Dh'OX YRIBON UCLEIC ACID-DIRECTED SYNTHESIS OF RIBONUCLEIC ACID BY AN ENZYME FROM ESCHERICHIA COLI* BY MICHAEL CHAMBERLINt AND PAUL BERG DEPARTMENT OF BIOCHEMISTRY, STANFORD UNIVERSITY SCHOOL OF MEDICINE Communicated by Arthur Kornberg, November 17, 1961 l'rotein structure is under genetic control;'-3 yet the precise mechanism by which DNA$ influences the formation of specific amino acid sequences in proteins is unknown. Several years ago, it was discovered that infection of Escherichia coli with certain virulent bacteriophages induces the formation of an RNA fraction possessing both a high metabolic turnover rate and a base composition correspond- ing to the DNA of the infecting vir~s.~-~ The existence of an analogous REA component in noninfected cells has also been demonstrated; in this instance, however, the base composition of the RNA resembles that of the cellular DNA.'. These observations focused attention on the possible role of this type of RNA in protein synthesis, and some of the evidence consistent with this view has recently been surnmari~ed.~ Until recently there was no known enzymatic mechanism for a DNA-directed synthesis of RNA. Polynucleotide phosphorylasel0, l1 although it catalyzes the synthesis of polyribonucleotides, does not by itself provide a mechanism for the formation of RNA with a specific sequence of nucleotides. The one instance in which a unique sequence of nucleotides is produced involves the limited addition of nucleotides exclusively to the end of pre-existing polynucleotide chains. 12-14 Our efforts were therefore directed toward examining alternate mechanisms for RNA synthesis, and in particular one in which DNA might dictate the nucleotide sequence of the RNA. In the present paper, we wish to report the isolation and some properties of an RNA polymerase from E. coli which, in the presence of DNA and the four naturally occurring ribonucleoside triphosphates, produces RNA with a base composition complementary to that of the DNA. Within the last year, several laboratories have reported similar findings with enzyme preparations from bacterial as well as from plant and animal source^.^^-^^ In the following paper, the effect of enzymatically synthesized RNA on the rate and extent of amino acid incorporation into protein by E. coli ribosomes in the presence of a soluble protein fraction is described. Unlabeled ribonucleoside di- and triphosphates were purchased from the Sigma Biochemical Corporation and the California Corporation for Biochem- ical Resenrch. 8-C14-1abeled ATP was purchased from the Schwartz Biochemical Company; the other, uniformly labeled, C1* ribonucleoside triphosphates were prepared enzymatically from the corresponding monophosphate derivatives55 isolated from the RN.4 of Chromatium grown on CL402 as sole carbon source.*6 CTP labeled with P31 in the eater phosphate was obtained by enzymatic phosphorylation of CMPS* prepared according to Hurwitz.27 The deoxyribonucleoside triphosphates were obtained by the procedure of Lehman et al.25 DNA from Aerobacter aerogenes, Mycobacterium phlei, and bacteriophages T2, T5, T6 was prepared as de- scribed previously.29 Unlabeled and P32 labeled DNA from E. coli were prepared as previously de~cribed.~' d-AT and d-GC poly- mers were prepared according to Schachman et al.32 and Radding et respectively. Trans- forming DNA from Ban'ZZus subliZisJ4 was a gift from E. %'. Nester, and DNA from phage 0X 81 Experimental Procedure.-MateriaZs: Calf thymus and salmon sperm DNA were isolated by the method of Kay et ~1.~8 DNA from Xdg phage was prepared as reported else~here.~a 82 BIOCHEMISTRY: CHAMBERLIN AND BERG PROC. N. A. s. 174 49 was generously supplied by R. L. Sinsheimer. Double-stranded 0X 174 DNA was syn- thesized using E. coli DNA polymerasez6 with single-stranded OX 174 DNA as primer.36. In this reaction, 2.7 times more DNA was synthesized than had been added as primer. RNA from tobacco mosaic virus was obtained from H. Frwenkel-Conrat, and ribosomal and amino acid- acceptor RNA were isolated from E. coli according to Ofengand et ~1.~~~ 37 Nucleic acid concen- trations are given as mpmoles of nucleotide phosphorus per ml. Glass beads, "Superbrite 100," obtained from the Minnesota Mining and Manufacturing Company, were washed as previously described.26 Streptomycin sulfate was a gift from Merck and Company, and protamine sulfate was purchased from Eli Lilly Company. DEAEcellulose was purchased from Brown and Company. Crystalline pancreatic RNase and pancreatic DNase were products of the Worthington Biochemical Co. The activities of E. coli-DNA p0lymerase,~6deoxyribonuclease~8 and -DNA dies- terase, 31 were determined as previously described and ribonuclease activity was measured by the disappearance of amino acid-acceptor RNA activity.f6 Polynucleotide phosphorylase was measured by P,32 exchange with ADP as reported by Littauer and Kornberg." Protein was determined by the method of Lowry et aLJg The standard assay for RNA polymerase measures the conversion of either CJ4 or P3z from the labeled ribonucleoside triphosphates into an acid-insoluble form. Enzyme dilutions were made with a solution containing 0.01 M Tris buffer, pH 7.9, 0.01 M MgC12, 0.01 M &mercaptoethanol, 5 X The reaction mixture (0.25 ml) contained: 10 pmoles of Tris buffer, pH 7.9, 0.25 pmole of MnClz, 1.0 pmole of MgClz, 100 mpmoles each of ATP, CTP, GTP, and UTP, 250 mpmoles of salmon sperm DNA, 3.0 pmoles of 8-mercaptoethanol, and 10 to 80 units of enzyme. One of the nucleo- side triphosphates was labeled with approximately 300 to 600 cpm per mpmole. After incubation at 37" for 10 min, the reaction mixture was chilled in ice, and 1.2 mg of serum albumin (0.03 ml) was added, followed by 3 ml of cold 3.5% perchloric acid (PCA). The precipitate was dispersed, centrifuged for 5 min at 15,000 X g, and washed twice with 3.0 ml portions of cold PCA. The residue was suspended in 0.5 ml of 2 AT ammonium hydroxide, transferred to an aluminum planchet. and after drying, counted in a windowless gas-flow counter. One unit of enzyme activity corresponds to an incorporation of 1 mpmole of CMPaz per hr under the conditions described above. The assay was proportional to the amount of enzyme added up to at least 80 units; thus 6.3, 12.5, and 25 pg of Fraction 4 enzyme incorporated 2.6, 5.1, and 10.0 mpmoles of CMP32. The rate of the reaction remained constant for approximately 20 min, and then derreased after this time. Since the radioactivity incorporated represents only one of the four nucleotides, the observed incorporation must be multiplied by a factor ranging from 3 to 5 for an estimate of the total amount of RNA synthesized. E. coli B was grown in continuous exponential phase culture40 with a glucose-mineral salts medium.41 Cells stored at -20" showed no loss of activity for over six months. The purifica- tion procedure and the results of a typical preparation are summarized in Table 1. Assays: M EDTA, and 1 mg per ml of crystalline bovine fierum albumin. The exact factor depends on the composition of the DNA primer used. Results.-Purification of RNA polymerase: (1) Cells: TABLE 1 PURIFICATION OF RNA POLYMERASE FROM E. coli Volume Specific activity Total activity Fraction (ml) (uni ta/mg) (UIlitZi) 1. Initial extract 260 40 370, OOO 2. Protamine eluate 37 1,600 205,000 153 , 000 4. Peak DEAE fraction 2 6,100 3. Ammonium sulfate 5 2,500 200,000 Unless noted otherwise, all operations were carried out at 4" and all centrifugations were at 30,000 X g for 15 min in an International HR-1 Centrifuge. Frozen cells (140 gm) were mixed in a Waring Blendor with 420 gm of glass beads and 150 ml of a solution (buffer A) containing 0.01 M Tris buffer, pH (2) Extract: VOL. 48, 1962 BIOCHEMISTRY: CHAMBERLIN AND BERG 83 7.9, 0.01 M MgCI2, and O.OOO1 M EDTA. After disruption of the cells at high speed for 15 min (maximum temperature lo"), a further 150 ml of buffer A was added and the glass beads were allowed to settle. The supernatant fluid was then decanted and the residue was washed with 75 ml of buffer A. The combined supernatant fluid and wash was centrifuged for 30 min and the resulting super- natant fluid collected (Fraction 1). (3) Streptomycin-protamine fractionation: Fraction 1 was centrifuged in the Spinco Model L preparative ultracentrifuge for 4 hr at 30,000 rpm in the No. 30 rotor. The protein concentration in the supernatant fluid was adjusted to about 12 mg per ml with buffer A, and /%mercaptoethanol was added to a final concentration of 0.01 M. To 350 ml of the diluted supernatant solution was added 17.5 ml of a 10% (w/v) solution of Streptomycin sulfate with stirring. After 15 min, the solution was centrifuged, and to 350 ml of the supernatant fluid was added 14.0 ml of a 1% (w/v) solution of protamine sulfate. The precipitate, collected by centrifugation, was washed by suspension in 175 ml of buffer A containing 0.01 M j3-mercapto- ethanol. The washed precipitate was then suspended in 35 ml of buffer A con- taining 0.01 M mercaptoethanol and 0.10 M ammonium sulfate, centrifuged for 30 min, and the supernatant fluid was collected (Fraction 2). To 37 ml of Fraction 2 was added 15.8 ml of ammonium sulfate solution (saturated at 25" and adjusted to pH 7 with ammonium hydroxide). The mixture was stirred for 15 min, and the precipitate was removed by centrifugation. To the supernatant liquid was added an addi- tional 16.2 ml of the saturated ammonium sulfate, and after 15 min the precipitate was collected by centrifugation for 30 min and dissolved in buffer B (0.002 M KP04, pH 8.4, 0.01 M MgC12, 0.01 M P-mercaptoethanol, and 0.0001 M EDTA) to a final volume of 5.0 ml (Fraction 3). Fraction 3 was diluted to a protein concentration of about 3 mg per ml with buffer B and passed onto a DEAE- cellulose column (10 cm X 1 cm2, washed with 150 ml of buffer B just prior to use) at a rate of about 0.5 ml per min. The column was washed with 10 ml of buffer B and then with enough of the same buffer containing 0.16 M KC1 to reduce the absorbency of the effluent at 280 mM to less than 0.05. The enzyme was eluted from the column with buffer B containing 0.23 M KCl. The activity appears within the first five ml of the latter eluant (Fraction 4). The specific activity of enzyme Fraction 4 was from 140 to 170 times greater than that of the initial extract. The puri- fication as described here has been quite reproducible, with specific activities in the final fraction ranging from 5,500 to 6,100. The enzyme preparation (Fraction 4) has a ratio of absorbencies at 280 and 260 ml of 1.5. Fraction 4, stored at 0 to 2", retains more than 90 per cent of its activity for up to two weeks and 40 to 60 per cent of the original activity after one month. En- zyme Fractions 1 through 3 are unstable, losing up to 30 per cent of their activity on overnight storage under a variety of conditions. Because of the marked in- stability of these earlier fractions, it is advisable to carry out the purification without stopping at intermediate stages. Contaminating enzymatic activities: Aliquots (100 pg) of Fraction 4 were assayed for contaminating enzymatic activities. This amount cf enzyme cata- (4) Ammonium sulfate fractionation: (5) Adsorption a,nd elution from DEAE-cellulose: (6) Properties of the purijied enzyme: (7) 84 BIOCHEMISTRY: CHAMBERLIN AND BERG PROC. N. A. S. lyzed an initial rate of incorporation of 2,000 mpmoles of nucleotide per hr. No detectable DNA polymerase was found (< 0.6 mpmole DNA per hr). DNase activity was barely detectable under conditions optimal for RNA polymerase. With either heated or unheated P3* DNA as substrate, no more than 0.13 mpmole of acid-soluble P32 was released during the course of a 30-min incubation. There was only slight RNase activity associated with Fraction 4. When 100 pg of the purified enzyme were incubated with 4 pmoles of purified acceptor RNA for 1 hr, there was no detectable inactivation of leucine-acceptor activity. Under similar conditions, 1 mg of enzyme produced a 30 per cent decrease in leucine-acceptor activity. With conditions optimal for RNA polymerase, sufficient polynucleotide phosphorylase activity was present to catalyze the exchange of 6.7 mpmoles of Pi32 into ADP per hour. Requirements for the RNA polymerase reaction: With the purified enzyme, RNA synthesis was dependent on the addition of DNA, a divalent cation, and the four ribonucleoside triphosphates (Table 2). In a later section, we shall describe a TABLE 2 TABLE 3 REOUIREMENTS FOR RNA SYNTHESIS THE REQUIREMENT FOR RIBONUCLEOSIDE Incorporation of CMPa* Components (mpmoles) Complete syst.em 7.3 minus Mn++ 4.3 minus Mg++ 5.6 minus Mn++ and Mg++ <0.03 minus DNA <0.03 minus enzyme <0.03 minus ATP, GTP, UTP 0.09 The standard system and assay procedure were used with 7.4 WK of Fraction 4 protein in each tube, ex- cept that MeCh was omitted from the enzyme diluent. TRIPHOSPHATES IN RNA SYNTHESIS Incorporation of CM-Paz Components (mpmoles) Complete system 4.6 minus ATP 0.08 minus UTP 98 per cent of the label to acid- soluble products which were electrophoretically identical with the 2`-(3') nucleo- side monophosphates. Treatment with pancreatic DNase or E. coli DNA di- esterasea1 produced no significant liberation of labeled acid-soluble products. M). We will designate this as "net synthesis." (2) Enzymatic and alkaline degradation of the product: VOL. 48, 1962 BIOCHEMISTRY: CHAMBERLIN AND BERG 87 TABLE 6 NET SYNTHESIS OF RNA Labeled Calculated Ratio of RNA Method DNA primer incorporated RNA formed* DNA added isolation Source of nucleotide amount of isolated to of (mrmoles) Calf thymus 81 200 2.0 -4 +X 174 phage 90 510 5.1 A T2 phage 78 360 3.6 A T2 phage 72 410 4.1 B T2 phage 150 460 4.6 C T5 phage 152 500 5.0 C d-AT copolymer 155 310 15.0 C CMP3* C !`-AM P * The amount of RNA in the isolated product was calculated from the amount of label incorporated and the base ratio of the primer DNA. Sunfhecria; Each tube contained in a final volume of 0.5 ml: 20 pmoles of Tris buffer, H 7.85; 8 pmoles of MgClt; 400 rnpmoles each of ATP, CTP, UTP, GTP; 6 rmolea of &mercaptoethanof; 100 mpmoles of DNA; and 100 pg of Fraction 4 protein. When d-AT was used as primer, only 20 mpmoles of primer were added and CTP and GTP were omitted from the mixture. The incubation mixture was heated for 10 min at 60° in 0.4 M NaCI. then di- alyzed 38 hr against 0.2 M NaCl-O.01 M Tru, pH 7.85. B. The reaction mixture waa extracted two times with phenol and the phenol fractions were washed two times with 0.4 M NaCl. The aqueous layers were pooled and dialyzed as in A. roduct was precipitated from the incubation mixture with a solu- tion containing 60 per cent ethanol an10.5 M NaCl at OD, washed once with the same solution. and dis solved in 1 ml of 0.2 M NaCl., then dialyzed aa in A. The incubation time waa 3 hr at 37". Product isohfion; A. C. The Treatment of 10 to 20 mpmoles of enzymatically prepared CMP32-labeled RNA with 0.1 pg of pancreatic RNase for 1 hr liberated 75 to 94 per cent of the P32 label as acid-soluble products. The amount of acid-insoluble P32 remaining after RNase treatment varied with different DNA primers and different methods of product isolation. Using 10 times the amount of RNase did not appreciably alter the results. The significance of this RNase resistant fraction is presently un- known. (3) Nucleotide composition: The nucleotide composition of the product was examined by two different methods. In the first method, four separate assays, each containing a different labeled nucleoside triphosphate, were performed with each DNA preparation, and the molar ratio in which the labeled nucleotides were incorporated was measured (Method A). The second method utilized electro- phoretic separation44 of the mononucleotides resulting from the alkaline degrad- ation of a "net synthesis" product in which all of the nucleoside triphosphates were labeled with C14 (Method B). The distribution of the label among isolated nucleotides wdtherefore a measure of the composition of the newly synthesized RNA. The results (Table 7) indicate that the gross composition of the product at all stages of synthesis was Complementary to that of the primer within the ac- curacy of the method. For double-stranded DNA, this complementary relation- ship becomes one of identity, since in the priming DNA adenine equals thymine and guanine equals cytosine. However, in the case of single-stranded @X 174 DNA (Table 8), the composition is indeed complementary to that of the DNA, and in this instance the amounts of AMP and UMP incorporation and of GMP and CMP incorporation are not equal. Furthermore, when double-stranded @X 174 DNA is used, the nucleotide composition of the resulting RNA is again iden- tical to that of the DNA primer. (4) Sedimentation telocity of the isolated product: The sedimentation velocity of the isolated RNA product was determined in the Spinco Model E analytical ultrac.entrifuge using ultraviolet optics. Values obtained in 0.2 M NaCl- 88 DNA primer d-GC polymer d-AT copolymer d-AT copolymer T2 phage T5 phage E. coli dl. phlei A. aerogenes BIOCHEMISTRY: CHAMBERLIN AND BERG PROC. N. A. s. TABLE 7 NUCLEOTIDE COMPOSITION OF THE RNA PRODUCT Method Primer* of -Nucleotide Composition-- E analysis AMP UMP GMP CMP G + C (mpmoles) A <0.03 <0.03 1.90 0.23 - B 21.7 20.0 - A 20.8 22.2 <0.03 <0.03 - B 7.7 7.4 4.3 4.3 1.76 B 4.8 5.0 3.6 3.4 1.56 - - A 1.8 1.9 1.9 2.0 1.01 A 3.7 4.0 7.9 8.5 0.48 A 1.8 1.7 2.3 2.2 0.80 Product Product A+U A+G G+C u+c - - 1.76 1.03 1.40 1.00 0.95 0.95 0.47 0.93 0.78 1.05 * The values given for the ratio A + T/G + C in the priming DNA are those found by Jome et a1.a" except in the case of phage T5 DNA.Ja Method A: For each DNA sample. four separate incubations were used. each containing a different ClLlabeled nucleotide. The amounts of DNA used in the various tests were as follows: 20 mpmoles of M. phlei, 50 mpmoles of A. aerooenea, 180 mpmoles of E. coli, 20 mpmoles of d-AT 20 mpmoles of d-GC. 12.5 pg of Fraction 4 enzyme were used in each incubation; all other conditions were tho& given for a standard assay. Mefhod B: The synthesis of the C24-labeled RNA was carried out under the following conditions. The reaction mixture (0.5 ml) contained: 20 pmoles of Tris buffer, pH 7.85, 0.5 pmole of MnCL. 2 pmoles of MgCk, 6 pmoles of 8-mercaptoethanol, 100 mpmoles each of CicATP. CicUTP, CiLGTP, CicCTP. 100 mpmolea of DNA, and 180 pg of Fraction 4 enzyme. Where d-AT primer was used, 20 mprnoles of primer were added and no CTP or GTP were added. After 180 min at 37", the product was precipitated and washed with cold 3 per cent PCA and incubated in 0.3 M KOH for 18 hr at 37'. An aliquot to which carrier nucleotides had been added was subjected to paper electrophoresis at pH 3.5 in 0.05 M citrate buffer. The individual nucleotides which were visualized with a UV lamp were eluted in 0.01 M HCI and counted. Recovery of the CIClabel in the eluted fractions was >95 percent. 180 mpmoles, 140 mpmoles, and 200 mpmoles of polyribonucleotide were produced in the reactions primed with T2 DNA, T5 DNA, and d-AT, respectively. TABLE 8 RNA SYNTHESIS COMPAR4TIVE BEHAVIOR OF SINGLE- AND DOUBLE-STR4NDED 0x 174 DNA AS PRIMER FOR --Nucleotide Composition of RNA- State of DN.4 AMP UMP GMP CMP used as primer (per cent) Si ?!le-stranded Predicted * 32 8 24 6 18 5 24 1 Found by method A 32 0 24 1 19 5 24 3 Found by method B 35 0 24 6 19 3 21 1 Double-stranded Predicted* 28 7 28 7 21 3 21 3 Found by method B 28 9 29 1 20 9 20 9 'I 'I ', 1L Ll Method A: Conditions as given in Table 7. 32 mpmoles of single-stranded PX 174 DNA were used in each incubation with 8 pg of Fraction 4 enzyme. Method B: Conditions as given in Table 7. With single-stranded DNA as primer, 25 mpmoles of, priming DNA were added, 71 mpmoles of RNA were produced in a 60 min incubation with 80 pg of Fraction 4 en- zyme. For the double-stranded DNA, 26 mamoles of priming DNA were added; 32 mpmoles of RNA were produced in a 60 min incubation with 40 pg of Fraction 4 enzyme. * The predicted values were calculated on the assum tion that the single-stranded 0X 174 DNA would yield RNA with a composition complementary to tEe composition reported by Sinsheimer.'9 Upon replication of 0X 174 DNA with DNA-polymerase it was aaaumed that the uroduct (presumably double- stranded DNA) had a base composition which is the average of the composition of the oriainal and of the newly synthesized strands. That this is a reasonable assumption is shown by unpublished studies of M. Swartz. T. Trautner, and A. Kornberg. When @X 174, DNA was used to prime limited (<30 per cent) or extensive (600 per cent) DNA synthesis. the composition of the newly formed DNA was: dAMP TMP dGMP dCMP Limited synthesis 31.0 24.1 20.1 24.5 Extensive synthesis 29.4 26.9 22.3 21 .s 0.01 M Tris, pH 7.9, ranged from 6 to 7.5 for 2- to 15-fold [[net synthesis" products prepared by phenol extraction or by salt-ethanol precipitation. As pointed out earlier, REA synthesis, as measured by the incorporation of either labeled CTP, UTP, or GTP did not occur in the absence of the other three nucleoside triphosphates or, in fact, in the absence of any one of the nucleoside triphosphates. It was therefore sur- prising to find that purified fractions of RNA polymerase catalyze the conversion DNA-dependent formation of polyadenylic acid: VOL. 48, 1962 BIOCHEMISTRY: CHAMBERLIN AND BERG 89 of C14-ATP to an acid-insoluble form in the absence of the other three ribonucleo- side triphosphates. The ratio of the activities AMP incorporated in the absence of UTP, CTP, GTP AMP incorporated in the presence of UTP, CTP, GTP increased from 0.5 to 10 as purification of the enzyme progressed. (1) Requirements for polyadenylic acid formation: Polyadenylic acid formation from ATP occurred only in the presence of DNA, a divalent cation, and the pur- ified enzyme (Table 9). Note that addition of unlabeled ADP produces only a TABLE 9 TABLE 10 FORMATION RNA POLYMERASE REQUIREMENTS FOR POLYADENYLIC ACID INCORPORATION OF SINGLE NUCLEOTIDES BY Incorporation of AMP System (mpmoles) Complete (with ATP as the only nucleoside triphosphate) 9.9 minus DNA ' of one chain along the other, lead to a DNA-dependent elongation of the The introduction of any other iiucleotidc into the growing lic acid chain. polyadenylic acid chain. Summary.-An RNA polymerase has been isolated from E. coli which in the pres- ence of the four ribonucleoside triphosphates, a divalent metal ion, and DNA synthesizes RNA with a base composition complementary to that of the priming DNA. Thus, while single- stranded @X 174 DNA yields RNA with a base composition complementary to that of the single-stranded form, double-stranded $JX 171 DXA (synt hcsized with DNA polymerase) primes the synthesis of RATA with a base composition virtually the same as that in both strands of the DNA. A novel feature of the RXA polymerase preparations is their ability to catalyze a DNA-dependent formation of polyadenylic acid in the presence of ATP alone. Neither UTP, GTP, nor CTP yields corre- t block or inhibit the sliding process and thereby terminate the growing Both strands of DNA can prime new KXA synthesis. VOL. 48, 1962 BIOCHEMISTRY: CHAMBERLIN AND BERG 93 sponding homopolymers; the DNA-dependent formation of polyadenylic acid is vir- tually completely inhibited by the presence of the other nucleoside triphosphates. * This work was supported by Public Health Service Research Grant No. RG6814 and Public t Pre-doctoral Fellow. f The abbreviations used in this paper are: RNA and DNA for rib+ and deoxyribonucleic acid, respectively; poly dT for polydeoxythymidylate; d-AT for the deoxyadenylate-thymidylate copolymer; d-GC for the deoxyguanylate-deoxycytidylate polymer: AMP, ADP and ATP for adenosine-5'-mono-, di-, and triphosphates, respectively. A similar notation is used for the cyti- dine (C), guanosine (G), and uridine (U) derivatives and their deoxy analogues (dA, dC, dG, dT) . Pi is used for inorganic orthophosphate, TMV for tobacco mosaic virus, and DNase and RNase for deoxyribo- and ribonuclease activities, respectively. 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