[142] DNA polymerase from T2-infected Escherichia coli

NUCLEIC ACID SYNTHESIS----/n Vitro

586

[142]

[ 142] D N A P o l y m e r a s e f r o m T 2 - I n f e c t e d Eschericlda coli 1

By H. VASXV,N AP0SHIAN "dTP PP ] [ dTP ] dGP*PP | [dGP* | dAP PP ] q- DNA -* DNA - dAP | q- 4(n) PP -dHMCP PP dHMCP~

Assay Method Reagents. The reaction mixture (total volume 0.3 ml) contains: Tris buffer, 0.07 M, pH 8.6 MgCI2, 0.007 M Mercaptoethanol, 0.01 M 3 X 10-6 M of each of the following: dHMCTP, dATP, dTTP, and dGTP-14C (1 X 106 c/m//~mole) or dGTP-a-82P Sodium Versenate, 7 H 10-6 M, pH 6.8 Heated salmon sperm DNA: 60 m/~moles of DNA-phosphorus A solution of salmon sperm DNA: 1 #mole DNA-phosphorus per ml of Tris-HCl buffer 0.02 M, pH 7.5, and 0.02 M NaCI is heated for 15 minutes at 100 ° and quickly cooled in ice water The other reagents are: GTP, 0.10 M Perchloric acid, 7?5

Procedure. This assay measures the conversion of 14C- or 32P-labeled deoxynucleoside triphosphates into an acid-insoluble product and was used to follow enzymatic activity during the purification procedure. After 30 minutes at 37 ° the reaction mixture is cooled in an ice bucket and 0.01 ml of 0.10M GTP is added. This is followed by the addition of 0.50 ml of cold 7% perchloric acid and 3 ml of cold water. The mixture is stirred and filtered through a GF/C Whatman glass filter paper (2.4 cm diameter). The tube and filter are washed three times with 4 ml of cold water. If a 6~P-labeled deoxynucleoside triphosphate is used, the filter paper is placed on a planchet, dried under an infrared lamp, and counted. Alternatively, the filter may be dried and then counted in a scintillation counter, especially if a 14C- or 3H-labeled substrate is used. A blank without enzyme is run with each set of assays, and its value is subtracted from each sample. One unit is defined as the amount catalyzing the incorporation of 1H. V. Aposhian and A. Kornberg, Y. Biol. Chem. 23'/, 519 (1962).

[142]

T2-DI~A POLYMERASE

587

10 millimieromoles of the labeled deoxynucleotide into the acid-insoluble product during the 30-minute incubation period. Enzyme specific activity is expressed as units of enzyme per milligram of protein. The radioactivity made acid-insoluble is proportional to the amount of enzyme added; thus, 0.01, 0.02, and 0.04 ml of a phosphocellulose fraction (diluted 1:100) resulted in the appearance of 388, 807, and 1611 cpm, respectively, in the acid-insoluble fraction. Enzyme dilutions are made in 0.05 M Tris buffer, pH 7.5, containing 0.01 M 2-mercaptoethanol and bovine serum albumin (1 mg/ml). Aliquots of the diluted enzyme solutions are added to the reaction mixture immediately after dilution and are then discarded. During the early stages of the purification and especially if crude extracts are being assayed it is necessary to use deoxyhydroxymethylcytidine triphosphate (dHMCTP) instead of dCTP in the assay mixture because of the presence of phage-induced dCTPase. Isolation Procedure

Growth of T2 Phage-Infected E. coli B. The phage-infected cells are grown and harvested as described by Dr. S. Zimmerman.TM In the fractionation procedures to be described, the temperature was maintained near 0 ° except where noted, and centrifugations were at 14,000 g for 20 minutes. The procedure is summarized in Table I. TABLE I PURIFICATION OF D N A

Fraction I. II. III. IV. V. VI. VII.

Cell extract Streptomycin Protamine Ammoniumsulfate Dialysis DEA_E-cellulose Phosphocellulose

POLYMERASE FROM T2 INFECTED E.

coli

Protein (mg/ml)

Specific activity (units/mg)

Total units

15.8 2.18 0.30 4.08 3.57 0.12 0.058

2.6 3.9 32.7 52.2 44.8 366 1576

20,850 14,280 6,272 5,325 4,000 1,866 1,554

Step 1. Preparation o/Extract. The extract is prepared by mixing 45 g of frozen cells, 150 g of glass beads (Superbrite lb No. 100), and 50 ml of a buffer solution (A) containing glycylglyeine (0.05 M, pH 7.0), sodium Versenate (0.002M), and reduced glutathione (0.002M) for ten 21~Vol. VI [30]. lbObtained from Minnesota Mining and Manufacturing Co.

588

NUCLEIC ACID SYNTHESIS--~n Vitro

[142]

minute periods in a 1-quart glass Waring blendor at two-thirds of maximal speed. The container and its contents are chilled between mixing periods by immersion in an ice water bath until the temperature of the suspension is below 10 °. After the last mixing period, 140 ml of buffer A is added, and mixing is continued at one-fifth of maximal speed for 10 minutes. The glass beads are allowed to settle for 3 minutes, the supernatant fluid is decanted, and the beads are briefly washed with 100 ml of buffer A. The supernatant and wash fluids are combined and centrifuged, and the resulting supernatant fluid is collected. The procedure is repeated with an additional 45 g of cells, and the supernatant fluids from these two batches are combined to give 500 ml of fraction I. Step 2. Streptomycin Precipitation. To 500 ml of fraction I are added 420 ml of buffer A and then, with stirring during a 10-minute period, 276 ml of streptomycin sulfate (5% solution). Stirring is continued for an additional 20 minutes. The precipitate, collected after eentrifugation, is homogenized in a Waring blendor at one-fifth of maximal speed for 30 minutes with 230 ml of a buffer solution (B) containing potassium phosphate (0.05 M, pH 7.4), sodium Versenate (0.002 M), and reduced glutathione (0.002 M). This suspension is mixed with 596 ml of buffer B and 840 ml of a solution containing sodium Versenate (0.002 M) and reduced glutathione (0.002 M) to produce fraction II. Step 3. Protoznine Precipitation and Elution. To 1680 ml of fraction II are added, with stirring, 210 ml of a 1% protamine sulfate solution. The addition requires 4 minutes. The suspension is divided into two equal parts, which are treated as separate batches. After centrifugation, the supernatant fluid is discarded, and the precipitate is transferred to a Waring blendor with 200 ml of potassium phosphate buffer (0.10 M, pH 7.0) containing sodium Versenate (O.O02M) and reduced glutathione (0.002 M), and homogenized at one-fifth of maximal speed for 10 minutes. An additional 140 ml of this buffer is added, and the mixing is continued for another 5 minutes. After eentrifugation, the supernatant fluid is collected. The supernatant fluids from both batches are combined (fraction III). Step ~. A.mmonium Sulfate Fractionation. To 640 ml of fraction III are added, with stirring, 131 g of ammonium sulfate. After 15 minutes, the precipitate is removed by centrifugation. To the supernatant fluid an additional 95 g of ammonium sulfate is added; after 15 minutes the precipitate is collected by centrifugation and dissolved in 25 ml of potassium phosphate buffer (0.025 M, pH 7.4) containing sodium Versenate (0.002 M) and reduced glutathione (0.002 M) to yield fraction IV. Step 5. Dialysis. Fraction IV, 20 ml, is dialyzed for 4 hours against

[142]

T2-DNA POLY~ERASE

589

K2HP04 (0.02M) containing 2-mercaptoethanol (0.01M), and the dialyzate is immediately used for the preparation of fraction VI. Step 6. DEAE-Cellulose Chromatography. A column of DEAE-cellulose (11 X 1.1 cm) is prepared and equilibrated with K2HP04 (0.02 M) containing 2-mercaptoethanol (0.01 M). Fraction V (20 ml) is passed through the column at the rate of 36 ml per hour. The column is then washed with 10 ml of the K2HPO4-mercaptoethanol solution. A constant gradient, the limits of which are 0.02 M and 0.30 M potassium phosphate buffer, pH 6.5, is applied. The buffers contained mercaptoethanol (0.01 M), and 150 ml of each buffer are used. Fractions are collected at 12-minute intervals. Elution of 63% of the activity occurred between 8.4 and 14.8 resin-bed volumes of effluent. The peak fraction is eluted in approximately 0.09 M phosphate. The peak fractions having specific activities of 172-564 are combined to yield 34 ml of fraction VI. Step 7. PhosphoceUulose Chromatography. A column of phosphocellulose (10 X 0.6 cm) is prepared and washed with potassium phosphate buffer (0.02M, pH 6.5) containing 2-mercaptoethanol (0.01M) until the pH of the effluent was 6.5. Fraction VI (5.0 ml) is passed through the column at a rate of 0.3 ml per minute. The following potassium phosphate buffers (pH 6.5) containing 2-mercaptoethanol (0.01 M) are used as stepwise eluents: 3 ml of 0.05 M, 6 ml of 0.10M, 6 ml of 0.15 M, 8 ml of 0.20 M, 2 ml of 0.50 M, and finally, 2 ml of 0.50 M. Approximately 83-99% of the enzyme activity applied to the phosphocellulose column is in the last cluate fraction. Comments. A summary of a typical preparation is shown in Table I. The specific activity of fraction VII has varied from 1000 to 2200 depending on the particular lot of infected cells used as the source of the enzyme. Features Distinguishing E. coli and T2 DNA Polymerase. The incubation of E. coli polymerase with rabbit antiserum prepared against purified E. coli polymerase results in virtually complete inhibition of the enzyme activity. Equivalent amounts of antiserum to E. coli polymerase do not inhibit T2 polymerase. Conversely rabbit antiserum prepared against T2 polymerase inhibits T2 polymerase but does not inhibit E. coli polymerase. Such antiserum can be used to inhibit the appropriate DNA polymerase activity in crude extracts of T2 infected cells. Levels of p-chloromercuribenzoate (1.7 X 10`4 M) which inhibit T2 DNA polymerase almost completely, inhibit the E. coli DNA polymerase only 27%. The two DNA polymerases have different requirements as to the source and conformation of the DNA template.

590

NUCLEIC ACID SYNTHESIS---/n Vitro

[142]

Finally, the two D N A polymerases can be separated b y chromatography on a phosphocellulose column. Properties of the E n z y m e

DNase Activities of T2 Polymerase Fractions. The last two fractions in the purification of the T2 D N A polymerase were assayed for endonuelease 12 and exonuclease I activities 3 as described by Lehman. The ratios of polymerase to endonuclease (millimieromoles of nucleotides rendered acid insoluble:millimicromoles rendered acid soluble) of fractions V I and V I I were 46 and 220, respectively. The p o l y m e r a s e ' e x o n u clease I ratios of fractions V I and V I I were 27 and 26, respectively. The endonucleolytic and phosphodiesterase activities were measured on native and heated E. colt D N A , respectively. Requirements ]or Reaction. T h e omission of a single one of the four deoxynucleoside triphosphates or of D N A or MgCl2 reduced the reaction TABLE II SPECIFICITY OF T2 D N A POLYMERASE FOR ~)EOXYNUCLEOSIDE TRIPHOSPHATESa

Analog, used in form of deoxynueleoside triphosphate 5-Bromouracil 5-Fluorouracil 5-Bromocytosine 5-Fluorocytosine 5-Hydroxymethylcytosine

Deoxynucleoside triphosphate replaced by analog dTTP

dATP

dCTP

dGTP

100 9 <2 <2 <2

<2 <2 <2 <2 <2

<2 <2 104 67 98

<2 <2 <2 <2 <2

Values are given as percent of control values. Control values were measured as radioactive deoxynucleotide incorporated into DNA in the presence of dTTP, dATP, dCTP and dGTP, but in the absence of the analog. The rates were measured in the standard reaction mixtures except that incubation was for 60 minutes. Fraction VII was used as the source of the enzyme. to undetectable levels. The purified enzyme was about half as active when 2-mercaptoethanol was omitted and was unaffected by the absence of sodium Versenate. Replacing MgC12 by MnC12 reduced the incorporation 90%. Specificity. With T2 polymerase, as is the case with the D N A polymerase of E. colt, analogs of the n a t u r a l l y occurring bases serve as substitutes in a manner dictated b y the hydrogen-bonding relationships of adenine to thymine and of guanine to cytosine (Table I I ) . 2I. R. Lehman, G. Roussos, and E. A. Pratt, J. Biol. Chem. 237, 819 (1962). sI. R. Lehman, J. Biol. Chem. 235, 1479 (1960).

[143]

CALF THYMUS POLYMERASES

591

TABLE I I I P~ELATIVE EFFECTIVENESS OF VARIOUS D N A PRIMERS FOR

T2 DNA POLY~ERASm~ Priming activity

Source of DNA Salmon sperm dG-dC homopolymer Calf thymus Salmon liver dAT copolymer E. coli

T2 bacteriophage "Activated" calf thymus

Heated DNA 100 58 20 15 11 10 8 1

Native DNA 5 -0.7 4 -0.3 0.8 --

a The rates w e r e measured in the standard reaction mixture, and fraction VII was used as the source of the enzyme.

DNA's isolated from E. coli, bacteriophage T'2, and animal tissues s u p p o r t e d o n l y 5 - 1 0 % of t h e p o l y m e r a s e a c t i v i t y when c o m p a r e d w i t h these s a m e D N A ' s w h e n t h e y h a d been h e a t e d ( T a b l e I I I ) .

[143] Deoxynucleotide Polymerizing Enzymes from Calf Thymus Gland 1, 2 By F. J. BOLLUM DNA polymerase (replicative deoxynucleotidyl transferase) uC-dATP dCTP T denatured DNA -+ native uC-DNA + PPi dGTP template dTTP Terminal deoxynucleotidyl transferase n uC-dATP q- d(pTpTpT) --. d(pTpTpT(pA)~) -k n PPI initiator

(1)

(2)

P r e p a r a t i o n of D e o x y n u c l e o t i d e P o l y m e r i z i n g E n z y m e s C a l f t h y m u s g l a n d is a source of t w o s e p a r a t e d e o x y n u c l e o t i d y l transferases: DNA polymerase and a terminal deoxynucleotidyl trans1 Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation. 2This manuscript was prepared while the author was at the Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee.