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An ATP-dependent deoxyribonuclease from Diplococcus pneumoniae
BIOCHIMICA ET BIOPHYSICA ACTA
29
BBA 96647
AN A T P - D E P E N D E N T DEOXYRIBONUCLEASE FROM DIPLOCOCCUS
PNEUMONIAE I. PARTIAL PURIFICATION AND SOME BIOCHEMICAL P R O P E R T I E S G E R A L D F. VOVIS* AND GI~RARD B U T T I N
Centre de Gdndtique Moldculaire du C.N.R.S., 91 Gi[-sur-Yvette; Service de Gdndtique Cellulaire de L'Institut Pasteur et du Coll~ge de France, Paris (France) (Received J u l y 9th, 197 o)
SUMMARY
I. A 5-step procedure is described for the partial purification of a nucleoside triphosphate-dependent deoxyribonuclease from Diplococcus pneumoniae. An overall purification of more than 5oo-fold was achieved. 2. The enzyme was found to be active under alkaline conditions (optimum pH from 9.25 through, at least, 9.8) and to require a metal ion and a nucleoside triphosphate for its activity on native DNA. Mg~+ and Mn 2+ and ATP were found to be the most effective at satisfying these requirements. 3. ATP is split during the course of DNA hydrolysis. 4- The zone sedimentation pattern of the DNA hydrolysis products is consistent with an enzyme that attacks preexisting free ends (exonuclease), liberating oligonucleotides among its products. 5. The enzyme is much more active on native DNA than on heat-denatured DNA. It attacks poly d(A-T) but exhibits no activity with rRNA.
INTRODUCTION
The discovery of an ATP-dependent deoxyribonuclease, which splits the required ATP during DNA hydrolysis and appears to be involved in the process of genetic recombination, was first reported by BUTTIN AND WRIGHT1 during investigations of deoxyribonuclease activities in Escherichia coli extracts. The existence of an enzyme with apparently similar biochemical properties in Micrococcus lysodeikticus 2 suggested that this type of enzyme might represent a particular class of enzymes which catalyzes a major step in bacterial recombination in various organisms. To explore this possibility, we chose to examine D. pneumoniae, an organism possessing a transformation system which is an ideal model system with which to study recombination at the molecular level because of the ability to make correlations between DNA structure and function. The initial experiments were undertaken Abbreviations: BBOT, 2,5-bis-[2-(5-tert.-butylbenzoxazolyl)]thiophene; d i m e t h y l - P O P O P , 1,4-bis-[2-(4-methyl-5-phenyloxazolyl)]benzene; PPO, 2,5-diphenyloxazole. * Present address: Genetics Laboratory, The Rockefeller University, New York, N. Y. 10021 U.S.A.
Biochim. Biophys. Acta, 224 (I97O) 29-41
3°
G . F . VOVIS, G. BUTTIN
to determine whether pneumococcus also possesses a nucleoside triphosphate-dependent deoxyribonuclease with the aim of investigating the enzymatic basis of genetic recombination in a transformable strain. This report describes the partial purification and some biochemical properties of such an enzyme in pneumococcus, and a companion report presents genetic evidence establishing its enzymatic involvement in bacterial recombination S. During the course of these investigations, various reports appeared concerning the properties of ATP-dependent deoxyribonucleases from several other different organisms. To what extent these enzymes appear to be similar in their biochemical properties will be discussed.
METHODS AND MATERIALS
Bacteria and growth media The strain, C1z str-r 41, used for the enzyme purification was a derivative of Clone 3 (ref. 4), which had been transformed to streptomycin resistance 3. The cells for the enzyme purification were grown at 37 ° in a medium prepared by combining the following solutions: I °/o Difco neopeptone (pH 7.7) containing 0.85 % NaC1; IO °/o Difco yeast extract (pH 7.6); 25 % glucose; 1.68 % L-glutamine; in the ratio of 50:4:2 :I, respectively. The inoculum, 1/8 the volume of the mass culture, was grown to a density of approx. 2.1o s colony-forming units/ml in the same medium but with 20 times less glucose. The pH of the mass culture was maintained near neutrality by titrating phenol red, present at 0.0002 ~o, with concentrated NaOH. DNA preparation To prepare 3H-labeled T 7 phage DNA, T 7 phage were used to infect a culture of E. eoli C6oo (ref. 5) growing in medium 63 lacking glycerol but containing vitamin B 1 (ref. 6) and supplemented per 1 with 20 ml 20 °/o glucose and 50 ml IO % Difco casein amino acids (vitamin free). 5 min later, EMe-3Hjthymidine (18 C/mmole) and deoxyadenosine were added to a final concentration of approx. 3 #C/ml and approx. 4 #g/ml, respectively. The 32p-labeled T 7 phage were prepared in low-phosphate minimal medium:, supplemented per 1 with 20 m120 °/o glucose, 5 ml IO °/o Difco casein amino acids (vitamin free), IO ml 1 % L-threonine, IO ml I °/o L-leucine and I ml 0.05 % vitamin B 1. The carrier-free Ha32po 4 was added at the time of infection to a final concentration of about 20/~C/ml. In both cases, the multiplicity of infection was about 5 and the infected cultures were shaken at 37 ° until complete lysis. The DNA was purified essentially by the method of RICHARDSON et al. s. To remove the residual phenol the DNA solution was dialyzed extensively against a o.oi M Tris, o.I lV[ NaC1 and o.oi M EDTA solution, whose pH was adjusted to 8 with NaOH, and stored at o °. The specific activity of the [3HIDNA was 4" lO4-1" lO5 counts/min per/,g DNA while that of [32p~DNA was initially 4.4" I°a counts/min per #g DNA, both as determined by counting diluted aliquots on glass-filter paper (Carl Schleicher and Schuell Co., Keene, N.FI.) in IO ml of 2,5-bis-E2-(5-tert.-butylbenzoxazolyl)lthiophene (BBOT) scintillation fluid (8 g/1 toluene) with the preset channels in the Packard Tri-Carb liquid scintillation spectrometer Model 3375. The heat-denatured DNA was prepared by heating [3HIDNA , in o.oi M Tris-HC1 (pH 8), containing Biochim. Biophys. Acta, 224 (197 o) 29--41
A DEOXYRIBONUCLEASE FROM D.
pneumoniae
31
o.oi M NaC1, for IO min in a boiling water bath followed by rapid cooling in ice water. E D T A was then added to a final concentration of o.oi M.
Enzyme assay The standard reaction mixture (o.3 ml) contained 6#moles glycine-NaOH (pH 9.3), 3/,moles MgC12, 0.3 #mole 2-mercaptoethanol, 8 nmoles T 7 phage E3H3DNA, and 7.5 nmoles ATP (sodium salt; the stock solution was neutralized to p H 7 with NaOH). The reaction was started b y adding to the prewarmed reaction mixture 20/,1 of the material to be assayed, containing 0.005-0.03 enzyme unit, and the resulting solution was incubated at 37 ° for 5 rain. The reaction was stopped b y removing to ice and immediately adding 0.2 ml cold salmon sperm DNA (2.5 mg/ml) and 0.5 ml cold 0. 7 M trichloroacetic acid. After at least IO rain at o °, the resulting precipitate was pelleted by a Io-min centrifugation at 20 ooo × g and 0.3 ml of the supernatant was counted in IO ml of modified Bray's solution (200 g naphthalene, 5 ° mg 1,4-bis-E2-(4-methyl-5-phenyloxazolyl)]-benzene (dimethyl-POPOP), 7 g 2,5diphenyloxazole (PPO), and dioxane to I l) with a Packard liquid scintillation spectrometer. To determine specifically the ATP-dependent activity, a blank was run in parallel without ATP. One unit of enzyme activity is defined as that amount of enzyme which hydrolyzes I nmole of DNA to acid-soluble fragments per min under the above conditions and corrected for the blank value. O n e / , a t o m of DNA phosphorus is considered to be equivalent to 300/~g DNA, and E~ ~ was taken to be 213 at 26o nm (ref. 9). Beginning with the material after Fraction I I I (see RESULTS), dilutions were carried out in 0.02 M potassium phosphate buffer (pH 6.5) containing 0.2 M NaC1, o.oi M 2-mercaptoethanol, 20 % glycerol (v/v), and 0.5 mg/ml bovine albumin powder. The other fractions were diluted in the extraction buffer. Under these conditions, the activity was proportional to the amount of protein added for all fractions.
Assay/or norit nonadsorbable material ET-3zP]ATP (activity 8oo mC/mmole, > 97 % purity) was diluted Io-fold with an equal molar solution of nonradioactive ATP, and the standard reaction mixture contained 4 nmoles of this latter solution. The reaction was carried out and terminated in the normal manner. After centrifugation, 0. 5 ml of the supernatant was transferred to a tube containing 0.35 ml water, 0.3 ° ml norit-carrier solution lo, and 0.60 ml norit suspension (30 % packed volume), all at o °. The mixture was shaken for I rain. After 5 rain at o °, the mixture was centrifuged, and the supernatant was transferred quantitatively to another tube containing o.Io ml norit-carrier solution and 0.60 ml norit suspension. After treating as described above, I.O ml of the supernatant was removed to an aluminum planchet, and I drop of 3 M NaOH was added. The sample was evaporated to dryness and the radioactivity was measured in a gas-flow counter.
Alkaline sucrose gradients o/enzyme hydrolyzates The enzyme reaction was run in twice the volume of the standard reaction mixture with 24 nmoles T7 phage ~32P]DNA and o.15 unit of Fraction V. After 5 rain at 37 °, the reaction was stopped b y chilling on ice; 300/,1 was removed to 65/,1 of a mixture prepared by combining 60/~1 o. 7 M NaOH, 16o/~1 0. 5 M E D T A (pH 8), and 40/~l T 7 phage EaH]DNA; and the amount of DNA hydrolyzed to acid-soluble fragments in the remaining reaction mixture was determined in the usual manner. Then Biochirn. Biophys. Acta, 224 (I97 o) 29-41
32
G . F . VOVIS, G. BUTTIN
I5o #1 of the above alkaline mixture was overlayered on 4.8 ml of 5-2o % sucrose prepared in 0. 7 M NaC1, 0.3 M NaOH, and o.ooi M EDTA and centrifuged at 4 ° for 4 h at 39 ooo rev./min in the Spinco SW-5oL rotor. The bottom of the cellulose nitrate tube was pierced and I2-drop fractions were collected on Whatman 3 mm paper discs. The paper discs were washed successively in the cold with 5 % trichloroacetic acid, 0.2 N HC1, and 80 °/o ethanol. The discs were air dried and counted in Io ml of BBOT scintillation fluid. Protein determination In all the pooled fractions except Fraction V, protein was determined by the method of LOWRY et al. 11 on the acid precipitable material. Protein in Fraction V was estimated by ultraviolet absorption 1~ with a Zeiss spectrophotometer Model PMQ I2. To determine tile specific activity of the enzyme in the individual fractions from the first two columns, the protein was estimated from the absorption at 280 nm as measured by a Gilson Model G2 ultraviolet spectrophotometer and recorder. The absorption values were calibrated by determining, with the method of LOWRY et al. 11, the protein present in the acid-precipitable material of those fractions immediately preceding and succeeding the enzyme peak. Bovine albumin powder was used as the protein standard. Materials All chemicals were reagent grade. (NH4)2SO 4 (ultrapure) was purchased from Mann Research Laboratories; Whatman DEAE-cellulose (DE I) and Whatman phosphocellulose iP II) from W. and R. Balston, Ltd.; hydroxyapatit (hypatit C) from Clarkson Chemical Co. ; dextran 500 from Pharmacia; polyethylene glycol 6000 from Touzart and Matignon, Paris; ATP, ADP, AMP, CTP, GTP, UTP, dCTP, dGTP, and d T T P (all sodium salts) from CalBiochem; dATP (sodium salt) from Sigma; poly d(AIMe-3HIT) (control 57-2-324; 25.1 mC/mmole phosphorus; minimal tool. wt. > 50 ooo) from Miles Laboratories; EMe-3H~thymidine, H33~P04 (carrier-free) and EV-32pIATP from le Commissariat ~ l'Energie Atomique, France; BBOT, PPO and dimethyl-POPOP from Packard Instrument Co., Inc.; bovine albumin powder (Fraction V from bovine plasma) from Armour Pharmaceutical Co. The I3~PIrRNA was a generous gift from Dr. H. Fukuhara.
RESULTS
Puri]ieation o[ the enzyme All steps, unless specifically noted, were carried out at o-3 ° . Preparation o] extract. The pneumococcal cells, harvested in late logarithmic phase (4 g of wet cells per I of culture), were washed twice with 0.85 % NaC1 (pH 7). 3° g of cells were resuspended in 47 ml of o.oi M Tris-HC1 buffer (pH 7.7) containing o.oi M 2-mercaptoethanol. The cells were sonicated with a Sonifier cell disruptor, Model WI85D (Heat Systems-Ultrasonics, Inc.) at a power setting of 9 ° W for 25 rain, while the glass centrifuge tube containing the cell suspension was maintained in a salt-ice-water bath (approx. --IO°). The sonicate was centrifuged twice at 25 500 ×g for IO rain. The supernatant (71 ml, Fraction I, Table I) was collected. Biochim. Biophys. Acta, 224 (197 o) 29-41
A DEOXYRIBONUCLEASE FROM D. TABLE
pneumoniae
33
I
PURIFICATION OF THE ATP-DEPENDENT DEOXYRIBONUCLEASE
Fraction and step
I. II. III. IV. V.
Crude e x t r a c t Phase separation DEAE-cellulose Phosphocellulose Hydroxyapatit
Units pooled per step Number
%
3146 2962 981 274
ioo 94 33 28 39
lO 7
Units recoverable per step (%)
Specific activity (units/rag protein)
Accumulative purification
IOO ioo 62 39 44
1.24 3.52 18. 4 4 °2 921
i .o 2.8 14.8 324 743
(-/ola)
Phase partition. To 17. 4 ml of Fraction I were added with mixing at o °, 2.o ml 20 °/o dextran (w/w) and 5.6 ml 30 % polyethylene glycol (w/w); the emulsion was mixed for 2 h at o °. After centrifuging at 34 7 °0 x g for IO rain, the clear yellow upper phase was removed, discarded and replaced by the upper phase from a mixture prepared by combining extraction buffer, dextran and polyethylene glycol in the same proportions as above and by mixing and centrifuging as above. Then, while stirring at o °, 6.52 g NaC1 were gradually added; the mixing was continued for 2 h at o °. The emulsion was centrifuged at 34 700 × g for 80 rain, and the slightly cloudy, pale yellow upper phase was removed and saved. This procedure was repeated three more times with the remaining portion of Fraction I. The four resulting upper phase fractions were combined (lO3 ml) and dialyzed against 4.5 1 of extraction buffer for 5 h. The dialysis procedure was repeated two more times against fresh extraction buffer. The dialyzed liquid (177 ml, Fraction II) was collected and fractionated on a DEAEcellulose column the same day. DEA E-cellulose chromatography. A DEAE-cellulose column (19.6 cm 2 × 14 cm) was prepared and equilibrated with the extraction buffer. Fraction II was adsorbed to the column at an average rate of 7 ° ml/h. The column was washed with 57 ° ml of the equilibration buffer, and then a linear gradient of elution, total volume 3 1, was applied with the equilibration buffer and the equilibration buffer containing 0. 4 M NaC1 as limiting concentrations. Fractions of 2o-21 ml were collected at an average flow rate of 14o ml/h. Approx. 60 % of the applied activity was eluted between 7 and 13.2 resin bed volumes of effluent. Peaks fractions containing specific activity greater than IO units/rag protein were pooled (159 ml, 49 % recovery of the applied activity) and dialyzed against 8 1 of 0.02 N[ potassium phosphate buffer (pH 6.5) containing o.oi M 2-mercaptoethanol and 5 % glycerol (v/v) for 6 h. The dialysis buffer was changed, and the dialysis was continued for another 6 h. The dialyzed liquid was collected (135 ml, Fraction III). Approx. 30 % of the activity was lost during dialysis. Phosphocellulose chromatography and (NH4)2S04 concentration. A phosphocellulose column (4.9 cm~ × 12.5 cm) was prepared and equilibrated with 0.02 M potassium phosphate buffer (pH 6.5) containing o.oi M 2-mercaptoethanol. Fraction III was added to the column at a rate of 123 ml/h and was washed into the column with 12o ml of the above buffer. A linear gradient of elution was applied, with 0.02 and 0.4 IV[ potassium phosphate buffer (pH 6.5) containing o.oi M 2-mercaptoethanol as the limiting concentrations; 450 ml of each buffer was used. Fractions of 9-1o ml Biochim.WBiophys. Acta, 224 (I96o) 29-41
34
G. F. VOVIS, G. BUTTIN
were collected at an average flow rate of 13o ml/h. About 4o% of the applied activity was eluted between 6.2 and 7-7 resin bed volumes of effluent. Peak fractions with a specific activity greater than 200 units/mg protein were pooled (36 ml) and concentrated by (NHi)~SO 4 precipitation (18 g). The (NH4)2SO 4 was added gradually with stirring at o °. The stirring was continued for 15 min at o °, and the mixture was centrifuged at 41 4 o o × g for 3 ° min. The precipitate was redissolved in 3.5 ml of 0.02 M potassium phosphate buffer (pH 6.5) containing o.2 M NaC1 and o.oi M 2mercaptoethanol by stirring at o ° overnight. Undissolved precipitate was removed b y centrifugation, and the solution was dialyzed for 4 h against this latter buffer to remove a n y residual (NH4)2SO t. The dialyzed liquid (Fraction IV, 28 ~/o of the activity applied to the phosphoeellulose column) was collected. Hydroxyapatit chromatography. A hydroxyapatit column (0.785 cm 2 ×3.5 cm) was prepared and equilibrated with 0.02 M potassium phosphate buffer (pH 6.5) containing 0.2 ~ NaC1 and o.oi M 2-mercaptoethanol. Fraction IV was added to the column and adsorbed over a I6.5-min period. The column was then washed successively with I ml of 0.02 ~ , 2 ml of 0.05 M, and 3 ml of o.I M potassium phosphate buffer (pH 6.5), each containing 0.2 M NaC1 and o.oi M 2-mercaptoethanol. A linear gradient of elution, total volume of 60 ml, was applied with o. I M and 0. 4 M potassium phosphate buffer (pH 6.5) containing 0.2 M NaC1 and o.oi M 2-mercaptoethanol as the limiting concentrations. Fractions of 1.5 ml were collected every 2 rain. To determine the recoverable activity, the fractions must be dialyzed to remove some inhibitory material (probably Ca2+), for assay of a given fraction reveals a 2-3-fold increase in enzyme activity after dialysis. About 45 % of the applied activity was eluted between 3-3 and 8.2 resin bed volumes of effluent. The enzyme-containing fractions all exhibited an absorbance at 280 nm of less than o.o18. The six fractions containing the highest activity were pooled (9 ml, 39 % of the applied activity) and dialyzed for 8 h against I 1 of 0.02 M potassium phosphate buffer (pH 6.5) containing 0.2 M NaC1, o.oi M 2-mercaptoethanol, and 20 ~o glycerol (v/v). The dialyzed liquid (5.4 ml, Fraction V) was collected and stored at o °. Fraction V had been stable (no detectable loss of activity) at o ° for over 7 months.
Properties o] Fraction V Proportionality. As shown in Fig. I, the enzyme activity in Fraction V is proportional to the amount of protein added through at least 15o ng. Fig. 2 shows that the enzyme activity is not proportional to the time of incubation at 37 ° for periods longer than 5 min. pH optimum. The enzyme is active under alkaline conditions (Fig. 3). The p H optimum in glycine-NaOH buffer, where the p H was measured at room temperature, begins at about 9.25 and extends at least through 9.8. Metal ion requirement. The enzyme requires a metal ion for its activity, and either Kg2+ or Mn z+ can satisfy this requirement (Fig. 4). The optimum range for 1rig2+ is from approx. 7" IO-a M to about 2-IO -~ M, while the optimum concentration for Mn 2+ is 4" lO-4 M. The sharp decline in the Mn 2- activity at higher concentrations corresponds with the formation of a precipitate in the standard reaction mixture. Ca 2+ at a concentration greater than I . IO-a M inhibits the enzyme activity as assayed under the standard conditions (I-lO -2 M Mg2+). At a Ca 2+ concentration greater than or equal to I . l O -3 M, a precipitate is also observed in the reaction mixture. Biochim. Biophys. Acta, 224 (197 o) 29-41
pneumoniae
A DEOXYRIBONUCLEASE FROM D.
o
~o 75
A At,
15
35
-i _0
x
5 50
~,c
o Z 0
"6
E
g 25 c
/
jo o/o/8p
Q_
I
0
I
I
50 I00 ng Prolein
150
0
I tO
t
20 Time (rain)
I. 30
Fig. i. E n z y m e activity as a function of protein concentration in Fraction V. The m e a s u r e m e n t s were m a d e u n d e r the s t a n d a r d conditions (see METHODS AND MATERIALS). Fig. 2. Hydrolysis of D N A into acid-soluble f r a g m e n t s as a function of time. The reaction was r u n in duplicate in 9 times the volume of the s t a n d a r d reaction m i x t u r e (see METHODS AND MATERIALS) with 0.52 u n i t of Fraction V. Aliquots of 0.3 ml were w i t h d r a w n at the indicated times and t r e a t e d in the usual m a n n e r to determine the D N A hydrolyzed to acid-soluble fragments.
~60 x
A
&~t^a
o 40 - X
~2o 0
I
I
I
8.5
I
9
9.5 pH
I
I0
Fig. 3. Effect of p H and buffer on the enzyme activity. The m e a s u r e m e n t s were made under the s t a n d a r d conditions (see METHODS AND MATERIALS) with the indicated buffers. To each reaction m i x t u r e was added 0.o 7 unit of enzyme activity from F r a c t i o n V. The p H of the buffers (0.024 M) was m e a s u r e d at room t e m p e r a t u r e , The p H change t h a t occurs between r o o m t e m p e r a t u r e and 37 ° is the same for stock solutions of b o t h buffers. /x, g l y c i n e - N a O H ; C), Tris-HC1.
%
)
x
4o
o
" % , ~
-o -oo <~ z
zo
"5 $ o
I
-5
i0 -4
Molorily
i0-3
10-2
Fig. 4. Effect of metal ions on the enzyme activity. The m e a s u r e m e n t s were made u n d e r the s t a n d a r d conditions (see METHODS AND MATERIALS) w i t h the indicated metal ions. 0.07 unit of enzyme activity from F r a c t i o n V was added to each reaction mixture. A, Mg~+; FI, 1V~nZ+; C), C a 2 + + o . o I M Mg z+.
Biochim. Biophys. Acta, 224 (I97 o) 29-41
36
G . F . VOVIS, G. BUTTIN
Nucleoside triphosphate requirement. Fraction V has an absolute requirement for a nucleoside triphosphate. In the absence of any such compound, there is no detectable activity on native DNA. Fig. 5 shows the dose-response curve observed with ATP. Under the conditions examined, ATP has been found to be the most effective in fulfilling this requirement. CTP has been found to be about 80 % as effective as ATP but only at concentrations IOO times greater than the optimal ATP concentration, i.e, about 1. 5. lO -3 M for CTP versus 1.25" IO-s M for ATP. It is not yet certain whether this nonphysiological range of activity for CTP is due to CTP or to some contaminant in the commercially available CTP. ~o60 x
-o
c
0 10-6
1 10-5
.aTP molorily
I IO-a
t
10-3
Fig. 5. E f f e c t of A T P on t h e e n z y m e a c t i v i t y . The m e a s u r e m e n t s were m a d e u n d e r t h e s t a n d a r d c o n d i t i o n s (see METHOBS A N D M A T E R I A L S ) w i t h t h e i n d i c a t e d A T P c o n c e n t r a t i o n s . To each react i o n m i x t u r e 0.o 7 u n i t of e n z y m e a c t i v i t y from F r a c t i o n V w a s added.
Table I I shows the activities of other nucleoside triphosphates and other compounds as compared to that of ATP. The measurements were made at I. lO -5 M, which is in the range of optimal ATP activity. The other nucleoside triphosphates are all inactive at this concentration. In addition, the specificity is apparently independent
TABLE
II
EFFECT OF NUCLEOSIDE TRIPHOSPHATES AND OTHER COMPOUNDS ON THE ENZYME ACTIVITY M e a s u r e m e n t s were m a d e u n d e r t h e s t a n d a r d c o n d i t i o n s (see METHODS A N D M A T E R I A L S ) w i t h t h e i n d i c a t e d c o m p o u n d , whose s t o c k s o l u t i o n h a d been n e u t r a l i z e d w i t h N a O H , a n d 0.07 u n i t of e n z y m e a c t i v i t y from F r a c t i o n V.
Compound (z.o. zo -5 M)
nmoles DNA hydrolyzed to acid-soluble ]ragments
ATP CTP GTP UTP dATP dCTP dGTP dTTP ADP AMP A T P b u t no e n z y m e No t r i p h o s p h a t e
0.472 0.002 0.003 0.004 0.458 0.004 0.o05 0.oo 5 0.004 o.oo2 0.003 o.oo2
Biochim. Biophys. A a a , 224 (197 o) 29-41
A DEOXYRIBONUCLEASE FROM D .
pneumoniae
37
of the sugar moiety, for d A T P is as active as ATP. However, it is a triphosphate that is required, for ADP and AMP are both inactive. A T P hydrolysis. To determine whether ATP is consumed during the course of DNA hydrolysis, the amount of [7-32PIATP converted into norit nonadsorbable material as a function of enzyme concentration was measured (Fig. 6). In the absence of DNA, there is no change in the amount of norit nonadsorbable material, a fact which indicates that Fraction V is probably free of contaminating phosphatases.In contrast, with DNA included in the reaction mixture, there is an increase in the amount of 32p that is norit nonadsorbable, an amount which increases in proportion to the enzyme concentration. Since it is known that the extent of DNA hydrolysis, as measured by the production of acid-soluble fragments, is itself proportional to the amount of enzyme added in this range of enzyme concentration (Fig. I), it is strongly suggested that there is a direct proportion between the amount of ATP split and the amount of acid-soluble fragments produced (Fig. 7; approx. 2 nmoles of ATP per nmole of DNA hydrolyzed to acid-soluble fragments). Furthermore, the absence of ATP splitting in the absence of DNA indicates that this splitting is apparently dependent upon the concomitant hydrolysis of the DNA. zx
2OO
45
g g
§30
:
c a_
~15 50 c
A
A-
I
.50
_A
•
i
t
I00
150
Protein(rig)
0
1
I
i
25 .50 75 nrno~es o! DNA ocid solublex10 2
Fig. 6. The splitting of A T P as a function of protein concentration in Fraction V. The measurem e n t s were made as described in METHODS AND MATERIALS with [7-32PIATP. Approx. 3 % of the 3~p in t h e [7-3~P]ATP stock solution was norit non-adsorbable. A, + D N A ; A, - - D N A . Fig. 7. The splitting of A T P as a function of D N A hydrolyzed into acid-soluble fragments. The d a t a are from the same e x p e r i m e n t as those in Fig. 6. The [7-32PIATP rendered norit nonadsorbable is corrected for the background.
Nature o/attack. To determine whether the nature of attack is endonucleolytic or exonucleolytic, T 7 [32PIDNA was incubated in reaction mixtures that contained ATP but no enzyme, ATP and enzyme, or enzyme but no ATP. An aliquot was sedimented in a preformed alkaline sucrose gradient, and another was assayed for the DNA hydrolyzed to acid-soluble fragments (Fig. 8). No difference was observed in the DNA sedimentation pattern when either enzyme or ATP was absent from the reacBiochim. Biophys. Acta, 224 (i97 o) 29-41
38
G . F . VOVIS, G. BUTTIN
tion mixture, and no acid-soluble fragments were found under these conditions. This shows that Fraction V does not contain any significant amount of either endonucleolytic or exonucleolytic activity in the absence of ATP. Following incubation in the presence of both ATP and enzyme, IO % of the DNA was acid soluble and two DNA bands were observed in the gradient. One is at a position which is not significantly different from that of unaltered T 7 DNA, while the other is located at the position where low molecular weight DNA digestion products would be expected. This latter sedimentation pattern suggests that the enzyme liberates small fragments from preexisting free ends of DNA molecules (exonuclease). If the enzyme was an endonuclease whose internal attack produced either single-strand or double-strand breaks, then with IO % of the DNA hydrolyzed into acid-soluble fragments, a significant amount of degradation products would sediment between the unaltered DNA peak and the peak of very low molecular weight material1; this is clearly not observed. In addition, the gradient pattern of the hydrolyzed DNA also suggests that the products of DNA hydrolysis are mainly oligonucleotides. The peak of slow-sedimenting material represents approx. 30 % of the recovered (24 % of the applied) 32p activity, yet only Io % of the applied radioactive label is acid soluble. Thus, more than 50 % of this very low molecular weight material would appear to be acid insoluble and, therefore, certainly not to consist of mononucleotides. This oligonucleotide estimate in the digestion products is a minimum since the fractions from the gradient were acid precipitated and washed. 6000 O. 2 000' ~-
"1"ATP
4000~
/~V/~
2000
I000
' L i
0~
.....:~'I
1
-....... "f---~/"
II
21
!I0 31
i
20 I
/
6000 -kATP 2000
i
4000
. "°
2000
1000~-
0~ I
~1
2000
3J
21
,
0 I
0
0 It
0
li 00 000
"t-frcclionV
I
o
/
.
~
"o I
2I
Froclion number
',000
3I
0
40
80 Protein(n9,
120
Fig. 8. Alkaline sucrose gradient p a t t e r n s of undigested and partially digested DNA. A s s a y s were r u n in reaction m i x t u r e s containing T 7 phage [32P]D2qA, and the indicated additional comp o n e n t s as described in METHODS AND MATERIALS. I n the alkaline sucrose gradients, T 7 phage [aH]DNA was added as a reference marker. The recovery, in all cases, was approx. 80 % for the 3zp activity and approx. IiO ~o for the 8H activity. The greater t h a n ioo % recovery of 3H is p r o b a b l y an artifact resulting from differential quenching of low-energy fl particles within paper. Fig. 9. Hydrolysis of various s u b s t r a t e s as a function of protein concentration in Fraction V. The m e a s u r e m e n t s were made u n d e r the s t a n d a r d conditions (see METHODS A N D M A T E R I A L S ) in the presence (closed symbols) or absence (open symbols) of A T P and with 4 nmoles of the following substrates: A - A , A--&, native T 7 phage D•A; C)-C), O - O , h e a t - d e n a t u r e d T 7 phage DNA; [~-[~, I - I , poly d ( A - T ) .
Biochirn. Biophys. Mcta, 224 (197 o) 29-41
A DEOXYRIBONUCLEASE FROM D.
pneumoniae
39
Substrate speci]icity. Fig. 9 shows the activity of Fraction V on various substrates. Under standard conditions, Fraction V exhibits limited activity on heatdenatured DNA. In contrast to what is observed with native DNA, some activity still can be detected in the absence of ATP. Whether this latter activity is due to the same enzyme that is active on native DNA has not be further investigated. Fraction V also attacks poly d(A-T). Under the standard conditions, the extent of hydrolysis, as measured b y the release of acid-soluble [3H]thymidylate, is about 50 % of that observed with native DNA. The activity on poly d(A-T) rules out a base specificity of attack for the production of oligonucleotides that would be governed b y guanine-cytosine base pairs. DISCUSSION
This work establishes the presence of a previously undescribed deoxyribonuclease in D. pneumoniae that is characterized b y its requirement for ATP, which is split during DNA hydrolysis. During the course of these investigations, independent reports appeared which demonstrated that nucleoside triphosphate-dependent deoxyribonucleases are also present in Bacillus laterosporus 13 and Mycobacterium smeg-
matisl4,15. The enzymes from these latter two organisms as well as those from D. pneumoniae, E. coli1,16,17 and M. lysodeikticus (refs 2, 18, 19; A. R6RSCH, personal communication) appear to be members of the same class of enzymes as judged b y their biochemical properties. They all are active under alkaline conditions. All have a metal ion requirement which is fulfilled by a relatively high concentration of Mg 2+ (approx. I . lO -2 M). The reported data are consistent with the fact that Mn 2+ can substitute at lower concentrations. In addition, the five enzymes are much more active on native DNA than on denatured DNA. Work with the enzymes from B. laterosporus, D. pneumoniae, E. coli, and M. lysodeikticus, all of which were purified at least 5oo-fold, demonstrates that there is no activity on native DNA in the absence of the nucleoside triphosphates. Under the conditions examined, ATP appears to be the most effective at satisfying this requirement for all five enzymes and probably is the physiological effector. The other nueleoside triphosphates have varying degrees of activity with the various enzymes. In addition, this nucleoside triphosphate requirement is independent of the sugar moiety for dATP, whenever tested, readily substitutes for ATP. With all the enzymes quoted so far, ATP is split during the course of DNA hydlolysis and when it has been investigated with highly purified enzyme preparations, the products of ATP splitting were found to be ADP and P1 (refs. 16, 13, 19). In all cases, there is proportionality between the amount of ATP split and the amount of DNA hydrolyzed to acid-soluble fragments. However, the stoichiometry of the reaction remains obscure, for the DNA hydrolysis products include oligonucleotides. The presence among the reaction products of oligonucleotides, which m a y be digested further as a function of time b y the same enzyme, makes it impossible so far to correlate the number of ATP molecules converted to ADP and Pl with the numbei of phosphodiester bonds broken in the DNA duplex. It m a y be pointed out here that the nature of the degradation products distinguishes this class of enzymes from the restriction endonuclease discribed b y MESELSON AND YUAN2°. Biochim. Biophys. Acta, 224 (197 o) 29-41
4°
G.F. VOVIS, G. BUTTIN
We concluded from the sedimentation pattern of partially digested DNA that the nature of attack by the pneumococcal enzyme is very likely to be exonucleolytic. From their studies, ANAl et al. is concluded that the attack by the M . lysodeikticus deoxyribonuclease is endonucleolytic. This m a y be, to some extent, a problem of semantics. We consider that exonucleolytic attack is characterized b y where the degradation is initiated (freeends)rather than by the nature of the digestion products (mononucleotides and/or oligonucleotides). Actually, the mode of digestion for the M . lysodeikticus enzyme as reported b y ANAI et al. is is not essentially different from that suggested by our own experiments with the pneumococcal enzyme. Clearly, in both systems, no degradation products of a size intermediate between unaltered DNA molecules and oligonucleotides is observed. This suggests either that the time required for complete degradation of a DNA molecule is short as compared to the time required for the fixation of an enzyme molecule on a new chain or that essentially most of the DNA molecules have been partially digested. Their sedimentation studies of increasingly digested DNA would seem to eliminate this latter interpretation for the micrococcal system. Their viscosity studies, however, do not demonstrate more than the gradient patterns; for although the comparison of residual viscosity with percent acid-soluble fragments indicates a more rapid loss of vicosity than that expected with a purely exonucleolytic attack, such a comparison does not take into account the fact that the oligonucleotides released are likely to be, in part, acid insoluble as suggested by our results. This leads to an underestimation of the actual extent of DNA degradation. If one assumes that the nature of the products formed is identical in the pneumococcal and micrococcal systems and that the amount of DNA digested is twice that found to be acid soluble as suggested by our own experiments, then the viscosity decrease observed by ANAI et al. is corresponds to the one reported for a purely exonucleolytic type of degradation. The fact that the ATP splitting is concomitant with DNA hydrolysis suggests, as has already been considered by others 16,1~,19, that the ATP is serving as an energy source rather than just as an allosteric effector. OISHIiv suggests that because the E. coli enzyme attacks single-stranded DNA in the absence of ATP, the energy m a y be required to cause a configurational change in the DNA substrate. However, it should be pointed out that if these enzymes isolated from various organisms really have a common mechanism of action, then the apparent inability of some of them 13'~s to attack denatured DNA without ATP m a y challenge this interpretation. A definitive conclusion concerning the significance of ATP splitting should be postponed until further experiments unambiguously demonstrate that hydrolysis of denatured DNA, when it is observed, is due to the activity of the same enzyme. In any case, one should not fail to think of the ATP requirement as one way whereby the cell exerts some control in the balance between the synthetic and the degradative processes which take place at the level of DNA. The similar biochemical properties of these five enzymes do strongly suggest that they actually play a similar physiological role in the cell. Studies with E. coli show that the enzyme is involved in bacterial recombination 16,17'2~. A companion report demonstrates that such is also the case in pneumococcus 3.
Biochim. Biophys. Acta. 224 (197o) 29-41
A DEOXYRIBONUCLEASE FROM D. p n e u m o n i a e
41
ACKNOWLEDGMENTS
W e are deeply i n d e b t e d to Dr. Boris Ephrussi for v e r y generously p r o v i d i n g l a b o r a t o r y space an d for his c o n s t a n t interest in this work. Th e d e d i c a t i o n a n d e x p e r t t e c h n i c a l assistance of Catherine F o u g e r e is most g r a t e f u l l y acknowledged. We also wish to t h a n k Marguerite DGchamps for p r e p a r i n g most of t h e r a d i o a c t i v e D N A an d Dr. H. F u k u h a r a for v e r y k i n d l y t r a n s l a t i n g one of the references from J a p a n e s e for us. The m a t e r i a l herein was t a k e n from the dissertation s u b m i t t e d to the G r a d u a t e School of Case W e s t e r n R e s e r v e U n i v e r s i t y b y G. F. V. in p a r t i a l fulfillment of t h e r e q u i r e m e n t s for the degree of D o c t o r of Philosophy in Biology. This research was s u p p o r t e d b y a g r a n t to the late Dr. H a r r i e t t E p h r u s s i - T a y l o r from the D~l~gation G6n~rale ~ la Recherche Scientifique et T e c h n i q u e ; a N a t i o n a l I n s t i t u t e s of H e a l t h p r e d o c t o r a l research fellowship 5-FoI-GM-36 194; an E u r o p e a n Molecular Biology Organization short t e r m fellowship; a n d grants from the Commissariat 5~ l ' E n e r g i e A t o m i q u e to both laboratories. A p r e l i m i n a r y report a n d a b s t r a c t were given at the E u r o p e a n S y m p o s i u m on the Molecular Mechanism of R e c o m b i n a t i o n in Micro-Organisms, L u n t e r e n , t h e N e t h e r l a n d s , Sept. 28-3 o, 1969, a n d at a m e e t i n g of t h e Soci6t6 Fran~aise de G6n6tique, Toulouse, France, April 24-25, 197o , respectively.
REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21
G. BUTTIN AND IV[. WRIGHT, Cold Spring Harbor Syrup. Quant. Biol., 33 (1968) 259. Y. TSUDAAND B. S. STRAUSS,Biochemistry, 3 (1964) 1678. G. F. Vovls AND G. BUTTIN, Biochim. Biophys. Acta, 224 (197o) 42. A. M. SICARD, Genetics, 5° (1964) 31. R. K. APPLEYARD,Genetics, 39 (1954) 44o. A. B. PARDEE, F. JACOBAND J. [k~IONOD,J. Mol. Biol., i (1959) 165. C. LEVINTHAL,E. R. SIGNERAND t~. FETHEROLF, Proc. Natl. Acad. Sci. U.S., 48 (1962) 123o. C. C. RICttARDSON, R. B. INMANAND A. KORNBERG,J. Mol. Biol., 9 (1964) 46. M. E. REICHMANN,S. A. RICE, C. A, THOMASAND P. DOTY, J. Am. Chem. Soc., 76 (1954) 3047• C. C. RICHARDSON, Procedures in Nucleic Acid Research, Harper and Row, New York, 1966, p.212. O. H. LOWRY, N. J. ROSENBROUGH,A. L. EARRAND R. J. RANDALL,J. Biol. Chem., 193 (1951) 265 . O. WARBURGAND W. CHRISTIAN,Biochem. Z., 31o (1941) 384 . M. ANAl, J. Japan. Biochem. Soc., 39 (1967) 167. F. G. WINDER AND M. P. COUGHLAN, Biochem. J., I i i (1969) 679. F. G. WINDER AND M. LAVlN, Biochem. J., 115 (1969) 6. M. WRIGHT AND G. BUTTIN, Bull. Soc. Chim. Biol., 51 (1969) 1373. M. OISHI, Proc. Natl. Acad. Sci. U.S., 64 (1969) 1292. M. ANAl, T. HIRAttASHIAND Y. TAKAGI, J. Biol. Chem., 245 (197o) 767 . M. ANAI, T. HIRAHASHI,M. YAMANAKAAND Y. TAKAGI,J. Biol. Chem., 245 (197o) 775. M. ~¢[ESELSONAND R. YUAN, Nature, 217 (1968) IIIO. S. D. ]3ARBOURAND A. J. CLARK, Proc. Natl. Acad. Sci. U.S., 65 (197o) 955. Biochim. Biophys. Acta, 224 (197o) 29-41
29
BBA 96647
AN A T P - D E P E N D E N T DEOXYRIBONUCLEASE FROM DIPLOCOCCUS
PNEUMONIAE I. PARTIAL PURIFICATION AND SOME BIOCHEMICAL P R O P E R T I E S G E R A L D F. VOVIS* AND GI~RARD B U T T I N
Centre de Gdndtique Moldculaire du C.N.R.S., 91 Gi[-sur-Yvette; Service de Gdndtique Cellulaire de L'Institut Pasteur et du Coll~ge de France, Paris (France) (Received J u l y 9th, 197 o)
SUMMARY
I. A 5-step procedure is described for the partial purification of a nucleoside triphosphate-dependent deoxyribonuclease from Diplococcus pneumoniae. An overall purification of more than 5oo-fold was achieved. 2. The enzyme was found to be active under alkaline conditions (optimum pH from 9.25 through, at least, 9.8) and to require a metal ion and a nucleoside triphosphate for its activity on native DNA. Mg~+ and Mn 2+ and ATP were found to be the most effective at satisfying these requirements. 3. ATP is split during the course of DNA hydrolysis. 4- The zone sedimentation pattern of the DNA hydrolysis products is consistent with an enzyme that attacks preexisting free ends (exonuclease), liberating oligonucleotides among its products. 5. The enzyme is much more active on native DNA than on heat-denatured DNA. It attacks poly d(A-T) but exhibits no activity with rRNA.
INTRODUCTION
The discovery of an ATP-dependent deoxyribonuclease, which splits the required ATP during DNA hydrolysis and appears to be involved in the process of genetic recombination, was first reported by BUTTIN AND WRIGHT1 during investigations of deoxyribonuclease activities in Escherichia coli extracts. The existence of an enzyme with apparently similar biochemical properties in Micrococcus lysodeikticus 2 suggested that this type of enzyme might represent a particular class of enzymes which catalyzes a major step in bacterial recombination in various organisms. To explore this possibility, we chose to examine D. pneumoniae, an organism possessing a transformation system which is an ideal model system with which to study recombination at the molecular level because of the ability to make correlations between DNA structure and function. The initial experiments were undertaken Abbreviations: BBOT, 2,5-bis-[2-(5-tert.-butylbenzoxazolyl)]thiophene; d i m e t h y l - P O P O P , 1,4-bis-[2-(4-methyl-5-phenyloxazolyl)]benzene; PPO, 2,5-diphenyloxazole. * Present address: Genetics Laboratory, The Rockefeller University, New York, N. Y. 10021 U.S.A.
Biochim. Biophys. Acta, 224 (I97O) 29-41
3°
G . F . VOVIS, G. BUTTIN
to determine whether pneumococcus also possesses a nucleoside triphosphate-dependent deoxyribonuclease with the aim of investigating the enzymatic basis of genetic recombination in a transformable strain. This report describes the partial purification and some biochemical properties of such an enzyme in pneumococcus, and a companion report presents genetic evidence establishing its enzymatic involvement in bacterial recombination S. During the course of these investigations, various reports appeared concerning the properties of ATP-dependent deoxyribonucleases from several other different organisms. To what extent these enzymes appear to be similar in their biochemical properties will be discussed.
METHODS AND MATERIALS
Bacteria and growth media The strain, C1z str-r 41, used for the enzyme purification was a derivative of Clone 3 (ref. 4), which had been transformed to streptomycin resistance 3. The cells for the enzyme purification were grown at 37 ° in a medium prepared by combining the following solutions: I °/o Difco neopeptone (pH 7.7) containing 0.85 % NaC1; IO °/o Difco yeast extract (pH 7.6); 25 % glucose; 1.68 % L-glutamine; in the ratio of 50:4:2 :I, respectively. The inoculum, 1/8 the volume of the mass culture, was grown to a density of approx. 2.1o s colony-forming units/ml in the same medium but with 20 times less glucose. The pH of the mass culture was maintained near neutrality by titrating phenol red, present at 0.0002 ~o, with concentrated NaOH. DNA preparation To prepare 3H-labeled T 7 phage DNA, T 7 phage were used to infect a culture of E. eoli C6oo (ref. 5) growing in medium 63 lacking glycerol but containing vitamin B 1 (ref. 6) and supplemented per 1 with 20 ml 20 °/o glucose and 50 ml IO % Difco casein amino acids (vitamin free). 5 min later, EMe-3Hjthymidine (18 C/mmole) and deoxyadenosine were added to a final concentration of approx. 3 #C/ml and approx. 4 #g/ml, respectively. The 32p-labeled T 7 phage were prepared in low-phosphate minimal medium:, supplemented per 1 with 20 m120 °/o glucose, 5 ml IO °/o Difco casein amino acids (vitamin free), IO ml 1 % L-threonine, IO ml I °/o L-leucine and I ml 0.05 % vitamin B 1. The carrier-free Ha32po 4 was added at the time of infection to a final concentration of about 20/~C/ml. In both cases, the multiplicity of infection was about 5 and the infected cultures were shaken at 37 ° until complete lysis. The DNA was purified essentially by the method of RICHARDSON et al. s. To remove the residual phenol the DNA solution was dialyzed extensively against a o.oi M Tris, o.I lV[ NaC1 and o.oi M EDTA solution, whose pH was adjusted to 8 with NaOH, and stored at o °. The specific activity of the [3HIDNA was 4" lO4-1" lO5 counts/min per/,g DNA while that of [32p~DNA was initially 4.4" I°a counts/min per #g DNA, both as determined by counting diluted aliquots on glass-filter paper (Carl Schleicher and Schuell Co., Keene, N.FI.) in IO ml of 2,5-bis-E2-(5-tert.-butylbenzoxazolyl)lthiophene (BBOT) scintillation fluid (8 g/1 toluene) with the preset channels in the Packard Tri-Carb liquid scintillation spectrometer Model 3375. The heat-denatured DNA was prepared by heating [3HIDNA , in o.oi M Tris-HC1 (pH 8), containing Biochim. Biophys. Acta, 224 (197 o) 29--41
A DEOXYRIBONUCLEASE FROM D.
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31
o.oi M NaC1, for IO min in a boiling water bath followed by rapid cooling in ice water. E D T A was then added to a final concentration of o.oi M.
Enzyme assay The standard reaction mixture (o.3 ml) contained 6#moles glycine-NaOH (pH 9.3), 3/,moles MgC12, 0.3 #mole 2-mercaptoethanol, 8 nmoles T 7 phage E3H3DNA, and 7.5 nmoles ATP (sodium salt; the stock solution was neutralized to p H 7 with NaOH). The reaction was started b y adding to the prewarmed reaction mixture 20/,1 of the material to be assayed, containing 0.005-0.03 enzyme unit, and the resulting solution was incubated at 37 ° for 5 rain. The reaction was stopped b y removing to ice and immediately adding 0.2 ml cold salmon sperm DNA (2.5 mg/ml) and 0.5 ml cold 0. 7 M trichloroacetic acid. After at least IO rain at o °, the resulting precipitate was pelleted by a Io-min centrifugation at 20 ooo × g and 0.3 ml of the supernatant was counted in IO ml of modified Bray's solution (200 g naphthalene, 5 ° mg 1,4-bis-E2-(4-methyl-5-phenyloxazolyl)]-benzene (dimethyl-POPOP), 7 g 2,5diphenyloxazole (PPO), and dioxane to I l) with a Packard liquid scintillation spectrometer. To determine specifically the ATP-dependent activity, a blank was run in parallel without ATP. One unit of enzyme activity is defined as that amount of enzyme which hydrolyzes I nmole of DNA to acid-soluble fragments per min under the above conditions and corrected for the blank value. O n e / , a t o m of DNA phosphorus is considered to be equivalent to 300/~g DNA, and E~ ~ was taken to be 213 at 26o nm (ref. 9). Beginning with the material after Fraction I I I (see RESULTS), dilutions were carried out in 0.02 M potassium phosphate buffer (pH 6.5) containing 0.2 M NaC1, o.oi M 2-mercaptoethanol, 20 % glycerol (v/v), and 0.5 mg/ml bovine albumin powder. The other fractions were diluted in the extraction buffer. Under these conditions, the activity was proportional to the amount of protein added for all fractions.
Assay/or norit nonadsorbable material ET-3zP]ATP (activity 8oo mC/mmole, > 97 % purity) was diluted Io-fold with an equal molar solution of nonradioactive ATP, and the standard reaction mixture contained 4 nmoles of this latter solution. The reaction was carried out and terminated in the normal manner. After centrifugation, 0. 5 ml of the supernatant was transferred to a tube containing 0.35 ml water, 0.3 ° ml norit-carrier solution lo, and 0.60 ml norit suspension (30 % packed volume), all at o °. The mixture was shaken for I rain. After 5 rain at o °, the mixture was centrifuged, and the supernatant was transferred quantitatively to another tube containing o.Io ml norit-carrier solution and 0.60 ml norit suspension. After treating as described above, I.O ml of the supernatant was removed to an aluminum planchet, and I drop of 3 M NaOH was added. The sample was evaporated to dryness and the radioactivity was measured in a gas-flow counter.
Alkaline sucrose gradients o/enzyme hydrolyzates The enzyme reaction was run in twice the volume of the standard reaction mixture with 24 nmoles T7 phage ~32P]DNA and o.15 unit of Fraction V. After 5 rain at 37 °, the reaction was stopped b y chilling on ice; 300/,1 was removed to 65/,1 of a mixture prepared by combining 60/~1 o. 7 M NaOH, 16o/~1 0. 5 M E D T A (pH 8), and 40/~l T 7 phage EaH]DNA; and the amount of DNA hydrolyzed to acid-soluble fragments in the remaining reaction mixture was determined in the usual manner. Then Biochirn. Biophys. Acta, 224 (I97 o) 29-41
32
G . F . VOVIS, G. BUTTIN
I5o #1 of the above alkaline mixture was overlayered on 4.8 ml of 5-2o % sucrose prepared in 0. 7 M NaC1, 0.3 M NaOH, and o.ooi M EDTA and centrifuged at 4 ° for 4 h at 39 ooo rev./min in the Spinco SW-5oL rotor. The bottom of the cellulose nitrate tube was pierced and I2-drop fractions were collected on Whatman 3 mm paper discs. The paper discs were washed successively in the cold with 5 % trichloroacetic acid, 0.2 N HC1, and 80 °/o ethanol. The discs were air dried and counted in Io ml of BBOT scintillation fluid. Protein determination In all the pooled fractions except Fraction V, protein was determined by the method of LOWRY et al. 11 on the acid precipitable material. Protein in Fraction V was estimated by ultraviolet absorption 1~ with a Zeiss spectrophotometer Model PMQ I2. To determine tile specific activity of the enzyme in the individual fractions from the first two columns, the protein was estimated from the absorption at 280 nm as measured by a Gilson Model G2 ultraviolet spectrophotometer and recorder. The absorption values were calibrated by determining, with the method of LOWRY et al. 11, the protein present in the acid-precipitable material of those fractions immediately preceding and succeeding the enzyme peak. Bovine albumin powder was used as the protein standard. Materials All chemicals were reagent grade. (NH4)2SO 4 (ultrapure) was purchased from Mann Research Laboratories; Whatman DEAE-cellulose (DE I) and Whatman phosphocellulose iP II) from W. and R. Balston, Ltd.; hydroxyapatit (hypatit C) from Clarkson Chemical Co. ; dextran 500 from Pharmacia; polyethylene glycol 6000 from Touzart and Matignon, Paris; ATP, ADP, AMP, CTP, GTP, UTP, dCTP, dGTP, and d T T P (all sodium salts) from CalBiochem; dATP (sodium salt) from Sigma; poly d(AIMe-3HIT) (control 57-2-324; 25.1 mC/mmole phosphorus; minimal tool. wt. > 50 ooo) from Miles Laboratories; EMe-3H~thymidine, H33~P04 (carrier-free) and EV-32pIATP from le Commissariat ~ l'Energie Atomique, France; BBOT, PPO and dimethyl-POPOP from Packard Instrument Co., Inc.; bovine albumin powder (Fraction V from bovine plasma) from Armour Pharmaceutical Co. The I3~PIrRNA was a generous gift from Dr. H. Fukuhara.
RESULTS
Puri]ieation o[ the enzyme All steps, unless specifically noted, were carried out at o-3 ° . Preparation o] extract. The pneumococcal cells, harvested in late logarithmic phase (4 g of wet cells per I of culture), were washed twice with 0.85 % NaC1 (pH 7). 3° g of cells were resuspended in 47 ml of o.oi M Tris-HC1 buffer (pH 7.7) containing o.oi M 2-mercaptoethanol. The cells were sonicated with a Sonifier cell disruptor, Model WI85D (Heat Systems-Ultrasonics, Inc.) at a power setting of 9 ° W for 25 rain, while the glass centrifuge tube containing the cell suspension was maintained in a salt-ice-water bath (approx. --IO°). The sonicate was centrifuged twice at 25 500 ×g for IO rain. The supernatant (71 ml, Fraction I, Table I) was collected. Biochim. Biophys. Acta, 224 (197 o) 29-41
A DEOXYRIBONUCLEASE FROM D. TABLE
pneumoniae
33
I
PURIFICATION OF THE ATP-DEPENDENT DEOXYRIBONUCLEASE
Fraction and step
I. II. III. IV. V.
Crude e x t r a c t Phase separation DEAE-cellulose Phosphocellulose Hydroxyapatit
Units pooled per step Number
%
3146 2962 981 274
ioo 94 33 28 39
lO 7
Units recoverable per step (%)
Specific activity (units/rag protein)
Accumulative purification
IOO ioo 62 39 44
1.24 3.52 18. 4 4 °2 921
i .o 2.8 14.8 324 743
(-/ola)
Phase partition. To 17. 4 ml of Fraction I were added with mixing at o °, 2.o ml 20 °/o dextran (w/w) and 5.6 ml 30 % polyethylene glycol (w/w); the emulsion was mixed for 2 h at o °. After centrifuging at 34 7 °0 x g for IO rain, the clear yellow upper phase was removed, discarded and replaced by the upper phase from a mixture prepared by combining extraction buffer, dextran and polyethylene glycol in the same proportions as above and by mixing and centrifuging as above. Then, while stirring at o °, 6.52 g NaC1 were gradually added; the mixing was continued for 2 h at o °. The emulsion was centrifuged at 34 700 × g for 80 rain, and the slightly cloudy, pale yellow upper phase was removed and saved. This procedure was repeated three more times with the remaining portion of Fraction I. The four resulting upper phase fractions were combined (lO3 ml) and dialyzed against 4.5 1 of extraction buffer for 5 h. The dialysis procedure was repeated two more times against fresh extraction buffer. The dialyzed liquid (177 ml, Fraction II) was collected and fractionated on a DEAEcellulose column the same day. DEA E-cellulose chromatography. A DEAE-cellulose column (19.6 cm 2 × 14 cm) was prepared and equilibrated with the extraction buffer. Fraction II was adsorbed to the column at an average rate of 7 ° ml/h. The column was washed with 57 ° ml of the equilibration buffer, and then a linear gradient of elution, total volume 3 1, was applied with the equilibration buffer and the equilibration buffer containing 0. 4 M NaC1 as limiting concentrations. Fractions of 2o-21 ml were collected at an average flow rate of 14o ml/h. Approx. 60 % of the applied activity was eluted between 7 and 13.2 resin bed volumes of effluent. Peaks fractions containing specific activity greater than IO units/rag protein were pooled (159 ml, 49 % recovery of the applied activity) and dialyzed against 8 1 of 0.02 N[ potassium phosphate buffer (pH 6.5) containing o.oi M 2-mercaptoethanol and 5 % glycerol (v/v) for 6 h. The dialysis buffer was changed, and the dialysis was continued for another 6 h. The dialyzed liquid was collected (135 ml, Fraction III). Approx. 30 % of the activity was lost during dialysis. Phosphocellulose chromatography and (NH4)2S04 concentration. A phosphocellulose column (4.9 cm~ × 12.5 cm) was prepared and equilibrated with 0.02 M potassium phosphate buffer (pH 6.5) containing o.oi M 2-mercaptoethanol. Fraction III was added to the column at a rate of 123 ml/h and was washed into the column with 12o ml of the above buffer. A linear gradient of elution was applied, with 0.02 and 0.4 IV[ potassium phosphate buffer (pH 6.5) containing o.oi M 2-mercaptoethanol as the limiting concentrations; 450 ml of each buffer was used. Fractions of 9-1o ml Biochim.WBiophys. Acta, 224 (I96o) 29-41
34
G. F. VOVIS, G. BUTTIN
were collected at an average flow rate of 13o ml/h. About 4o% of the applied activity was eluted between 6.2 and 7-7 resin bed volumes of effluent. Peak fractions with a specific activity greater than 200 units/mg protein were pooled (36 ml) and concentrated by (NHi)~SO 4 precipitation (18 g). The (NH4)2SO 4 was added gradually with stirring at o °. The stirring was continued for 15 min at o °, and the mixture was centrifuged at 41 4 o o × g for 3 ° min. The precipitate was redissolved in 3.5 ml of 0.02 M potassium phosphate buffer (pH 6.5) containing o.2 M NaC1 and o.oi M 2mercaptoethanol by stirring at o ° overnight. Undissolved precipitate was removed b y centrifugation, and the solution was dialyzed for 4 h against this latter buffer to remove a n y residual (NH4)2SO t. The dialyzed liquid (Fraction IV, 28 ~/o of the activity applied to the phosphoeellulose column) was collected. Hydroxyapatit chromatography. A hydroxyapatit column (0.785 cm 2 ×3.5 cm) was prepared and equilibrated with 0.02 M potassium phosphate buffer (pH 6.5) containing 0.2 ~ NaC1 and o.oi M 2-mercaptoethanol. Fraction IV was added to the column and adsorbed over a I6.5-min period. The column was then washed successively with I ml of 0.02 ~ , 2 ml of 0.05 M, and 3 ml of o.I M potassium phosphate buffer (pH 6.5), each containing 0.2 M NaC1 and o.oi M 2-mercaptoethanol. A linear gradient of elution, total volume of 60 ml, was applied with o. I M and 0. 4 M potassium phosphate buffer (pH 6.5) containing 0.2 M NaC1 and o.oi M 2-mercaptoethanol as the limiting concentrations. Fractions of 1.5 ml were collected every 2 rain. To determine the recoverable activity, the fractions must be dialyzed to remove some inhibitory material (probably Ca2+), for assay of a given fraction reveals a 2-3-fold increase in enzyme activity after dialysis. About 45 % of the applied activity was eluted between 3-3 and 8.2 resin bed volumes of effluent. The enzyme-containing fractions all exhibited an absorbance at 280 nm of less than o.o18. The six fractions containing the highest activity were pooled (9 ml, 39 % of the applied activity) and dialyzed for 8 h against I 1 of 0.02 M potassium phosphate buffer (pH 6.5) containing 0.2 M NaC1, o.oi M 2-mercaptoethanol, and 20 ~o glycerol (v/v). The dialyzed liquid (5.4 ml, Fraction V) was collected and stored at o °. Fraction V had been stable (no detectable loss of activity) at o ° for over 7 months.
Properties o] Fraction V Proportionality. As shown in Fig. I, the enzyme activity in Fraction V is proportional to the amount of protein added through at least 15o ng. Fig. 2 shows that the enzyme activity is not proportional to the time of incubation at 37 ° for periods longer than 5 min. pH optimum. The enzyme is active under alkaline conditions (Fig. 3). The p H optimum in glycine-NaOH buffer, where the p H was measured at room temperature, begins at about 9.25 and extends at least through 9.8. Metal ion requirement. The enzyme requires a metal ion for its activity, and either Kg2+ or Mn z+ can satisfy this requirement (Fig. 4). The optimum range for 1rig2+ is from approx. 7" IO-a M to about 2-IO -~ M, while the optimum concentration for Mn 2+ is 4" lO-4 M. The sharp decline in the Mn 2- activity at higher concentrations corresponds with the formation of a precipitate in the standard reaction mixture. Ca 2+ at a concentration greater than I . IO-a M inhibits the enzyme activity as assayed under the standard conditions (I-lO -2 M Mg2+). At a Ca 2+ concentration greater than or equal to I . l O -3 M, a precipitate is also observed in the reaction mixture. Biochim. Biophys. Acta, 224 (197 o) 29-41
pneumoniae
A DEOXYRIBONUCLEASE FROM D.
o
~o 75
A At,
15
35
-i _0
x
5 50
~,c
o Z 0
"6
E
g 25 c
/
jo o/o/8p
Q_
I
0
I
I
50 I00 ng Prolein
150
0
I tO
t
20 Time (rain)
I. 30
Fig. i. E n z y m e activity as a function of protein concentration in Fraction V. The m e a s u r e m e n t s were m a d e u n d e r the s t a n d a r d conditions (see METHODS AND MATERIALS). Fig. 2. Hydrolysis of D N A into acid-soluble f r a g m e n t s as a function of time. The reaction was r u n in duplicate in 9 times the volume of the s t a n d a r d reaction m i x t u r e (see METHODS AND MATERIALS) with 0.52 u n i t of Fraction V. Aliquots of 0.3 ml were w i t h d r a w n at the indicated times and t r e a t e d in the usual m a n n e r to determine the D N A hydrolyzed to acid-soluble fragments.
~60 x
A
&~t^a
o 40 - X
~2o 0
I
I
I
8.5
I
9
9.5 pH
I
I0
Fig. 3. Effect of p H and buffer on the enzyme activity. The m e a s u r e m e n t s were made under the s t a n d a r d conditions (see METHODS AND MATERIALS) with the indicated buffers. To each reaction m i x t u r e was added 0.o 7 unit of enzyme activity from F r a c t i o n V. The p H of the buffers (0.024 M) was m e a s u r e d at room t e m p e r a t u r e , The p H change t h a t occurs between r o o m t e m p e r a t u r e and 37 ° is the same for stock solutions of b o t h buffers. /x, g l y c i n e - N a O H ; C), Tris-HC1.
%
)
x
4o
o
" % , ~
-o -oo <~ z
zo
"5 $ o
I
-5
i0 -4
Molorily
i0-3
10-2
Fig. 4. Effect of metal ions on the enzyme activity. The m e a s u r e m e n t s were made u n d e r the s t a n d a r d conditions (see METHODS AND MATERIALS) w i t h the indicated metal ions. 0.07 unit of enzyme activity from F r a c t i o n V was added to each reaction mixture. A, Mg~+; FI, 1V~nZ+; C), C a 2 + + o . o I M Mg z+.
Biochim. Biophys. Acta, 224 (I97 o) 29-41
36
G . F . VOVIS, G. BUTTIN
Nucleoside triphosphate requirement. Fraction V has an absolute requirement for a nucleoside triphosphate. In the absence of any such compound, there is no detectable activity on native DNA. Fig. 5 shows the dose-response curve observed with ATP. Under the conditions examined, ATP has been found to be the most effective in fulfilling this requirement. CTP has been found to be about 80 % as effective as ATP but only at concentrations IOO times greater than the optimal ATP concentration, i.e, about 1. 5. lO -3 M for CTP versus 1.25" IO-s M for ATP. It is not yet certain whether this nonphysiological range of activity for CTP is due to CTP or to some contaminant in the commercially available CTP. ~o60 x
-o
c
0 10-6
1 10-5
.aTP molorily
I IO-a
t
10-3
Fig. 5. E f f e c t of A T P on t h e e n z y m e a c t i v i t y . The m e a s u r e m e n t s were m a d e u n d e r t h e s t a n d a r d c o n d i t i o n s (see METHOBS A N D M A T E R I A L S ) w i t h t h e i n d i c a t e d A T P c o n c e n t r a t i o n s . To each react i o n m i x t u r e 0.o 7 u n i t of e n z y m e a c t i v i t y from F r a c t i o n V w a s added.
Table I I shows the activities of other nucleoside triphosphates and other compounds as compared to that of ATP. The measurements were made at I. lO -5 M, which is in the range of optimal ATP activity. The other nucleoside triphosphates are all inactive at this concentration. In addition, the specificity is apparently independent
TABLE
II
EFFECT OF NUCLEOSIDE TRIPHOSPHATES AND OTHER COMPOUNDS ON THE ENZYME ACTIVITY M e a s u r e m e n t s were m a d e u n d e r t h e s t a n d a r d c o n d i t i o n s (see METHODS A N D M A T E R I A L S ) w i t h t h e i n d i c a t e d c o m p o u n d , whose s t o c k s o l u t i o n h a d been n e u t r a l i z e d w i t h N a O H , a n d 0.07 u n i t of e n z y m e a c t i v i t y from F r a c t i o n V.
Compound (z.o. zo -5 M)
nmoles DNA hydrolyzed to acid-soluble ]ragments
ATP CTP GTP UTP dATP dCTP dGTP dTTP ADP AMP A T P b u t no e n z y m e No t r i p h o s p h a t e
0.472 0.002 0.003 0.004 0.458 0.004 0.o05 0.oo 5 0.004 o.oo2 0.003 o.oo2
Biochim. Biophys. A a a , 224 (197 o) 29-41
A DEOXYRIBONUCLEASE FROM D .
pneumoniae
37
of the sugar moiety, for d A T P is as active as ATP. However, it is a triphosphate that is required, for ADP and AMP are both inactive. A T P hydrolysis. To determine whether ATP is consumed during the course of DNA hydrolysis, the amount of [7-32PIATP converted into norit nonadsorbable material as a function of enzyme concentration was measured (Fig. 6). In the absence of DNA, there is no change in the amount of norit nonadsorbable material, a fact which indicates that Fraction V is probably free of contaminating phosphatases.In contrast, with DNA included in the reaction mixture, there is an increase in the amount of 32p that is norit nonadsorbable, an amount which increases in proportion to the enzyme concentration. Since it is known that the extent of DNA hydrolysis, as measured by the production of acid-soluble fragments, is itself proportional to the amount of enzyme added in this range of enzyme concentration (Fig. I), it is strongly suggested that there is a direct proportion between the amount of ATP split and the amount of acid-soluble fragments produced (Fig. 7; approx. 2 nmoles of ATP per nmole of DNA hydrolyzed to acid-soluble fragments). Furthermore, the absence of ATP splitting in the absence of DNA indicates that this splitting is apparently dependent upon the concomitant hydrolysis of the DNA. zx
2OO
45
g g
§30
:
c a_
~15 50 c
A
A-
I
.50
_A
•
i
t
I00
150
Protein(rig)
0
1
I
i
25 .50 75 nrno~es o! DNA ocid solublex10 2
Fig. 6. The splitting of A T P as a function of protein concentration in Fraction V. The measurem e n t s were made as described in METHODS AND MATERIALS with [7-32PIATP. Approx. 3 % of the 3~p in t h e [7-3~P]ATP stock solution was norit non-adsorbable. A, + D N A ; A, - - D N A . Fig. 7. The splitting of A T P as a function of D N A hydrolyzed into acid-soluble fragments. The d a t a are from the same e x p e r i m e n t as those in Fig. 6. The [7-32PIATP rendered norit nonadsorbable is corrected for the background.
Nature o/attack. To determine whether the nature of attack is endonucleolytic or exonucleolytic, T 7 [32PIDNA was incubated in reaction mixtures that contained ATP but no enzyme, ATP and enzyme, or enzyme but no ATP. An aliquot was sedimented in a preformed alkaline sucrose gradient, and another was assayed for the DNA hydrolyzed to acid-soluble fragments (Fig. 8). No difference was observed in the DNA sedimentation pattern when either enzyme or ATP was absent from the reacBiochim. Biophys. Acta, 224 (i97 o) 29-41
38
G . F . VOVIS, G. BUTTIN
tion mixture, and no acid-soluble fragments were found under these conditions. This shows that Fraction V does not contain any significant amount of either endonucleolytic or exonucleolytic activity in the absence of ATP. Following incubation in the presence of both ATP and enzyme, IO % of the DNA was acid soluble and two DNA bands were observed in the gradient. One is at a position which is not significantly different from that of unaltered T 7 DNA, while the other is located at the position where low molecular weight DNA digestion products would be expected. This latter sedimentation pattern suggests that the enzyme liberates small fragments from preexisting free ends of DNA molecules (exonuclease). If the enzyme was an endonuclease whose internal attack produced either single-strand or double-strand breaks, then with IO % of the DNA hydrolyzed into acid-soluble fragments, a significant amount of degradation products would sediment between the unaltered DNA peak and the peak of very low molecular weight material1; this is clearly not observed. In addition, the gradient pattern of the hydrolyzed DNA also suggests that the products of DNA hydrolysis are mainly oligonucleotides. The peak of slow-sedimenting material represents approx. 30 % of the recovered (24 % of the applied) 32p activity, yet only Io % of the applied radioactive label is acid soluble. Thus, more than 50 % of this very low molecular weight material would appear to be acid insoluble and, therefore, certainly not to consist of mononucleotides. This oligonucleotide estimate in the digestion products is a minimum since the fractions from the gradient were acid precipitated and washed. 6000 O. 2 000' ~-
"1"ATP
4000~
/~V/~
2000
I000
' L i
0~
.....:~'I
1
-....... "f---~/"
II
21
!I0 31
i
20 I
/
6000 -kATP 2000
i
4000
. "°
2000
1000~-
0~ I
~1
2000
3J
21
,
0 I
0
0 It
0
li 00 000
"t-frcclionV
I
o
/
.
~
"o I
2I
Froclion number
',000
3I
0
40
80 Protein(n9,
120
Fig. 8. Alkaline sucrose gradient p a t t e r n s of undigested and partially digested DNA. A s s a y s were r u n in reaction m i x t u r e s containing T 7 phage [32P]D2qA, and the indicated additional comp o n e n t s as described in METHODS AND MATERIALS. I n the alkaline sucrose gradients, T 7 phage [aH]DNA was added as a reference marker. The recovery, in all cases, was approx. 80 % for the 3zp activity and approx. IiO ~o for the 8H activity. The greater t h a n ioo % recovery of 3H is p r o b a b l y an artifact resulting from differential quenching of low-energy fl particles within paper. Fig. 9. Hydrolysis of various s u b s t r a t e s as a function of protein concentration in Fraction V. The m e a s u r e m e n t s were made u n d e r the s t a n d a r d conditions (see METHODS A N D M A T E R I A L S ) in the presence (closed symbols) or absence (open symbols) of A T P and with 4 nmoles of the following substrates: A - A , A--&, native T 7 phage D•A; C)-C), O - O , h e a t - d e n a t u r e d T 7 phage DNA; [~-[~, I - I , poly d ( A - T ) .
Biochirn. Biophys. Mcta, 224 (197 o) 29-41
A DEOXYRIBONUCLEASE FROM D.
pneumoniae
39
Substrate speci]icity. Fig. 9 shows the activity of Fraction V on various substrates. Under standard conditions, Fraction V exhibits limited activity on heatdenatured DNA. In contrast to what is observed with native DNA, some activity still can be detected in the absence of ATP. Whether this latter activity is due to the same enzyme that is active on native DNA has not be further investigated. Fraction V also attacks poly d(A-T). Under the standard conditions, the extent of hydrolysis, as measured b y the release of acid-soluble [3H]thymidylate, is about 50 % of that observed with native DNA. The activity on poly d(A-T) rules out a base specificity of attack for the production of oligonucleotides that would be governed b y guanine-cytosine base pairs. DISCUSSION
This work establishes the presence of a previously undescribed deoxyribonuclease in D. pneumoniae that is characterized b y its requirement for ATP, which is split during DNA hydrolysis. During the course of these investigations, independent reports appeared which demonstrated that nucleoside triphosphate-dependent deoxyribonucleases are also present in Bacillus laterosporus 13 and Mycobacterium smeg-
matisl4,15. The enzymes from these latter two organisms as well as those from D. pneumoniae, E. coli1,16,17 and M. lysodeikticus (refs 2, 18, 19; A. R6RSCH, personal communication) appear to be members of the same class of enzymes as judged b y their biochemical properties. They all are active under alkaline conditions. All have a metal ion requirement which is fulfilled by a relatively high concentration of Mg 2+ (approx. I . lO -2 M). The reported data are consistent with the fact that Mn 2+ can substitute at lower concentrations. In addition, the five enzymes are much more active on native DNA than on denatured DNA. Work with the enzymes from B. laterosporus, D. pneumoniae, E. coli, and M. lysodeikticus, all of which were purified at least 5oo-fold, demonstrates that there is no activity on native DNA in the absence of the nucleoside triphosphates. Under the conditions examined, ATP appears to be the most effective at satisfying this requirement for all five enzymes and probably is the physiological effector. The other nueleoside triphosphates have varying degrees of activity with the various enzymes. In addition, this nucleoside triphosphate requirement is independent of the sugar moiety for dATP, whenever tested, readily substitutes for ATP. With all the enzymes quoted so far, ATP is split during the course of DNA hydlolysis and when it has been investigated with highly purified enzyme preparations, the products of ATP splitting were found to be ADP and P1 (refs. 16, 13, 19). In all cases, there is proportionality between the amount of ATP split and the amount of DNA hydrolyzed to acid-soluble fragments. However, the stoichiometry of the reaction remains obscure, for the DNA hydrolysis products include oligonucleotides. The presence among the reaction products of oligonucleotides, which m a y be digested further as a function of time b y the same enzyme, makes it impossible so far to correlate the number of ATP molecules converted to ADP and Pl with the numbei of phosphodiester bonds broken in the DNA duplex. It m a y be pointed out here that the nature of the degradation products distinguishes this class of enzymes from the restriction endonuclease discribed b y MESELSON AND YUAN2°. Biochim. Biophys. Acta, 224 (197 o) 29-41
4°
G.F. VOVIS, G. BUTTIN
We concluded from the sedimentation pattern of partially digested DNA that the nature of attack by the pneumococcal enzyme is very likely to be exonucleolytic. From their studies, ANAl et al. is concluded that the attack by the M . lysodeikticus deoxyribonuclease is endonucleolytic. This m a y be, to some extent, a problem of semantics. We consider that exonucleolytic attack is characterized b y where the degradation is initiated (freeends)rather than by the nature of the digestion products (mononucleotides and/or oligonucleotides). Actually, the mode of digestion for the M . lysodeikticus enzyme as reported b y ANAI et al. is is not essentially different from that suggested by our own experiments with the pneumococcal enzyme. Clearly, in both systems, no degradation products of a size intermediate between unaltered DNA molecules and oligonucleotides is observed. This suggests either that the time required for complete degradation of a DNA molecule is short as compared to the time required for the fixation of an enzyme molecule on a new chain or that essentially most of the DNA molecules have been partially digested. Their sedimentation studies of increasingly digested DNA would seem to eliminate this latter interpretation for the micrococcal system. Their viscosity studies, however, do not demonstrate more than the gradient patterns; for although the comparison of residual viscosity with percent acid-soluble fragments indicates a more rapid loss of vicosity than that expected with a purely exonucleolytic attack, such a comparison does not take into account the fact that the oligonucleotides released are likely to be, in part, acid insoluble as suggested by our results. This leads to an underestimation of the actual extent of DNA degradation. If one assumes that the nature of the products formed is identical in the pneumococcal and micrococcal systems and that the amount of DNA digested is twice that found to be acid soluble as suggested by our own experiments, then the viscosity decrease observed by ANAI et al. is corresponds to the one reported for a purely exonucleolytic type of degradation. The fact that the ATP splitting is concomitant with DNA hydrolysis suggests, as has already been considered by others 16,1~,19, that the ATP is serving as an energy source rather than just as an allosteric effector. OISHIiv suggests that because the E. coli enzyme attacks single-stranded DNA in the absence of ATP, the energy m a y be required to cause a configurational change in the DNA substrate. However, it should be pointed out that if these enzymes isolated from various organisms really have a common mechanism of action, then the apparent inability of some of them 13'~s to attack denatured DNA without ATP m a y challenge this interpretation. A definitive conclusion concerning the significance of ATP splitting should be postponed until further experiments unambiguously demonstrate that hydrolysis of denatured DNA, when it is observed, is due to the activity of the same enzyme. In any case, one should not fail to think of the ATP requirement as one way whereby the cell exerts some control in the balance between the synthetic and the degradative processes which take place at the level of DNA. The similar biochemical properties of these five enzymes do strongly suggest that they actually play a similar physiological role in the cell. Studies with E. coli show that the enzyme is involved in bacterial recombination 16,17'2~. A companion report demonstrates that such is also the case in pneumococcus 3.
Biochim. Biophys. Acta. 224 (197o) 29-41
A DEOXYRIBONUCLEASE FROM D. p n e u m o n i a e
41
ACKNOWLEDGMENTS
W e are deeply i n d e b t e d to Dr. Boris Ephrussi for v e r y generously p r o v i d i n g l a b o r a t o r y space an d for his c o n s t a n t interest in this work. Th e d e d i c a t i o n a n d e x p e r t t e c h n i c a l assistance of Catherine F o u g e r e is most g r a t e f u l l y acknowledged. We also wish to t h a n k Marguerite DGchamps for p r e p a r i n g most of t h e r a d i o a c t i v e D N A an d Dr. H. F u k u h a r a for v e r y k i n d l y t r a n s l a t i n g one of the references from J a p a n e s e for us. The m a t e r i a l herein was t a k e n from the dissertation s u b m i t t e d to the G r a d u a t e School of Case W e s t e r n R e s e r v e U n i v e r s i t y b y G. F. V. in p a r t i a l fulfillment of t h e r e q u i r e m e n t s for the degree of D o c t o r of Philosophy in Biology. This research was s u p p o r t e d b y a g r a n t to the late Dr. H a r r i e t t E p h r u s s i - T a y l o r from the D~l~gation G6n~rale ~ la Recherche Scientifique et T e c h n i q u e ; a N a t i o n a l I n s t i t u t e s of H e a l t h p r e d o c t o r a l research fellowship 5-FoI-GM-36 194; an E u r o p e a n Molecular Biology Organization short t e r m fellowship; a n d grants from the Commissariat 5~ l ' E n e r g i e A t o m i q u e to both laboratories. A p r e l i m i n a r y report a n d a b s t r a c t were given at the E u r o p e a n S y m p o s i u m on the Molecular Mechanism of R e c o m b i n a t i o n in Micro-Organisms, L u n t e r e n , t h e N e t h e r l a n d s , Sept. 28-3 o, 1969, a n d at a m e e t i n g of t h e Soci6t6 Fran~aise de G6n6tique, Toulouse, France, April 24-25, 197o , respectively.
REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 18 19 20 21
G. BUTTIN AND IV[. WRIGHT, Cold Spring Harbor Syrup. Quant. Biol., 33 (1968) 259. Y. TSUDAAND B. S. STRAUSS,Biochemistry, 3 (1964) 1678. G. F. Vovls AND G. BUTTIN, Biochim. Biophys. Acta, 224 (197o) 42. A. M. SICARD, Genetics, 5° (1964) 31. R. K. APPLEYARD,Genetics, 39 (1954) 44o. A. B. PARDEE, F. JACOBAND J. [k~IONOD,J. Mol. Biol., i (1959) 165. C. LEVINTHAL,E. R. SIGNERAND t~. FETHEROLF, Proc. Natl. Acad. Sci. U.S., 48 (1962) 123o. C. C. RICttARDSON, R. B. INMANAND A. KORNBERG,J. Mol. Biol., 9 (1964) 46. M. E. REICHMANN,S. A. RICE, C. A, THOMASAND P. DOTY, J. Am. Chem. Soc., 76 (1954) 3047• C. C. RICHARDSON, Procedures in Nucleic Acid Research, Harper and Row, New York, 1966, p.212. O. H. LOWRY, N. J. ROSENBROUGH,A. L. EARRAND R. J. RANDALL,J. Biol. Chem., 193 (1951) 265 . O. WARBURGAND W. CHRISTIAN,Biochem. Z., 31o (1941) 384 . M. ANAl, J. Japan. Biochem. Soc., 39 (1967) 167. F. G. WINDER AND M. P. COUGHLAN, Biochem. J., I i i (1969) 679. F. G. WINDER AND M. LAVlN, Biochem. J., 115 (1969) 6. M. WRIGHT AND G. BUTTIN, Bull. Soc. Chim. Biol., 51 (1969) 1373. M. OISHI, Proc. Natl. Acad. Sci. U.S., 64 (1969) 1292. M. ANAl, T. HIRAttASHIAND Y. TAKAGI, J. Biol. Chem., 245 (197o) 767 . M. ANAI, T. HIRAHASHI,M. YAMANAKAAND Y. TAKAGI,J. Biol. Chem., 245 (197o) 775. M. ~¢[ESELSONAND R. YUAN, Nature, 217 (1968) IIIO. S. D. ]3ARBOURAND A. J. CLARK, Proc. Natl. Acad. Sci. U.S., 65 (197o) 955. Biochim. Biophys. Acta, 224 (197o) 29-41