in M-9 medium containingcasamino acids (2 gm per liter) to a cell density of 2 X 109 cells per ml. Ltryptophan to a finalconcentration of 1 ,ug per ml and T4r + phage at a multiplicity of 4 were added. Twenty minutesafter infection the biogen was quickly cooled to 5', and the infected cells were collected by cen-trifugation. The packed cells (120 gm) were stored at -20' and showed no loss of activity after6 months. Frozen cells (23 gm) were suspended in 110 ml of 0.05 M Tris buffer (pH 7.4) contain-ing 0.001 M glutathione. After disruption of the cells by sonication (Branson sonicator, modelS-57), cell debris was removed by centrifugation. The supernatant fluid was collected and suffi-cient buffer (10 ml of above buffer) was added to yield a solution whose optical density at 260 m1Awas 105 (Fraction I).

(ii) Streptomycin precipitation: To 138 ml of extract were added, with stirring, 27.6 ml of 5%streptomycin sulfate over a 30-min period. After an additional 15 min the suspension was cen-trifuged and the supernatant fluid discarded. The precipitate was suspended in 138 ml of 0.1 Mpotassium phosphate buffer (pH 7.5) containing 0.002 M glutathione (Fraction II).

(iii) Autolysis: To 143 ml of Fraction II was added 0.43 ml of 1 M MgCl2. The suspension wasincubated at 37' for 1-2 hr, until 90% of the ultraviolet-absorbing material at 260 mA was ren-dered acid-soluble.'7 The autolysate was chilled to 0' and the protein precipitate was removedby centrifugation. The supernatant (Fraction III) was immediately subjected to ammonium sul-fate fractionation.

(iv) Ammonium sulfate fractionation: To 130 ml of Fraction III 13 gm of ammonium sulfatewas added, with stirring, over a 30-min period. After an additional 20 min the precipitate was re-moved by centrifugation and an additional 25 gm of ammonium sulfate were added to the super-natant fluid over a 30-min period. Twenty minutes later, the precipitate was collected by centrif-ugation and dissolved in 10 ml of 0.1 M potassium phosphate buffer (pH 7.5) containing 0.002 Mglutathione (Fraction IV).

(v) DEAE-cellulose fractionation: A column of DEAE-cellulose (8 cm2 X 12 cm) was preparedand washed with 5 liters of 0.01 M potassium phosphate buffer (pH 7.5) containing 0.01 M 2-mercaptoethanol. Fraction IV (200 mg of protein) was diluted to 30 ml with the same buffer,dialyzed against 1 liter of the equilibrating buffer for 5 hr, and applied to the column. The ad-sorbent was washed with 200 ml of the same buffer and the enzyme was then eluted with 0.05 Mpotassium phosphate buffer (pH 7.5) containing 0.01 M 2-mercaptoethanol. The 0.05 M eluatewas collected in 10-ml fractions. Approximately 30% of the enzyme applied to the adsorbent wasobtained in 20 ml of the 0.05 M eluate. The fractions which contained enzyme of specific activitygreater than 7000 units per mg were pooled (Fraction V).

(vi) Phosphocellulose fractionation: A column of phosphocellulose (1 cm2 X 10 cm) was pre-pared and washed with 1 liter of 0.05 M potassium phosphate buffer (pH 7.5) containing 0.01 M2-mercaptoethanol. Twenty ml of Fraction V (2.5 mg of protein) was applied to the column. Afterthe resin was washed with 10 ml of the same buffer, elution of the enzyme was accomplished by20-ml portions of 0.05 M potassium phosphate buffer (pH 7.5) containing 0.01 M 2-mercapto-ethanol plus the following concentrations of KCl: 0.05 M, 0.1 M, and 0.25 M. Two-ml fractionswere collected and approximately 70% of the activity applied to the adsorbent was recovered inthe 0.25 M eluate in a volume of 6 ml (Fraction VI).The phosphocellulose fraction was approximately 1300-fold purified over the extract and con-

tained 15% of the activity initially present. In all the experiments to be described, Fraction VIwas used as the enzyme source. Fraction VI has been stored for 6 months at 0' without loss ofactivity (less than 10%).

Results.-Appearance of polynucleotide kinase after infection with phage T4:Phosphorylation of 5'-hydroxyl-terminated DNA by ATP could be detected inextracts of T4 phage-infected E. coli about 5 min after infection (Fig. 2). A maxi-mal level of activity was observed approximately 20 min after infection.

Properties of the purified enzyme: Using the purified enzyme, the activity was

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dependent upon the addition of 5'-hydrox- TABLE 1yl-terminated DNA, a divalent cation, PURIFICATION OF POLYNUCLEOTIDE KINASEand a sulfhydryl compound (Table 2). Total Specificactivity activityUnder the conditions of the standard assay Fraction (units) (units/mg)I.Extract 80,000 45the optimal Mg++ concentration was 1 X II. Streptomycin 73,000 6710-2 M. Mn++ could only partially re- III. Autolysis 110,000 180

IV. Ammonium sul-place Mg++. At an optimal concentra- fato 93,000 440tion of 3.3 X 10-3 M only 50 per cent of V. DEAE-cellulose 20,000 9,150the maximal activity obtained with Ig++ VI. Phosphocellulose 12,000 62,000has been observed. In the absence of 2-mercaptoethanol 2 per cent of maximalactivity was observed. Equal protection was obtained with 0.1 M glutathione.The optimal pH range for the enzyme is 7.4 to 8.0 in Tris buffer. At pH 7.6 ineither 0.07 M sodium or potassium phosphate buffer 5 per cent of the value observedin Tris buffer was obtained. Addition of 2 Mmoles of potassium or sodium phos-phate buffer (pH 7.6) to the standard reaction mixture, buffered with Tris, resultedin a 52 per cent inhibition.

2.0-

40

16

30 1

E 1.2

20-Additional 80minmoles08 of DNA subsateaf

tO 04-

So mu moles of DNA substrate00 20 40 60 80 100 120

00 10 20 30 40 Time in minutes

Time after infection, minutes

FIG. 2.-Appearance of polynucleotide FIG. 3.-Extent of phosphorylation of 5'-hy-kinase after infection with phage T4. droxyl-terminated DNA. The incubations wereE. coli cells were grown in modified M-9 carried out under standard assay conditions inmedium (see Methods). At a cell titer of several tubes, with the addition of 1 unit of Fraction1 X 109/ml, L-tryptophan and 4 T4r+ VI to each. At 60 min an additional 80mts molefphage per cell were added ("zero mi- of substrate was added and the subsequent incor-utes"). At intervals, 50-wialiquots were poration of Pi was followed. Additional enzymepipetted onto ice centrifuged, resus- resulted in no further release of Pi.pended in buffer, and disrupted by sonica-tion. Kinase activity was measured inthe standard assay.

The purified enzyme (30 units) released no acid-soluble radioactivity from na-tive or heat-denatured H3'-T7 DNA under assay conditions. In the absence of anacceptor DNA there was no detectable hydrolysis of P12-ATP (less than 0.1%)during a 2-hr incubation with 60 units of Fraction VI.

Studies on enzyme specificity: (1) Quantitative phosphorylation of 5'-hydroxyltermini: With the addition of excess enzyme or with prolonged incubation, theincorporation of P32-radioactivity could be shown to reach a limit (Fig. 3). When

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TABLE 2 TABLE 3REQUIREMENTS FOR KINASE ACTIVITY STOICHIOMETRY OF POLYNUCLEOTIDE KINASE

Formation of acid- REACTIONinsoluble P32 PhosphorylatedComponents (mpmoles) Time ATP ADP DNA

Complete system 0.49 (min) (mumoles) (mimoles) (mumoles)Minus 5'-hydroxyl- 0 6.6 <0.01 <0.04

terminated DNA <0.005 30 5.3 1.2 1.3Minus Mg++ <0.005 A -1.3 +1.2 +1.3Minus 2-mercapto-

ethanol 0.01 The incubation was carried out under standardMinus enzyme <0. 005 assay conditions with 160 mjsmoles of 5'-hydroxyl-

terminated salmon sperm DNA, 6.6 miAmoles of ATP,Conditions of the standard assay were employed and 1.5 units of Fraction VI. The ATP solution con-

with 0.5 units of Fraction VI in each tube. In tained 8-C1-ATP and -y-P32-ATP. Time 0 was beforetesting the omission of 2-mercaptoethanol from the addition of enzyme. At 0 and 30 min 0.03-mlthe reaction mixture, the enzyme dilution was aliquots were subjected to electrophoretic analysismade in the absence of this compound. (see Methods).

additional DNA substrate was added to the reaction mixture, a further incorpora-tion occurred. As shown in Table 4, dinucleotides could also be phosphorylated,and to the extent expected from the number of moles of free 5'-hydroxyl terminipresent.

(2) Stoichiometry of the forward reaction and identification of ADP as product:As shown below, the kinase catalyzes the formation of phosphomonoester linkages atthe 5'-hydroxyl termini of polynucleotides. The other product of the re-action was shown, by paper electrophoresis, to be ADP (Table 3). The loss ofATP was approximately equal to the amount of ADP and of phosphorylated DNAformed.

(3) Requirement for 5'-hydroxyl termini on DNA: Hydrolysis of several nativeDNA's with micrococcal nuclease to produce 5'-hydroxyl end groups increased theircapacity to accept P32-orthophosphate several hundredfold (Table 4). In con-trast, hydrolysis of DNA with E. coli endonuclease I to produce 5'-phosphoryl endgroups led to only a small increase in its ability to accept phosphate. However,dephosphorylation of the latter DNA by alkaline phosphatase increased the extent

TABLE 4SPECIFICITY OF POLYNUCLEOTIDE KINASE

Amount of Ratesubstrate (mlsmoles/- Extent

Compound (mgmoles) min/mg) (mismoles)1. Native T7 phage DNA 100 - 0.003*2. 5 '-Hydroxyl-terminated (micrococcal nuclease-

treated)T7 pha';e DNA 80 2200 1.2E. coliDNA 80 2000 0.9Salmon sperm DNA 80 2200 1.0Salmon sperm DNA (denatured) 80 1100 1.1

3. 5'-Phosphoryl-terminated (endonuclease I-treated)

E. coli DNA 80 0.03E. coli DNA (phosphatase-treated) 80 600 0.8-

4. 5'-Hydroxyl-terminated RNA 50 500 0.45. X,,Y 10 1900 9.46. 3'-AMIP 10 1200 8.3

2'-AMP 10 95 0.67. Adeno in:, cytidine 10 <10 <0.5Each compound was tested in the standard assay at the concentration shown. The extents of phosphorylation

were determined in a 1-hr incubation with the addition of 30 units of Fraction VI at 0 and 30 min. Measure-ment of radioactivity in products was determined as described in Methods. XpY represents the dinucleotidesbearing a 5'-hydroxyl end group. Polynucleotide concentrations are given in mjsmoles of DNA or RNA-P.

* In order to measure the extent of phosphorylation of native T7 DNA, ATP'2 having a specific activity of1 X 109 cpm per jsmole was used. The amount of Pi incorporated is approximately that expected for T7 DNA(see text).

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TABLE 5IDENTIFICATION OF THE p32 AT THE 5'-TERMINUS OF THE

POLYNUCLEOTIDE CHAINP32a released

Enzyme treatment (%)1. None 12. Alkaline phosphatase >953. Snake venom phosphodiesterase 24. 5'-Nucleotidase 15. Phosphodiesterase + 5'-nucleotidase >95

P32-labeled DNA (kinase product) was prepared as described in Methods. The incu-bation mixture (0.3 ml) for the alkaline phosphatase consisted of 8 mAmoles of DNA,30 moles of Tris buffer (pH 8), and 5 pg of phosphatase. The incubation mixtures(0.3 ml) for the phosphodiesterase, the 5'-nucleotidase, or both consisted of 12 mpmolesof DNA, 20 ;&moles of glycine buffer (pH 9.2), 2 jmoles of MgC12, and either 5 units ofphosphodiesterase, or 4 units of 5'-nucleotidase, or a combination of both. Incubationin each case was for 30 min at 37°. The P32i released was measured as previously de-scribed14 as acid-soluble, Norit nonadsorbable P"2.

of phosphorylation 30-fold, and the rate of phosphorylation by more than 50-fold(Table 4).

(4) Activity of the enzyme on various nucleic acid substrates: The purified en-zyme was tested on various compounds for both rates of reaction and extents ofmaximal phosphorylation (Table 4). Native T7 phage DNA was phosphorylatedto an extent of 1 mole of phosphate per 3.3 X 104 moles of DNA-P. This valueapproximates that expected for a single strand of T7 DNA, assuming that the strandis intact and bears a 5'-hydroxyl terminus. (See Note added in proof.) Heat-denatured 5'-hydroxyl-terminated DNA was phosphorylated at a slower rate but tothe same extent as the same DNA prior to heat-denaturation. The lack of specific-ity for secondary structure in the acceptor was shown by the ability of the enzymeto phosphorylate sRNA, dinucleotides, and 3'-AMP. The activity observed with2'-AMP is most likely due to contaminating 3'-AMP not removed by chromatog-raphy. Adenosine and cytidine were not phosphorylated.

(5) Identification of p32 at the 5'-terminus of a polynucleotide: Treatment of theP32-labeled kinase product with alkaline phosphatase resulted in the release of morethan 95 per cent of the radioactivity as P, (Table 5), demonstrating that the poly-nucleotide chain had been terminally labeled. The quantitative (>95%) releaseof Pl2" from the product by the successive action of snake venom phosphodiesteraseand 5'-nucleotidase showed that this terminal radioactivity was present as a 5'-phosphomonoester (Table 5). Incubation with either enzyme alone did not re-sult in the release of P12 . These results indicate that the y-phosphate of ATP wastransferred to the terminal 5'-hydroxyl group of the polynucleotide acceptor.The location of the p32 on the product was confirmed by utilizing a specific and

unique property of E. coli exonuclease I, its inability to hydrolyze the phosphodiesterbond of a dinucleotide.18 As illustrated schematically in Table 6, exonuclease Iinitiates a stepwise hydrolysis from the 3'-hydroxyl terminus of a single-strandedDNA molecule, liberating 5'-mononucleotides'8 and leaving the terminal 5'-di-nucleotide. As seen in Table 6, 95 per cent of the p32 could be identified in thedinucleotides resulting from extensive exonuclease I hydrolysis of the kinaseproduct. This experiment clearly established the location of the p32 at the 5'-terminus of the DNA molecule.

P32-labeled sRNA (kinase product) was also shown to be labeled at the 5'-terminus by an experiment in which the kinase product was hydrolyzed with

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TABLE 6IDENTIFICATION OF p32 IN 5'-TERMINAL DINUCLEOTIDE OF

POLYNUCLEOTIDE KINASE PRODUCTScheme of exonuelease I

action 32pXpX(px)npx 32PXPX + pX + n(pX) + pX

Compound 5 '-P32-terminated Dinucleotides MononuicleotidesDNA

p32 (m/moles) 1.6 1.5 0.1P32-labeled DNA, kinase product, was prepared as described in Methods. The incubation mixture for E.

coli exonuclease I consisted of 200 mjsmoles of heat-denatured P"-labeled DNA (kinase product), 40 jsmoles ofglycine buffer (pH 9.2), 4 jsmoles of MgC12, and 500 units of exonuclease I in a volume of 0.6 ml. After inctu-bation for 2 hr at 370, the digest was chromatographed on DEAE-cellulose'0 in the presence of unlabeled mono-and dinucleotides. The radioactivity eluted in the mono- and dinucleotide fractions is recorded above.

alkali (0.2 N NaOH, 370 for 16 hr) and then subjected to electrophoresis. All ofthe radioactivity (>90%) migrated as the nucleoside diphosphate.

(6) Identification of the terminal phosphorylated nucleotide: The purified kinaseis capable of phosphorylating all four of the nucleotides found in E. coli DNA whentheir 5'-hydroxyl groups are terminally located. When E. coli DNA is hydrolyzedwith an enzyme having little base specificity, E. coli endonuclease I, all four basescan be identified at the 5'-termini of the oligonucleotides formed.'3 As shown inTable 7, all of these terminal residues can be phosphorylated by the kinase once the5'-phosphoryl groups initially present have been removed. The value of thistechnique for end group analysis of DNA was shown by phosphorylating DNAwhich had been partially degraded with micrococcal nuclease, an enzyme which hasbeen shown to yield only adenylate and thymidylate as the 5'-terminal nucleotides. 9As seen in Table 7, only dAMP and dTMP contained radioactivity after hydrolysisof the DNA by phosphodiesterase and chromatography of the mononucleotides.

Discussion and Summary.-T4 phage infection of E. coli leads to the appearanceof an enzyme, polynucleotide kinase, which catalyzes the transfer of orthophosphatcfrom ATP to the 5'-hydroxyl termini of a wide variety of nucleic acid compounds.The specificity of the enzyme permits the phosphorylation of DNA, RNA, smalloligonucleotides, and even nucleoside 3'-monophosphates. This provides an en-zymatic reagent for the study of nucleic acid structure. The natural occurrenceand the number of 5'-hydroxyl end groups can be determined, and the terminalnucleotides can be isolated and identified. These applications permit an examina-

TABLE 7P32-NUCLEOTIDES ISOLATED AFTER VENOM DIESTERASE

TREATMENT OF PHOSPHORYLATED DNAEndonuclease Treatment prior to Phosphorylation

E. coli endonuclease Micrococcal nucleaseNucleotides % of TotaldAMP 14 44dTMP 38 54dGMP 17 <2dCMP 31 <2E. coli DNA was partially digested with either E. coli endonuclease I (and dephosphoryl-

ated with alkaline phosphatase) or with micrococcal nuclease (see Methods). After phos-phorylation of 100 mjumoles of each by P'2-ATP in a standard kinase reaction containing 30units of Fraction VI, the ATP was removed by dialysis. The 5'-P32-DNA's were digestedto mononucleotides by the action of snake venom phosphodiesterase (see Table 5) and theproducts chromatographed (see Methods). The per cent of total radioactivity found in eachof the four mononucleotides is given above for each endonuclease treatment. Recovery ofradioactivity was greater than 90% in both experiments.

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tion of the initial sites of hydrolysis by various endonucleases and provide a sensi-tive assay for enzymes which hydrolyze phosphodiester bonds to an extent whereacid-soluble products are not produced.

Determination of the function of this enzyme in vivo is complicated by its abilityto phosphorylate a number of compounds, and a delineation of its role in nucleicacid metabolism will require further study. The well-established metabolic path-ways involving other kinases lead to the synthesis of nucleoside-5'-triphosphates.This raises the question as to whether polynucleotide kinase, together with otherenzymes, might lead to the synthesis of a polynucleotide chain bearing an activated5'-terminus. This, in turn, could result in the condensation of preformed poly-nucleotide chains.

Finally, the presence of 5'-phosphoryl termini may serve to prevent the initiationof hydrolysis by exonucleases. The presence of 3'-phosphoryl end groups in DNAhas been shown to prevent exonuclease attack effectively'8 and, in addition, toeliminate their template activity for DNA polymerase.20

Note added in proof: Subsequent studies have revealed that only 70% of the strands of the T7DNA used in this experiment were intact. The nature and number of the end groups in this DNAmust await isolation of the intact strands.

The author acknowledges the valuable technical assistance of Miss Ann Dolan.The designations for a polynucleotide chain and the abbreviations used in this paper are those

described in J. Biol. Chem.

* This work was supported by USPHS grant AI-06045.1 Kornberg, A., in Enzymatic Synthesis of DNA (New York: John Wiley, 1961), p. 69.2 Fleissner, E., and E. Borek, these PROCEEDINGS, 48, 1199 (1962).3Gold, M., and J. Hurwitz, J. Biol. Chem., 239, 3858 (1964).4 Hecht, L. I., P. C. Zamecnik, M. L. Stephenson, and J. F. Scott, J. Biol. Chem., 233, 954

(1958).5 Adler, J., I. R. Lehman, M. J. Bessman, E. S. Simms, and A. Kornberg, these PROCEEDINGS,

44, 641 (1958).6Krakow, J. S., C. Coutsogeorgopoulos, and E. S. Canellakis, Biochem. Biophys. Res. Commun.,

5, 477 (1961).7Novogrodsky, A., and J. Hurwitz, Federation Proc., 24, 602 (1965).8 Glynn, I. M., and J. B. Chappell, Biochem. J., 90, 147 (1964).9 Cohn, W. E., in The Nucleic Acids, ed. E. Chargaff and J. N. Davidson (New York: Aca-

demic Press, Inc., 1955), vol. 1, p. 229.10 Lehman, I. R., J. Biol. Chem., 235, 1479 (1960)."1 Richardson, C. C., R. B. Inman, and A. Kornberg, J. Mol. Biol., 9, 46 (1964).12 Keller, E. B., Biochem. Biophys. Res. Commun., 17, 412 (1964).13 Lehman, I. R., G. G. Roussos, and E. A. Pratt, J. Biol. Chem., 237, 819 (1962).14 Richardson, C. C., and A. Kornberg, J. Biol. Chem., 239, 242 (1964).15 Schildkraut, C. L., C. C. Richardson, and A. Kornberg, J. Mol. Biol., 9, 24 (1964).16 Markham, R., and J. D. Smith, Biochem. J., 52, 552 (1952).'7 Richardson, C. C., C. L. Schildkraut, H. V. Aposhian, and A. Kornberg, J. Biol. Cheml., 239,

222 (1964).18 Lehman, I. R., and A. L. Nussbaum, J. Biol. Chem., 239, 2628 (1964).19 Sulkowski, E., and M. Laskowski, Sr., J. Biol. Chem., 237, 2620 (1962).20 Richardson, C. C., C. L. Schildkraut, and A. Kornberg in Synthesis and Structure of AMacro-

molecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 9.

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