THE JOURNAL OB BIOLOGICAL CHEMISTRY Vol. 243, No. 23, Issue of December 10, PP. 6222-6233,1968 Printed in U.S. A. A Deoxyribonucleic Acid Polymerase from Micrococcus Zuteus (Micrococcus lysodeikticus) Isolated on Deoxyribonucleic Acid-Cellulose* ROSE M. LITMAN (Received for publication, March 12, 1968) From the Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 372031 SUMMARY A deoxyribonucleic acid polymerase from Micrococcus luteus has been isolated by allowing it to bind to its substrate, DNA, attached to a solid matrix of celluIose and subsequent detachment from the DNA by high salt concentrations. This approach has permitted the isolation of a highly purified en- zyIpe by easy and reproducible methods from a partially purified preparation in which the polymerase is only a minor constituent. The enzyme preparations are quite active with DNA primers, almost entirely free of endo- or exonucleases, and extremely stable in the presence of DNA and other nu- cleotide polymers, or in high salt solution. At low salt con- centrations, however, activity is rapidly lost. The active polymerase in high salt has a sedimentation rate of about 7 S. Kinetic studies indicate that the interactions between the polymerase-DNA structure and the deoxynucleoside triphos- phates and Mgf+ or Mn++ are complex. It can be calcu- lated from DNA saturation data that there are only one or two sites of initiation on helical linear DNA molecules, and that reinitiation on a synthetic complex doesnot occur. DNA synthesis stops when a doubling of the added primer is reached. DNA polymerases (deoxynucleoside triphosphate :DNA deoxynucleotidyl transferase, EC 2.7.7.7) from several sources have been isolated, in most cases by procedures which are classi- cal in the field of enzyme purification. In the present work, however, I report the purification of a bacterial DNA polymerase through binding to its substrate, DNA, which has been immobi- lized on cellulose by irradiation with ultraviolet light. A par- tially purified preparation of DNA polymerase percolated through a column of DNA-cellulose results in the unimpeded outflow of almost all of the protein as well as other extraneous material, * This work was supported by the National Institutes of Health, United States Public Health Service Grant GM-13767. $ This work was begun in the Department of Biophysics, Uni- versity of Colorado Medical Center, Denver, Colorado. while the polymerase and perhaps a few other proteins bind to the column. Polymerase activity can then be removed by elu- tion with a high concentration of NaCl. This procedure has been applied to the isolation of a DNA polymerase from Micro- coccus luteus (formerly M. Zy~odeikticus),~ because of the com- mercial availability of dried cells and their ease of lysis. The techniques evolved should, however, be applicable to the isolation of any enzyme capable of binding to DNA. The polymerase thus isolated resembles in its general enzy- mological aspects the M. luteus DNA polymerase previously isolated and described by Zimmerman (2) as well as those iso- lated from Escherichia coZi (3, 4) and BaciZZus subtilis (5). It is, however, different in its requirements for stability, in the DNA structures which it synthesizes, in the termination of synthesis of DNA at a doubling of the added primer, and in the almost total absence of contaminating nucleases. A study of the properties of the DNA synthesized by this enzyme will be pre- sented in a later publication. The procedures for the prepara- tion of DNA-cellulose and for the isolation of the polymerase thereon are presented in this paper, followed by a description of the properties of the polymerase and its enzymatic reaction. EXPERIMENTAL PROCEDURE Bacteria Materials M. Zuteus was purchased in the spray-dried form from Miles Laboratories, Inc., Elkhart, Indiana. DipZococcus pwumoniae, strain 14, resistant to streptomycin and to Optochin and isolated in the laboratory of L. S. Lerman, was utilized as the main source of DNA. D. pneumoniae, strain 84, isolated by Morse (6), which requires uracil for growth and is sensitive to both streptomycin and Optochin, was used as the recipient strain in transformation assays. The procedures utilized in transforma- tion assays will be described in detail in a later publication. Nucleotides Unlabeled deoxyribonucleoside triphosphates were purchased from Calbiochem. dATP-8% and dCTP-*H were purchased 1 M. lysodeikticus has been reclassified by the International Committee on Bacterial Nomenclature as M. Zuteus (1). by guest on November 23, 2020 http://www.jbc.org/ Downloaded from
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THE JOURNAL OB BIOLOGICAL CHEMISTRY Vol. 243, No. 23, Issue of December 10, PP. 6222-6233,1968
Printed in U.S. A.
A Deoxyribonucleic Acid Polymerase from Micrococcus Zuteus (Micrococcus lysodeikticus) Isolated on Deoxyribonucleic Acid-Cellulose*
ROSE M. LITMAN
(Received for publication, March 12, 1968)
From the Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 372031
SUMMARY
A deoxyribonucleic acid polymerase from Micrococcus luteus has been isolated by allowing it to bind to its substrate, DNA, attached to a solid matrix of celluIose and subsequent detachment from the DNA by high salt concentrations. This approach has permitted the isolation of a highly purified en- zyIpe by easy and reproducible methods from a partially purified preparation in which the polymerase is only a minor constituent. The enzyme preparations are quite active with DNA primers, almost entirely free of endo- or exonucleases, and extremely stable in the presence of DNA and other nu- cleotide polymers, or in high salt solution. At low salt con- centrations, however, activity is rapidly lost. The active polymerase in high salt has a sedimentation rate of about 7 S. Kinetic studies indicate that the interactions between the polymerase-DNA structure and the deoxynucleoside triphos- phates and Mgf+ or Mn++ are complex. It can be calcu- lated from DNA saturation data that there are only one or two sites of initiation on helical linear DNA molecules, and that reinitiation on a synthetic complex does not occur. DNA synthesis stops when a doubling of the added primer is reached.
DNA polymerases (deoxynucleoside triphosphate :DNA deoxynucleotidyl transferase, EC 2.7.7.7) from several sources have been isolated, in most cases by procedures which are classi- cal in the field of enzyme purification. In the present work, however, I report the purification of a bacterial DNA polymerase through binding to its substrate, DNA, which has been immobi- lized on cellulose by irradiation with ultraviolet light. A par- tially purified preparation of DNA polymerase percolated through a column of DNA-cellulose results in the unimpeded outflow of almost all of the protein as well as other extraneous material,
* This work was supported by the National Institutes of Health, United States Public Health Service Grant GM-13767.
$ This work was begun in the Department of Biophysics, Uni- versity of Colorado Medical Center, Denver, Colorado.
while the polymerase and perhaps a few other proteins bind to the column. Polymerase activity can then be removed by elu- tion with a high concentration of NaCl. This procedure has been applied to the isolation of a DNA polymerase from Micro- coccus luteus (formerly M. Zy~odeikticus),~ because of the com- mercial availability of dried cells and their ease of lysis. The techniques evolved should, however, be applicable to the isolation of any enzyme capable of binding to DNA.
The polymerase thus isolated resembles in its general enzy- mological aspects the M. luteus DNA polymerase previously isolated and described by Zimmerman (2) as well as those iso- lated from Escherichia coZi (3, 4) and BaciZZus subtilis (5). It is, however, different in its requirements for stability, in the DNA structures which it synthesizes, in the termination of synthesis of DNA at a doubling of the added primer, and in the almost total absence of contaminating nucleases. A study of the properties of the DNA synthesized by this enzyme will be pre- sented in a later publication. The procedures for the prepara- tion of DNA-cellulose and for the isolation of the polymerase thereon are presented in this paper, followed by a description of the properties of the polymerase and its enzymatic reaction.
EXPERIMENTAL PROCEDURE
Bacteria
Materials
M. Zuteus was purchased in the spray-dried form from Miles Laboratories, Inc., Elkhart, Indiana. DipZococcus pwumoniae, strain 14, resistant to streptomycin and to Optochin and isolated in the laboratory of L. S. Lerman, was utilized as the main source of DNA. D. pneumoniae, strain 84, isolated by Morse (6), which requires uracil for growth and is sensitive to both streptomycin and Optochin, was used as the recipient strain in transformation assays. The procedures utilized in transforma- tion assays will be described in detail in a later publication.
Nucleotides
Unlabeled deoxyribonucleoside triphosphates were purchased from Calbiochem. dATP-8% and dCTP-*H were purchased
1 M. lysodeikticus has been reclassified by the International Committee on Bacterial Nomenclature as M. Zuteus (1).
from Schwarz BioResearch, dTTP-21% from New England Nuclear Corporation, and poly rA and poly rU* from Miles Lab- oratories, Inc.
DNA-Calf thymus DNA was purchased from Worthington. Bacterial DNAs were isolated by a modification of the procedure of Berns and Thomas (7) followed by treatment with heated ribonuclease and passage over particles of Permutit Carbo-Dur (Matheson Coleman and Bell) or dialysis to remove the RNA breakdown products. After alcohol precipitation, the DNA was redissolved in 1 or 10 mM NaCl. M. Zu.%us DNA was fur- ther purified by isopycnic centrifugation in CsCl followed by dialysis against low salt concentration. aH-Labeled pneumo- coccal DNA was isolated by a similar procedure from cells of strain 14 which had been grown in the presence of tritium- labeled thymidine. Bacteriophage T4 DNA isolated according to the procedure of MacHattie et al. (8) was kindly supplied by Dr. T. Solie. Unless otherwise noted, DNA concentrations or amounts are always expressed in nucleotide equivalents.
Enzymes
Pancreatic ribonuclease, deoxyribonuclease I, and lysozyme were purchased from Worthington.
Methods
Cobrimetrk Determinations
Protein concentrations were measured by the method of Lowry et al. (9) with reduced volumes and bovine serum albumin (Pentex, Inc., Kankakee, Illinois) standards. DNA was meas- ured by the Burton diphenylamine test (10).
Measurements of DNA Synthesis
Standard Assay-Unless otherwise noted, polymerase activity was measured by the amount of dATP-S-i4C rendered acid-insol- uble under the following standard conditions. The reaction mixture contained, in 0.2 ml, 20 pmoles of Tris-HCl buffer, pH 7.7, 500 nmoles of MgC12, 25 nmoles of native pneumococcal DNA, a total of 25 nmoles of the four deoxyribonucleoside tri- phosphates added in a ratio approximately equivalent to that of the base composition of the DNA, and a tracer amount of dATP-81%. Enzyme dilutions when needed were made into 10 mu potassium phosphate buffer, pH 6.8. The volume of the enzyme added to the reaction mixture was never greater than 20 ~1 and usually in the range of 2 to 10 ~1. After the addition of the enzyme, the mixture is incubated at 37” for 60 min, then chilled, 0.03 ml of calf thymus DNA (2 mg per ml) is added as a carrier, and finally 0.5 ml of cold N HClOd. After standing for a few minutes on ice, about 3 ml of cold Hz0 are added, and the precipitate is collected by filtration with suction on a 2.4-cm diameter glass fiber paper (Whatman GF/C) and washed three times with about 8 ml of cold water. The papers are then dried and put into 2 ml of a standard toluene base scintillation solu- tion and counted in a Packard scintillation counter. In this procedure, the blank values (either no enzyme or minus one or more of the triphosphates) are of the order of 0.4% of the added radioactivity.
One unit of polymerase activity is defined as the amount of enzyme which can polymerize nucleotides at an initial rate of 25 nmoles per hour under standard assay conditions. (The actual number of polymerase units added is specified in the
2 The abbreviations used are: poly rA, polyadenylic acid; poly rU, polyuridylic acid.
description of specific experiments.) Specific activity is ex- pressed in terms of units of polymerase activity per mg of pro- tein.
Diethylaminoethyl Cellulose Technique-In some experiments, the amount of dATP-814C converted into polymeric form was measured by the DEAE-cellulose binding technique of Altman and Lerman (11) .a An aliquot of a reaction mixture is spotted onto DEAE-cellulose paper (Whatman Chromedia DE 81) and dried. The paper is then washed several times with 5% Nap- HPOI and finally rinsed with water. After drying, the paper is cut into rectangles which are immersed in scintillation fluid and counted. DNA remains bound to the DEAE-cellulose paper while the nucleoside triphosphates are totally removed. The advantages of this procedure over the precipitation method described above are that (a) the blank values are of the order of less than 0.1% of the added radioactivity, (5) recoveries with small aliquots are greatly improved, and (c) a very large number of samples can be handled simultaneously. However, while the glass fiber papers are transparent in the scintillation fluid and affect the efficiency of counting only slightly, the DEAE-cellu- lose papers are opalescent and decrease the efficiency of counting in DNA bound to the surface of the DEAE-cellulose paper by about 15% compared to the glass fiber papers. Appropriate corrections are therefore made in calculating results obtained by a combination of these two techniques.
Preparcbn of DNA-Cellulose
Cellulose-First 15 g of Solka-Floe (Brown Company, Berlin, New Hampshire) are washed by stirring for 10 min in 306 ml of N HCl, filtered on Whatman No. 1 paper in a buchner funnel, and rinsed thereon with water. The cellulose is then stirred with another 206 ml of N HCl for 10 min, again filtered and washed exhaustive1.y with water until no trace of acid remains, and finally spread out to dry in air.
DNA-Cellulose-Acid-washed cellulose, 0.75 g, is mixed with a desired amount of DNA; in the work reported here, 6 ml of calf thymus DNA, 2 mg per ml, in 1 or 10 mM NaCl were used. The two are kneaded together with a spatula to achieve adequate mixing, then spread thinly over the surface of a 150-ml beaker and dried under a blower (cool air) for several hours, and finally allowed to stand in air overnight. The material is then scraped from the sides of the beaker and suspended by stirring for several minutes in 20 ml of absolute ethanol. The alcohol suspension is placed under a low pressure mercury lamp (Mineralight, Ultra-Violet Products, Inc., South Pasadena, California) at a distance of about 10 cm from the surface of the alcohol and exposed to 15 min of irradiation (about 100,000 ergs per mm*) with continuous slow stirring. The alcohol is then removed by filtration with suction on Whatman No. 2 paper and the DNA- cellulose is washed three times by stirring in 50 ml of 1 mM NaCl for 10 min followed by filtration, and finally spread out to dry in air. The amount of DNA bound is calculated from the ultra- violet absorbance of the washes and also determined by the Burton diphenylamine test (10) on an appropriate weight of the dry DNA-cellulose. About 10% of the DNA is bound to the cellulose in the absence of irradiation and 90% after irradiation. The DNA-cellulose used in this work contained 30 pmoles of deoxyribonucleotides per g of cellulose. These preparations are stable at room temperature for periods of several years.
Except for the lysis procedure, the following is an adaptation of a procedure described in the isolation of E. coli DNA poly- merase (4, 12). Other procedures have been tried, but the following steps lead to reproducible results in high yield. Ex- cept where otherwise indicated, all steps were carried out at about 4”.
Lysis and Sonic Oscillation-A 25-g portion of spray-dried M. luteus is suspended in 500 ml of 50 mM glycine ethyl ester buffer, pH 7.0, with the aid of a Waring blendor. Then 85 mg of lysozyme are added and the mixture is held at 30” for 15 min at which time lysis has begun. Between 0.2 and 0.5 ml of 5y0 so- dium deoxycholate is added, and the suspension is kept at 30” for another 15 min at which time lysis is complete. The very viscous yellow-orange suspension is chilled and subjected in lOO-ml aliquots to sonic oscillation with the Branson probe sonic oscillator at maximum setting for 30 set, thereby reducing the viscosity. The suspension is centrifuged at 16,000 x g for 10 min, and the large soft orange-brown pellets are discarded. The supernatant is turbid, yellow-brown, and only slightly viscous (lysate).
Streptomycin Precipitation-the lysate is diluted with 1 volume of 50 InM Tris-HCl buffer, pH 7.5, and a volume of 5% strepto- mycin sulfate (E. R. Squibb and Sons, New Brunswick, New Jersey) equal to 12.5% of this total volume is then added over a period of about 10 min with constant stirring. After standing for 30 to 60 min, the mixture is centrifuged at 16,000 x g for 30 min, and the turbid supernatant is discarded. The somewhat soft orange-yellow pellet is placed in the Waring Blendor along with a volume of 50 mM potassium phosphate buffer, pH 7.4, equal to 10% of the volume of the previous step. The mixture is homogenized at low speed for 5 min, then allowed to remain on ice for 10 min, and the blending and cooling periods are re- peated twice more. The resulting suspension is left in the cold overnight, and finally centrifuged at 80,000 x g for 2 hours. The large soft orange-brown pellet is discarded. The super- natant is slightly turbid, yellow, and rather viscous (streptomy- cin extract).
DNase and RNase Treatment-To the streptomycin extract 50 pg of DNase I, 100 pg of RNase, and MgClz to a concentration of 3 mM, are added. The mixture is incubated at 30”, and at intervals the fraction of ultraviolet-absorbing material rendered acid-soluble is measured. When this value reaches about 80% of the total 260 rnp absorbance (usually within 60 min), the solution, which at this stage is no longer viscous, but is fairly turbid, is centrifuged at 16,000 X g for 20 min. The small yellow pellet is discarded and the clear yellow supernatant (nuclease fraction) is carried to the next step.
Ammonium Sulfate Fractionation-To the nuclease fraction are added glutathione (reduced) and sodium EDTA to 1 mM each, and then with stirring 25 g of ammonium sulfate per 100 ml of solution. The suspension is stirred for 15 mm, allowed to stand for 15 min, and is then centrifuged at 48,000 x g for 20 min. The yellow pellets are resuspended in 10 ml of 50 mM potassium phosphate buffer, pH 7.4, resulting in a yellow and turbid suspension (ammonium sulfate Fraction I). Another 16 g of ammonium sulfate are added per 100 ml of supernatant. Again the suspension is stirred for 15 min, allowed to stand for 15 min, and finally centrifuged at 48,000 x g for 20 min. The supernatant which is clear and colorless is discarded while the pellets are dissolved in 10 ml of 50 mM potassium phosphate buffer, pH 6.8, to give a yellow but almost clear solution (ammo- nium sulfate Fraction II).
RESULTS AND DISCUSSION
The protein content and polymerase activity of the various steps in the preparation of the partially purified enzyme are presented in Table I, from which it can be seen that the bulk of the activity is in ammonium sulfate Fraction II. This fraction is stable at 4” for at least several months. It is also to be noted that the addition of nucleases increases the apparent polymerase activity; the nuclease fraction, which has 1.5 times the specific activity of the streptomycin extract, differs from the latter mainly in the addition of DNase and RNase. Since the en- hancing effect is also observed when the RNase treatment is omitted, this increase in apparent polymerase activity can be attributed to the addition of DNase.
Several procedures have been applied for the further purifica- tion of the polymerase. It proved impossible to use DEAE- cellulose, on which all activity was lost, but fractionation on hydroxyapatite yielded a 20-fold purification. However, even after hydroxyapatite adsorption and elution, and other steps, the preparations were contaminated with endonuclease activity and with ultraviolet-absorbing material of nonprotein nature.
In preliminary sedimentation studies, it was found that poly- merase in impure preparations binds to DNA, in solutions of low salt concentrations, even in the absence of Mg++ or nucleo- side triphosphates.4 To take advantage of this specific binding for polymerase isolation it was desirable to immobilize DNA on some solid medium. For this purpose we have employed an adaptation of a technique of Britten (13),5 in which DNA is bound to cellulose as a result of irradiation by ultraviolet light. The preparation of DNA-cellulose is described under “Methods,” and the protein content and polymerase activity of the most active fractions isolated thereon are indicated in Table I.
Fractionation of Polymerase on DNA-Cellulose
The procedure is carried out at room temperature with solu- tions which are initially at 4”. DNA-cellulose, 0.5 g, is stirred with 20 ml of 10 mM potassium phosphate buffer, pH 6.8, for several minutes, poured into a 1.3-cm diameter column with a cotton stopper at the bottom to a height of 5 cm, and washed with another 30 ml of buffer. A 2-ml portion of Fraction II diluted to 4 ml with the same buffer is added and allowed to pass into the DNA-cellulose (4 ml is the approximate interstitial volume of 0.5 g of DNA-cellulose). Flow is shut off and the diluted Fraction II is allowed to remain in contact with the DNA-
4 R. M. Litman, unpublished results. 6 Modified by L. S. Lerman.
cellulose for 15 min. Flow is then resumed, and the column is washed first with 40 to 50 ml of the buffer and then is eluted with a total of 25 ml of 0.4 M NaCl and finally with 20 ml of 0.7 M
NaCI, both buffered with 10 mM potassium phosphate, pH 6.8. The polymerase used in this work begins to be eluted after about 4 ml of the 0.7 M NaCl solution have passed through the column.
As can be seen from Table II, almost all of the protein is re- moved by the buffer alone. About 30% of the polymerase activity and, although not shown, the bulk of the yellow material and other extraneous matter are also contained in the buffer washes. The fractions eluted with 0.4 M NaCl contain 6.5% of the protein and 10% of the activity. The 0.7 M NaCl frac- tions have 2a/c of the protein and 15 to 20% of the activity. Pro- tein recoveries are therefore of the order of 100~o but polymerase activity is recovered to only 50 to 60%. Fraction II contains DNase, carried over from a previous step, the bulk of which is eluted in the buffer washes. The addition of DNase to either Fraction II or to the buffer fractions leads to no .enhancement of polymerase activity, whereas the addition of DNase to either the 0.4 M or 0.7 M NaCl fractions increases apparent polymerase activity by 1.5- to a-fold (Table II). The recovery of polymerase activity determined in the presence of added DNase is of the order of 85%.
The presence of substantial DNase or other nucleolytic activ- ity in the material added to the column is essential for the suc- cess of this fractionation procedure. Binding of polymerase which had been purified on hydroxyapatite to the DNA-cellulose occurred only if exogenous DNase was included. Although no Mg++ is added to the Fraction II that is applied to DNA-cellu- lose, the distilled water used throughout this work contains a hlg++ concentration of 0.01 to 0.1 rnM,e which should be suffi- cient to support a low rate of nuclease activity. It should be possible, in principle, to first treat the DNA-cellulose with DNase and Mg++, but this possibility has not yet been explored.
No DNA is removed from the DNA-cellulose by the poly- merase isolation procedure. Furthermore, the same DNA-cellu- lose may be used for repeated enzyme isolations with identical results, provided that the high salt solution is washed out before the addition of more ammonium sulfate Fraction II. The isola- tion procedure is also reproducible with different batches of DNA-cellulose, and the purified polymerase preparations are stable in the high salt solutions over a period of at least several months at 4”. As indicated in Table I, the 0.7 M NaCl peak fractions have a specific activity of 900 units per mg of protein, equivalent to a synthetic rate of polymerization of 20 to 25 nmoles of nucleotides per pg of protein per hour. Unless other- wise stated, all succeeding work is performed with the enzyme purified to this step.
Because of the limitations of knowledge concerning the param- eters which control polymerase enzyme activities, it does not seem useful to compare apparent specific activities of the bac- terial DNA polymerases prepared by other investigators. How- ever, the best reported specific activities, when assayed with the poly d(A-T) template, do not differ by as much as an order of magnitude from the present enzyme.
Properties of Enzyme
Stability of Polymerase
Polymerase activity is stable in 0.7 M NaCl or solutions of higher salt concentrations over periods of at least several months
B L. S. Lerman, personal communicat~ion.
TABLE II Fractionalion of ammonium sulfate Fraction II on Dh’A-cellulose
Fractions Protein
Added to column, Frac- tion II.
Eluted from column Buffer. , NaCl, 0.4 M.. . . NaC1, 0.7 M. _.
0 Polymerase activity tested in standard procedure in the pres- ence of 0.005 rg of DNase.
TABLE III E$ecl on polymerase activity of dialysis against low or high salt in
presence or absence of DNA
First, 0.5 ml of polymerase in 0.5 M NaCl containing 160 rg of protein per ml and having a specific activity of 260 units per mg was dialyzed in the presence or absence of 82.5 rg of pneumococcal DNA against 50 ml of 10 mM potassium phosphate buffer, pH 6.8, with or without 0.5 M NaCl as indicated for 2 hours at 4”. The bags were opened and the contents were analyzed immediately for protein and polymerase activity. After 24 hours, the samesamples were again tested for polymerase activity. The recoveries of protein and of polymerase activity are expressed in relation to the respective values obtained before dialysis.
I I Recovery after dialysis
Addition to pOlpl~~~S~
Additio% $eialysate Specific activity u
Protein Immediate “zi:,‘,’
1. None None 2. None Cl.5 M NaCl 3. DNA None 4. DNA 0.5 M NaCl
% 70 70 80 70
% 53 85 90 85
% 11 85 90 80
at 4”. Removal of the salt by dialysis or other means results in a rapid and irreversible loss of activity. When polymerase in 0.5 M NaCl is dialyzed for 2 hours against a lOO-fold volume of 10 mM potassium phosphate buffer, pH 6.8, there is little loss in protein, but an immediate loss of 30% in specific activity, and a continued drop in activity until only 10 to 20% of polymerase activity remains 24 hours after the dialysis (Table III, Line 1). The possibility of the dissociation of a low molecular weight “cofactor” from the polymerase by high salt and its subsequent
loss during dialysis is excluded by the following: (a) the continued loss of polymerase activity after removal from the dialysis bag (Table III, Line l), and (b) dialysis against the same high salt concentration results in no loss in activity (Table III, Line 2). These results do not, however, exclude a possible requirement for a cofactor which may be dissociable in low salt concentrations.
The loss of polymerase activity by dialysis against low salt concentrations can be completely prevented by adding DNA to the polymerase solution (Table III, Lines 3 and 4). A stable
6226 DNA Polymerase Isolated on DNA-Cellulose Vol. 243, No. 23
11.3s 7s 1 i I 1800 1600-2 - & 5 -” 2 o 1400 - > 1200- m n
? : b
3
2
I
0
FRACTION FIN. 1. Sedimentation of concentrated polymerase and of
catalase in D20 gradients. Either 0.2 ml of Diaflo ultrafiltered polymerase (30 rg of protein) or 0.2 ml of fungal catalase w&9 layered on top of a linear 90 to 26% DPO (Bio-Rad) gradient con- taining 0.6 M NaCl and 10 rnr.r potassium phosphate buffer, pH 6.8, in polyallomer tubes, total volume about 5 ml. After 163 hours of centrifugation at 45,060 rpm at 5” in the Spinco SW5OL rotor, 22 fractions were collected. For catalase, enzyme activity wasdeter- mined as described by Chance and Maehly (14); lOtI’% of the ac- tivity was recovered. Polymerase activity was measured with lop1 of each fraction and is plotted as the number of acid-precipi- table counts per min in a standard assay after 2 hours at 37”. Absorbance at 230 m was measured in a Gary spectrophotom- eter model 16 in microcuvettes. The remainder of each frac- tion was used in the Lowry reaction for protein. These latter values are expressed in microgram equivalents of bovine serum albumin.
DNA-polymerase complex is presumably formed which main- tains polymerase activity even in 10 mu potassium phosphate buffer. Furthermore, DNA or salt added to the dialyzed poly- merase at any time will prevent any further loss in enzyme ac- tivity, but neither DNA nor salt restores the activity lost prior to their addition. The potential protective effect of the addi- tion of other proteins has not been studied, since it was felt undesirable to introduce possible contamination by other en- zymes.
Zone Sedimeniation
A comparison was made between the sedimentation rate of the polymerase and that of catalase in a DzO gradient. In some experiments, to ensure the presence of a sufficient amount of protein in the polymerase preparation for detection by the method of Lowry el al. (9) after centrifugation, several milliliters of polymerase were first concentrated by ultrafiltration through
a Diaflo membrane (Amicon Corporation, Cambridge, Massa- chusetts). Unfortunately these membranes are sensitive to high salt concentrations, and some material was, during the concentration procedure, extracted from the membrane which then appeared in solution as nonspecific ultraviolet-absorbing material. The distribution of protein (material reacting with Lowry reagent), ultraviolet absorbance at 230 rnp and of poly- merase activity after centrifugation of the Diaflo-concentrated polymerase in a DzO gradient in the presence of 0.5 M NaCl is shown in Fig. 1. The position of fungal catalase (Fermco Chemi- cal Company, Chicago) centrifuged under identical conditions is also indicated.
It can be seen that there is a major protein peak approximately coincident with the single peak of polymerase activity. On the basis of an s value for fungal catalase of 11.3 (15, IS), an s value of approximately 7 is calculated for polymerase. With the assumptions of spherical shape and a partial specific volume of 0.75, this sedimentation coefficient corresponds to a molecular weight of 100,000 to 150,000. The active M. luteus polymerase, therefore, has a molecular weight similar to those of the E. coli and T4 DNA polymerases (17). Smaller proteins with no ac- tivity and an average calculated sedimentation value of 2.2 S are also present in this gradient. When sedimentation is carried out in the absence of added salt, conditions in which no poly- merase activity is obtained, little or no protein is detectable in the 7 S position, but proteins sedimenting at lower rates are present in greater quantity. The relation of these smaller pro- teins to the active polymerase is currently under investigation. Although all of the substance reacting with Lowry reagent introduced into the gradient is recovered, only 37% of the added polymerase activity is present in the fractions. This loss in activity may be due to the somewhat low NaCl concentration (0.5 M) present in this gradient.
Materials reacting with the Lowry reagent were also obtained at the bottom and at the meniscus of the gradient. In both cases, they were accompanied by high absorbance at 230 rnb, and neither contained any detectable polymerase activity. With most proteins, a 10 pg per ml solution has an absorbance at 230 rnM of 0.05 to 0.075. The peak fractions of the polymerase pro- tein and the 2.2 S protein both of which have protein concentra- tions of about 10 g per ml or less have an absorbance at 230 rnp in the neighborhood of 0.05. The material at the extremities of the gradient, however, for the equivalent of 10 &ml of material reacting with Lowry reagent exhibit Az3,, of 0.5 at the bottom of the tube and 0.2 at the top of the tube. It suggested, therefore, that these components are either mixtures of protein and nonprotein substances or entirely nonproteinaceous and that they may correspond to the material mentioned above extracted from the Diaflo membrane.
In other experiments, polymerase preparations were concen- trated by ultrafiltration through collodion bags (Schleicher and Schuell) and then similarly subjected to sedimentation in DzO gradients. In the experiment of Fig. 2, the concentration of NaCl was increased to 1 M, and the recovery of activity as tested with the d(A-T) copolymer was of the order of 100%. Again a protein peak, accounting for 60% of the protein recovered from the gradient, is coincident with the peak of polymerase activity, and although material reacting with Lowry reagent was also found elsewhere in the gradient, there was no obvious accumulation at either the bottom or the top of the centrifuge tube.
Endonuclease-The presence of extremely low endonuclease activity in the polymerase preparations was detected by,a very slow inactivation of the transforming activity of pneumococcal DNA. Standard assay conditions were used except for the omission of one or more of the deoxynucleoside triphosphates to prevent DNA synthesis. At various times, samples were removed, diluted, and tested for transforming activity. With 2.4 pg of polymerase protein (1.32 units), 83.6% of the trans- forming activity was recovered after 4 hours of reaction at 37” with native DNA. This extent of survival is also obtained with 6 x 10-S pg of pancreatic DNase. There are, therefore, 2.5 X 10vs pg eq of DNase per pg of polymerase protein. This value is of the same order of magnitude as that obtained by Zimmer- man (2) in his polymerase preparation, with a different procedure for measuring endonuclease activity. With heat-denatured transforming DNA, no drop was detectable over long periods of time. The transforming activity of such DNA is, however, quite low, and the accuracy in these measurements is poor compared to that with native DNA.
Another endonuclease is detectable when ultraviolet-irradiated DNA is employed. In an experiment designed to test the effi- ciency of heavily irradiated DNA as a primer, it was found that while the unirradiated DNA after 2 hours in the presence of 0.4 pg of polymerase protein (0.28 unit) had more than 95% of its original transforming activity, the irradiated DNA activity had decreased to 80% of its original value. Again in terms of pancreatic DNase, this represents 2.5 x 10eb pg eq per pg of polymerase protein, an amount 10 times greater than that detect-
FRACTION FIG. 2. Sedimentation of concentrated polymerase in DzO gra-
dient + 1 M N&l. Polymerase, 0.2 ml, concentrated by ultrafil- tration in a collodion bag, was layered onto a linear 90 to 20% DzO (Bio-Rad) gradient containing 1 M NaCl and 50 mM Tris-HCl, pH 7.7, in polyallomer tubes, total volume about 5 ml. After 16 hours of centrifugation at 45,006 rpm at 20’ in the Spinco SW5OL rotor, 20 samples were collected. For polymerase activity, 5 ~1 of each fraction were added to 0.2 ml containing 20 pmoles of Tris-HCl, pH 7.7, 250 nmoles of MgC12, 5 nmoles of poly d(A-T), 6 nmoles each of dATP and dTTP, and a tracer amount of dATP-81°C. The mixtures were incubated at 37” for 60 min, at which time carrier DNA and HC104 were added, and the precipitates were fil- tered and counted as indicated under standard assay procedure in “Methods.” Protein determinations with the Lowry reagent were carried out on 0.2 ml of each fraction and are expressed as micro- gram equivalents of bovine serum albumin.
TABLE IV Exonucleolytic activity of polymerase preparation
The reaction mixtures (0.2 ml) contained 20 Nmoles of Tris- HCl, pH 7.7,500 nmoles of MgCla (for native DNA) or 1250 nmoles of MgClz (for denatured DNA), 25 nmoles of nonradioactive pneu- mococcal DNA, and 20,006 cpm of *H-pneumococcal DNA. The denatured samples were heated to 100” for 2 min and cooled on ice before the addition of MgClt. Then the four deoxynucleoside triphosphates or Na4Pz0, were added to the samples indicated, and finally 0.36 unit of polymerase. After 2 hours of incubation at 37”, all samples were cooled on ice, 0.1 ml of carrier calf thymus DNA, 2 mg per ml, was added, and finally 0.5 ml of cold 1 N HCIO4. After remaining on ice for 10 min the precipitates were removed by centrifugation, and the clear supernatants were placed in 10 vol- umes of a scintillation fluid composed of 4 g of 2,5-diphenyloxa- zole, 50 mg of 1,4-bis[2-(5-phenyloxazolyl)]benzene, and 120 g of naphthalene per liter of dioxane, and were counted in a Packard scintillation counter. The efficiency of counting of 3H in this solution was found to be 27% that of the glass fiber paper method used to measure total radioactivity and the values reported have been corrected for this difference.
able on control DNA. This endonuclease may be similar to the one first described by Strauss (18),’ and its presence in the polymerase preparations may be attributable to the fact that the polymerase is isolated on ultraviolet-irradiated DNA.
Ezonuclease-In a search for exonucleolytic activity, tracer amounts of 3H-labeled pneumococcal DNA (about 20,000 cpm) were added to the usual components of a reaction mixture minus the nucleoside triphosphates. After an appropriate incubation time, carrier DNA was added, the samples were precipitated with acid, centrifuged, and the radioactivity of the acid-soluble supernatant was determined. The results of such an experi- ment carried out with native and heat-denatured DNA and the effect of the addition of the four deoxynucleoside triphosphates or of pyrophosphate are presented in Table IV.
It can be seen from Table IV that a small amount of the 3H- DNA native or heat-denatured is in the acid-soluble superna- tant even in the absence of enzyme. With native DNA, no further counts are rendered acid-soluble unless either the four deoxynucleoside triphosphates or Na&‘207 are added. The amount of DNA solubilized is of the order of 20 to 50 times less than that reported by Zimmerman (2), under the same condi- tions. With heat-denatured DNA, a very small acid solubiliza- tion is observed in the absence of deoxynucleoside triphosphates, and this is increased by the addition of the nucleotides, although to a lesser degree than with native DNA. The addition of Na4-
6228 DNA Polywwrase Isolated on DNA-Cellulose Vol. 243, No. 23
100 ~- r---- A 1 B I
PNEUMOCOCCUS
CALF THYMUS _
"0 12 3 4 5 6 0 I2 3
HOURS AT 37"
FIG. 3. Rates of DNA synt.hesis. A, Native and denatured pneumococcal DNA primers: 0.2 ml of standard reaction mixtures containing either native (AT) or heat-denatured DNA (D) was in- cubated at 37” with 0.25 unit of polymerase. B, DNA primers from different sources: 0.2 ml of standard reaction mixtures con- taining native pnenmococcal, calf thymus, or phage T4 DNA was incubated at 37” with 0.4 unit of polymerase. III both A and H, at indicated times, 40-J aliquots were spotted on DEAR-cellulose paper, and washed and handled as described under “Methods.” y0 DNA s@hesis, (nanomolcs of DNA polymeriaed)/(nanomoles of DNA primer added).
P201 leads to about the same amount of acid-soluble material as obtained with native DNA.
Since the acid-soluble materials produced in the experiment of Table IV have not been characterized, it is not possible to state that the acid solubilization of the 3H-DNA is due specifically to exonucleolytic activity or to pyrophosphorolysis. Small oligonucleotides (less than 10 nucleotides long), the production of which would require an endonuclease, would also be acid- soluble. However, the acid solubilization of DNA, although occurring to an extent less than 2% of that of Zimmerman’s preparations (2), has similar requirements, i.e. the need for deoxyribonucleoside triphosphates or Na4P201, and it is plausible to suppose that it represents exonucleolytic activity and pyro- phosphorolysis similar to those described and discussed by this author.
To compare rates of synthesis and of depolymerization, a tracer amount of dATP-@PC was added to samples identical with Experiment 3 of Table IV, and DNA synthesis was measured by the usual procedure after 2 hours of incubation. The values obtained were 16.2 nmoles of nucleotides polymerized with native DNA and 10.7 nmoles with heat-denatured DNA. In that time, 0.72y0 of the 3H-labeled native DNA had become acid- soluble (Table IV, Experiment 3) of which, since 16.2 nmoles of pyrophosphate were formed from synthesis, 0.09% must have been the result of pyrophosphorolysis-like solubilization (as calculated from the results of Experiment 4, Table IV). I f we assume that the 3H-labeled and the nonradioactive primer DNA molecules are depolymerized at the same rate and that the newly synthesized product DNA is also as susceptible to degradation as the labeled primer, we can estimate that after the P-hour incubation, 0.63% of the total amount of DNA present (25 nmoles of primer plus 16.2 nmoles of product) or at most 0.25 nmole of DNA was degraded by exonuclease action. The ap- parent exonucleolytic activity on native DNA in the presence
of deoxyribonucleoside triphosphates is thus less than 1.6% of the concomitant polymeric activity. With heat-denatured DNA, this value falls to less than 1%. It is possible, however, that the different types of DNA present in these experiments are not degraded at equal rates. 3H decay leads to breakage of phosphodiester linkages in DNA, and it is likely that the 3H- DNA used in these studies by virtue of its more abundant ends (it was 2 to 3 months old at the time of these experiments) might be a better substrate for exonuclease activity than either the nonradioactive primer or the synthesized product DNA molecules. It is probable, therefore, that the calculated ratio of degradation to polymerization of DNA is an upper limit, and that the prevailing degradation with a homogeneous primer may be less.
Properties of Polymerase Reaction
In general the requirements for polymerase activity are those defined by Kornberg (20) for DNA polymerases: buffer, divalent cation, and the four deoxyribonucleoside triphosphates. The addition of mercaptoethanol or other sulfhydryl protectors, however, has no effect on either the activity or the stability of the enzyme.
pH Dependence
In Tris-HCl buffer, polymerase activity is optimal from about pH 7.6 to 8.2 and drops to 66% of maximum at pH 7.0 and 83% at pH 8.5. At optimal pH, the optimal Tris-HCl concentration iS 0.1 M. The pH of all buffers was measured at 25”. The actual pH used in this work (7.7 at 25”) at 37” is about 7.3 (21).
Rates of Synthesis
Under standard conditions, as in Fig. 3A, rates of DNA syn- thesis at 37” are constant for the first 30 to 60 min of reaction, after which they gradually decrease. Synthesis continues for long periods of time, but does not generally proceed beyond a doubling of the added primer. The addition of excess deoxy- ribonucleoside triphosphates at any time during synthesis has no effect on reaction rates. Synthetic rates are generally greater with native than with denatured DNA (Fig. 3A), and greater with heat-denatured than with NaOH-denatured DNA (not shown in Fig. 38). Large differences in synthetic rates are also observed with DNA primers from different sources (Fig. 3B).
Plots of nucleotides polymerized at 60 min against DNA added, with native and alkali-denatured pneumococcal DNA primers, are presented in Fig. 4. The pneumococcal DNA is polydisperse and is calculated to have, at neutral pH, a number average molecular weight of 9.3 x 106. (Molecular weights were cal- culated from sedimentation rates according to Studier (22). The weight average molecular weight, which is the value usually reported, is 17.4 x 106 as calculated from the same sedimenta- tion data.) With native DNA, 0.165 pg of polymerase protein (0.125 unit) reaches saturation at about 30 nmoles of added DNA. The apparent K,, calculated from the half-maximal rate is 3.75 x lop5 M expressed in terms of nucleotides in the DNA primer and 1.33 x 10mg M in DNA molecules. Although the enzyme preparation was not analyzed for its content of polymerase protein by sedimentation, it has about twice the specific activity of that presented in Fig. 2, in which 60% of the protein sedimented in the polymerase peak. It is likely, there- fore, that almost all of the protein in the polymerase preparation used in the experiment of Fig. 4 is active polymerase. At 30
nmoles of added DNA, there are 1 to 2 polymerase molecules (molecular weight, 125,000 daltons) for each double-stranded DNA molecule.
The data of Fig. 4 for alkali-denatured DNA suggest the occurrence of two reactions, one saturating at a low substrate concentration close to that at which native DNA saturates, and the other saturating at well above 100 nmoles of DNA. Since the DNA was allowed to react with NaOH at a concentra- tion of close to 1 mg per ml and subsequently neutralized, a substantial amount of helical structure of some sort may have re-formed. It is suggested that it is with this latter material that the reaction which saturates at a DNA concentration close to the saturating value for native DNA occurs. The second reaction, however, appears to saturate at a DNA concentration at which the number of DNA strands exceeds the number of protein molecules. (The number average molecular weight of pneumococcal DNA at pH 12.5 is 2.2 x 106, while the weight average molecular weight is 5.8 x 106.)
Although as indicated in Fig. 38, only initial velocities (up to 60 min) are linear, similar saturation kinetics are obtained when DNA synthesis is allowed to proceed for much longer times. As can be seen from Fig. 5, with a constant amount of polymerase, a linear relation between the amount of M. Zuteus DNA added and that synthesized is obtained up to about 10 nmoles of added DNA after either 1 or 18 hours of synthesis; beyond 10 nmoles of DNA, both curves are no longer linear. It is also to be noted that in the linear portion of the B-hour curve, the amount of DNA synthesized is, within the limit of experi- mental and calculation errors, equal to the amount of DNA added. Therefore, although a sufficient concentration of deoxy- ribonucleoside triphosphates are present to support further synthesis, DNA synthesis stopped at a doubling of the primer.
To explain nonlinear rates of synthesis and the cessation of
--CO
3-
NANOMOLES DNA ADDED
FIG. 4. Saturation of polymerase with native or alkali-de- natured pneumococcal DNA: Each sample of 0.2 ml containing 20 lrmoles of Tris-HCl. DH 7.7. 0.4 rrmole of M&12, 40 nmoles of deoxyribonucleoside triphosphites -(mixed in <he ;atio found in the DNA and including a tracer amount of dCTPJH), indicated amounts of nneumococcal DNA (denoted in nucleotide equiva- lents), and 6.165 pg of polymerase protein (0.125 unit) was in- cubated for 60 min at 37” and then handled as indicated under “Methods” for determinine DNA svnthesis. --, native DNA (AT). - - -, alkali-denatured DNA (D), concentrated native DNA diluted into NaOH to a final alkali concentration of 0.125 M. After standing for 15 min at room temperature, the solution was neutralized with HCI. The same NaCl concentration present in the denatured DNA samples was also introduced into the na- tive DNA samples. Different symbols represent results from sep- arate experiments, but with the same DNA and polymerase pre- parations.
“0 IO 20 30
NANOMOLES DNA ADDED
FIG. 5. DNA svnthesis at two different times as a function of added M. luteus-DNA. Each sample of 0.2 ml containing 20 umoles of Tris-HCl. DH 7.7. 30 nmoles of MnCL. 30 nmoles of heoxyribonucleoside triphosphates (mixed in the-ratio found in the DNA and including a tracer amount of dCTP-3H), indicated amounts of M. Zuteus DNA (denoted in nucleotide equivalents), and 0.25 unit of polymerase was incubated at 37” for either 1 or 18 hours and then handled as indicated under “Methods” for deter- mining DNA synthesis. l , l-hour incubation; 0, B-hour in- cubation; - - -, expected values for doubling of added DNA.
synthesis after a doubling of the added primer, the following facts must be taken into account. (a) The small number of protein molecules required to achieve maximal rates of synthesis
with helical DNA suggests that there is only a small number (perhaps one or two) of initiation sites for DNA synthesis on
any one DNA molecule. (b) Once synthesis is started, reinitia- tion does not take place. In Fig. 5, at low DNA concentrations, there is an excess of polymerase molecules and of deoxynucleo-
side triphosphates, yet only a doubling of the added DNA is achieved. The complex of the original and product DNA mole- cules does not apparently contain initiation sites. (4 Any polymerase not bound to DNA in the low salt concentrations used in the synthesis reactions will eventually be inactivated (Table III). (d) Product strands lengthen with time until they eventually attain approximately the same polydisperse distribu- tion as the original added DNA.48 * If it is assumed that syn-
thesis is initiated at ends of double-stranded DNA molecules, that all product polymers are initiated simultaneously, and that they are lengthened at the same rate, then the over-all rate of synthesis should decline according to the completion schedule defined by the distribution of template ends. Further, since no reinitiation occurs, synthesis stops when these ends are reached.
Divalent Cation Dependence
DNA synthesis is obtained in the presence of either Mgf+ or Mn++, but not with Zn++, Cu++, or Ca+f. The effect of Mg* or Mn* depends on the source as well as on the state of the DNA used as a primer. In the experiments of Fig. 6, the DNA and the deoxynucleoside triphosphates were held constant while
*This subject will be discussed more fully in a later com- munication.
6230 DNA Polymerase Isolated on DNA-Cellulose Vol. 243, No. 23
I.0
On8
0.6
0#4
OS2
0
on4
i
0 I 20 O,I 0.2
MgClz MnCl2 p moles
FIG. 6. Effect of Mg++ or Mn++ concentration on DNA synthesis with pneumococcal or phage T4 DNA primers. The samples (0.2 ml) contained 2Opmoles of Tris-HCl, pH 7.7,25 nmoles of pneumo- coccal DNA native or heat-denatured (3 min at 100°, cooled on ice) or 28 nmoles of Dhane T4 DNA native or heat-denatured (3 min at kNY’, cooled on*iceK 35 nmoles of total deoxynucleoside‘triphos- phates including dATP-8-l%, MgCL, or MnClz in the amounts indicated on the abscissa, and 0.35 unit of polymerase. After appropriate incubation, the samples were precipitated and han- dled as usual. The extent of synthesis with native pneumococcal DNA at its Mg++ optimum is equated to 1.0, and all other values are expressed relative to this. a and b, pneumococcal DNA; c and d, phage T4 DNA; a and c, MgCL; b and d, MnCL N, native DNA (O-O); D, heat-denatured DNA (X---X).
the divalent cation concentration was varied. With native DNA primers, an excess of either cation beyond an optimal value decreases polymerase activity, whereas with heat-denatured DNA primers large concentrations of the cations are not con- sistently inhibitory. Phage T4 DNA (native or heat-denatured) is a very poor primer in the presence of Mg++ (about 12 y0 of the activity with pneumococcal DNA, Fig. 6, a and c), while with Mn* synthetic rates equal to about half those with pneumococ- cal DNA are attained (Fig. 6, b and d). The differences in reac- tion rates with native calf thymus DNA with Mg* or Mn* reported by Zimmerman (2) were not observed in the present investigations.
The results of other experiments in which both the divalent cation and the deoxynucleoside triphosphate concentrations were varied while the DNA (native pneumococcal) was held constant are presented in Figs. 7 and 8. In these plots poly- merase activity is compared to the concentrations in the reac- tion mixtures of the metal ion and the deoxynucleoside triphos- phates in the associated complexes or unassociated forms. These values were calculated with the use of the association constants obtained by Walas (23) of 5.6 x lo4 M for Mn++ and 1.1 x 104~ for Mg*. The contribution of metal ion binding to DNA on the calculated concentration of free cation is neglected since the association constants for Mg* or Mn++ binding to DNA are of the order of 10 times less than that to the deoxynucleoside tri- phosphates (24). It is further assumed that the binding of the cations to the enByrne is much weaker than to the triphos- phates .
As can be seen from Figs. 7 and 8, as the concentration of deoxynucleoside triphosphates is increased, polymerase activity rises to a maximum and then falls with the depletion of the free cation concentration. In Fig. 8B where the Mg++ is present in great excess, only a small depletion of the free Mg* is effected by the concentrations of deoxynucleoside triphosphates used, and there is no decrease in polymerase activity after the maxi- mum is reached. With Mn* (Fig. 7), optimal rates of DNA synthesis are attained at ratios of total added Mnfe to total added triphosphates of 1 :l, with lowered activity on either side of this ratio. When the concentrations of added deoxynucleo- side triphosphates and of added Mn* are kept equal, normal saturation kinetics are obtained (Fig. 9), and the apparent in- hibition seen with an excess of either (Fig. 7) is eliminated. The K, for the deoxynucleoside triphosphate-Mn complexes, cal- culated from the data of Fig. 9, is 4 x lcb M. Except for the first two points in Fig. 7A, all of the concentrations of triphos- phate-Mn complexes in Fig. 7 are above the K,. With Mg++ (Fig. 8), optimal rates of synthesis are attained when a certain concentration of triphosphate-Mg complexes can be formed in- dependent of the over-all Mg* concentration. The K,,,, cal- culated for the deoxynucleoside triphosphate-Mg complexes from the half-maximal velocity data of Fig. 8, is 4.5 X lO+ M.
While it has been shown that DNA interacts with the poly- merase to form a stable structure, it is not obvious from the data Figs. 7 and 8 which the other constituents of the polymerase reaction are. The results appear to be explained by either of the following two reaction mechanisms. Mechanism 1: The deoxyribonucleoside triphosphate-cation complexes are the sub-
240 220 A-25nmoles MI?' B-125nmoles Mntt
200-
180
160
60
40
20
0 0 100 xl00 100 200 3000 too 200
NANOMOLES ADDED DEOXYNUCLEOSIDE TRIPHOSPHATE
FIG. 7. Effect of varying deoxynucleoside triphosphate and Mnft concentrations on DNA synthesis with native pneumococcal DNA primer. The samples (0.2 ml) contained 20 rmoles of Tris- HCl, pH 7.7; 25 nmoles of DNA; MnClz, 25 nmoles (A), 125 nmoles (I?), or 250 nmoles (C) ; an amount of deoxyribonucleoside triphos- phates (mixed in the ratio present in the DNA and including dTTP-8-‘4C) indicated on the abscissa; and 0.25 unit of polymer- aae. All samples were incubated for 60 min at 37” and precipi- tated and handled aa described under “Methods.” The Zeft ordi- nate indicates the calculated number of nanomoles of: l , deoxy- ribonucleoside triphosphate-cation complexes; 0, cation not complexed to triphosphates; or X, triphosphates not complexed to cation, at the start of incubation. The right ordinate indi- cates the number of nanomoles of nucleotides polymerized at 60 min, q - - -0.
skates that interact with the active site of the DNA-enzyme structure, and both uncomplexed cation and uncomplexed tri- phosphates somehow inhibit this interaction. If, for example, a second cation is needed for the reaction, the depletion of the ion by excess triphosphates might account for the apparent
300 2 s g 200
5 z
100
0 -0 100 200 -0 100 200 -”
NANOMOLES ADDED DEOXYNUCLEOSIDE TRIPHOSPHATE
FIQ. 8. Effect of varying deoxynucleoside triphosphate and Mg++ concentrations on DNA synthesis with native pneumococcal DNA primer. The samples (0.2 ml) contained 20 &moles of Tris- HCl, pH 7.7; 25 nmoles of DNA; MgC12, 350 nmoles (A) or 2500 nmoles (I?); an amount of deoxyribonucleoside triphosphates (mixed in the ratio present in the DNA and including dTTP-8-W) indicated on the abscissa; and 0.25 unit of polymerase. All sam- ples were incubated for 60 min at 37” and precipitated and handled as indicated under “Methods.” The left ordinates number the calculated nanomoles of: l , deoxynucleoside triphosphate-cation complexes; 0, cation not complexed to triphosphates) or X, tri- phosphates not complexed to cation, at the start of incubation. The left ordinate in B refers to the calculated number of nanomoles of Mg++ not complexed to triphosphates in this section of the figure only. All other nanomole values are to be read from the Eeft ordinate in A. The right ordinate indicates the number of nanomoles of nucleotides polymerized at 60 min (IJ- - -0).
31 I I , I
0 50 100 150 200
NANOMOLES dNTP-Mn ADDED
Pro. 9. Dependence of DNA synthesis on the concentration of equimolar mixtures of deoxynucleoside triphosphates (dNTP) and Mn++. Each sample (0.2 ml) containing 26 pmoles of Tris- HCl, pH 7.7, 25 nmoles of pneumococcal native DNA, 0.125 unit of polymerase, and the indicated equal amounts of MnClp and of deoxyribonucleoside triphosphates (mixed in the ratio found in the DNA and including a tracer amount of dCTP-*H) was incu- bated for 60 min at 37” and then handled as described under “Meth- ods” for the determination of DNA synthesis.
MOLARITY OF ADDED SALT FIG. 10. Stimulation of DNA synthesis by chloride salts with
pneumococcal DNA in the presence of Mn++. To standard assay reaction mixtures, but with optimal concentrations of MnCb re- placing MgCls, are added the three halide salts in the concentra- tions indicated, and 0.32 unit of polymerase. After 60 min of incubation at 37” each sample was precipitated and handled as described under “Methods.” The extent of synthesis is expressed in relation to the value obtained in the absence of the three added chloride salts.
inhibition by high triphosphate concentrations. It is also imaginable that uncomplexed triphosphates might be an essen- tial adjunct to the synthetic reaction and that the depletion of these by excess cation similarly may account for the apparent inhibition by high cation concentrations. Note that excess tri- phosphates inhibit only in the presence of limiting Mnft or Mg++ and that excess ion apparently inhibits only with low or limiting concentrations of triphosphates. Mechanism 2: The uncomplexed cation and uncomplexed deoxynucleoside triphos- phates are the actual substrates for the enzyme and the triphos- phate-cation complexes are present simply by virtue of their high association constants. The apparent inhibition by high added concentrations of either the cations or the triphosphates would result from the depletion of the uncomplexed triphos- phates or cations, respectively. The value of K,,, for the uncom- plexed triphosphates as substrates would have to be at least 10 times lower than those for the complexes, assumed as substrates in Mechanism 1. Several attempts to fit these data mathemati- cally to simple formal schemes have been made, but agreement has been marginal at best, thereby preventing the selection of one mechanism over any other.
E$ect of Other Ions
Inhibition and Stimulation-The effect of the chloride salts NaCI, KCl, and NH&l on DNA synthesis with native pneumo- coccal DNA in the presence of optimal concentrations of Mn++ is shown in Fig. 10. All three salts begin to interfere with the reaction above 0.08 M to 0.1 M concentrations. Below these concentrations, however, NaCl is without effect, KC1 enhances activity by about lo%, and NH&l by about 40%. If Mg* is used, all three halides begin to inhibit synthesis at about 0.04 M, and there is no stimulation at any concentration. Inorganic phosphate is a strong inhibitor of DNA synthesis. Again a differential effect is found in the presence of either Mg* or M&f. Potassium phosphate, 0.04 M (pH 7.7) leads to a 68 and 40% inhibition in the presence of Mg++ and Mn++, respectively. As indicated above, this concentration of the chloride salts is not inhibitory with either cofactor divalent cation.
6232 DNA Polymerase Isolated on DNA-Cellulose Vol. 243, Ko. 23
I I I I I f I I I I II
012345 0 I234
HOURS AT 37'
FIG. 11. Primed and unprimed synthesis of poly d(A-T). Re- action mixtures (0.5 ml) contained 50 rmoles of Tris-HCl, pH 7.7, 5 rmoles of MgC12, 150 nmoles each of dATP and dTTP, and 1.1 units of polymer&e. The primed reaction also contained 7.5 nmoles of ~olv d (A-T). The change in absorbance at 260 w was followed in a” Giiford’ recording s<ectrophotometer with the cu- vette compartment at 37”. After 5 hours at 37” the mixtures were allowed to stand at 25’ for 20 hours, at which time the absorbance of both the primed and unprimed reactions wasunchanged. Then, to the originally unprimed reaction (X), 7.5 nmoles of poly d(A-T) were added, whereae another 50 to 60 nmoles each of dATP and dTTP were added to the originally primed reaction (0). The left-hand ordinate applies to the change in absorbance for the un- primed reaction throughout, and the primed reaction for the first 5 hours. The right-hand ordinate, which applies to the last 4 hours of the originally primed reaction, represents the drop in absorbance at 260 rnp of the extra added dATP and dTTP.
Synthesis of Polymers of Deoxyribonucleotides
The synthesis of poly (dG) . (dC), poly d(A-T), poly (dA) . (dT), in the presence or absence of primers was attempted, but of these only a primed synthesis of presumed poly d(A-T) was obtained with the purified enzyme (Fig. 11). Unlike the re- sults with the &. coli polymerase (4, 25), once a maximum amount of poly d(A-T) has been formed there is no detectable degradation of this product (Fig. 11). In view of the finding that polymerase is not stable in the low salt concentrations needed for the synthetic reactions, it is not surprising that un- primed syntheses are not observed. Indeed as indicated in Fig. 11, after overnight incubation of an unprimed reaction, no polymerase activity is demonstrable even when primer poly d(A-T) is subsequently added, whereas with a similar but origi- nally primed reaction, the enzyme appears fully active some 20 hours after the synthetic reaction has stopped (Fig. 11). Zim- merman (2) similarly reported a lack of unprimed d(A-T) syn- thesis with his polymerase preparations, although he did observe such synthesis after a very long lag in the presence of y-globulin. When either poly rA or poly rU is added to a mixture containing dATP and dTTP at 37”, a polymer is made after a long lag, which on the basis of its buoyant density in alkaline CszSOc is identical with alternating poly d(A-T) (26). The ribohomo- polymers presumably serve to protect the polymerase from inac- tivation or dissociation but do not serve as templates. No polymer is synthesized in the presence of poly (rA) * (rU).
The above does not, however, explain the lack of synthesis in a primed poly (dG) . (dC) reaction, but it has also been shown with the E. coli polymerase that poly (dG) . (dC) synthesis is
not obtained with highly purified preparations, unless in the primed reaction some endonuclease is included (4). Again in the present wgrk, it has been found that with less purified frac- tions, such as those isolated on hydroxyapatite or even Fraction II, primed and unprimed synthesis of poly (dG) .(dC) or d(A-T) take place.
GENERAL CONCLUSIONS
The procedure developed in this work for the isolation of a DNA enzyme on DNA-cellulose should be extendable to other enzymes concerned with DNA synthesis, breakdown, or recom- bination. A conceivable disadvantageous complication to this procedure is that the cellulose-bound DNA is heavily irradiated with ultraviolet light. An endonucleolytic activity specific for ultraviolet-irradiated DNA was found to be present in the polymerase preparations, although heavily irradiated IINA itself does not serve as a primer for the M. Z&us DNA poly- merase.4 It is plausible to suppose, however, that the require- ments for DNA binding and for DNA primer activity are differ- ent. Polymerase binding to DNA occurs in the absence of the deoxyribonucleoside triphosphates and divalent cation required for synthesis. Further, polynucleotides like poly rA and poly rU which are not used by the polymerase as templates do pro- vide for enzyme stability which may be the result of binding. Therefore, although the procedure described here was developed for the isolation of a polymerase, it should be possible, with judicious application of variations of ionic conditions and in the physical or chemical state of the DNA, to make the DNA- cellulose method applicable to the isolation of various nucleuses, ligases, polymerases, as well as repressor molecules capable of binding to DNA.
Acknowledgments-I wish to express my gratitude to Pro- fessor L. S. Lerman for his invaluable advice, discussions, and help in the preparation of this manuscript, and to h9rs. Bess Stephens for her technical assistance in some of this work.
REFERENCES
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