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OF BIOCHEMISTRY

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BIOPHYSICS

167, 547-559 (197%

The DNA Polymerases Subcellular

Distribution

DONALD Maws McLean

Department

of Chinese

and Properties

J. ROUFA,2 R. E. MOSES3

Hamster

Cells1

of Two DNA Polymerases AND

of Biochemistry

SUSAN J. REED

and Department of Medicine, Houston, Texas 77025

Baylor

College of Medicine,

Received October 11, 1974 We have fractionated homogenates of Chinese hamster cells grown in tissue culture, and found that > 80% of those cells’ DNA-dependent DNA polymerase appears localized in the soluble cytoplasm. The Chinese hamster cytoplasmic DNA polymerase is very similar to DNA polymerases from several mammalian sources: it is large and heterogeneous (165,06021X~,069daltons), sensitive to sulfhydryl-blocking reagents and absolutely requires double stranded templates containing free 3’-OH primers. Two distinct species of DNA polymerase also have been isolated from purified Chinese hamster nuclei. One nuclear DNA polymerase appeared to be identical to DNA polymerase found in the cells’ soluble cytoplasm. The second polymerase, comprising 1.5-3% of the total DNA polymerase activity, was found only in nuclear extracts. That enzyme is resistant to sulfbydryl-blocking reagents and has an apparent molecular weight of 49,090. The data discussed in this report suggest that Chinese hamster cells, like other mammalian cell types, possess at least two DNA-dependent DNA polymerases that might participate in replicative DNA biosynthesis.

cytoplasmic and nuclear subcellular fractions. A number of investigators have demonstrated that mammalian tissues and cells growing in tissue culture possess multiple species of DNA-dependent DNA polymerase (3-9). In general, when fresh or frozen cell pellets are extracted with aqueous buffers, most of the cells’ DNA polymerase is in the form of a large enzyme localized primarily in the soluble cytoplasmic fraction. A second, smaller polymerase is found among the chromatin proteins of the cells’ nuclei. A third DNA polymerase, very similar if not identical to the cytoplasmic enzyme, also has been noted in the nuclear proteins (5). Some investigators have questioned the cytoplasmic location of the major mammalian DNA polymerase and have fractionated lyophilized cell pellets in nonaqueous buffers (10). They observed that the large DNA polymerase remains associated with

We are isolating and characterizing Chinese hamster cell clones that are unable to replicate DNA at elevated temperatures. We expect that such mammalian clones will be as useful in discerning biochemical mechanisms involved in mammalian replicative DNA biosynthesis as have collections of bacterial temperature-sensitive mutants (1, 2). To compare these to the wild-type enzyme phenotype, we have examined the DNA polymerases contained by an established tissue culture cell line of Chinese hamster cells (VT9). In this report we summarize our observations on the enzymatic and physical properties of two DNA polymerases derived from the soluble ‘This research has been supported by grants NP-135 from the American Cancer Society and GB-41117 from tbe National Science Foundation. 2Author to whom requests for reprints should be addressed. 3R.E.M. is the recipient of Career Development Award GM-70314-02 from the USPHS. 547 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

548

ROUFA,

MOSES

the nuclei during fractionation in nonaqueous solvents, and have suggested that assignment of the enzyme to the cytoplasm might be an artifact of cell swelling and/or the hypotonic aqueous buffers routinely used to make extracts. On the other hand, cytoplasmic DNA polymerase has been purified from enucleated rabbit reticulocytes, suggesting that the enzyme indeed is contained within the cytoplasm of that cell type (11). Inasmuch as mammalian cell fractionation procedures have been worked out in detail, emphasizing the use of particular aqueous buffer components to maintain integrity of the subcellular compartments (reviewed in 12), we have applied these conditions to fractionate freshly harvested Chinese hamster cell homogenates and to determine the subcellular distribution of DNA-dependent DNA polymerase activities in a tissue culture cell line useful for genetic studies. EXPERIMENTAL

METHODS

Materials The Chinese hamster lung cells used in these studies are cloned derivatives of the established line, V79, originally isolated by Yerganian (13). The clone used for these studies, HT-1, was isolated from V79 as previously described (14). Dulbecco’s modification of Eagle’s minimal medium (DMEM-HG)’ used in monolayer cultures and Eagle’s minimal medium for suspension cultures were purchased as premixed dry powders from Grand Island Biological Co. Fetal calf serum and a lyophilized mixture of antibiotics (penicillin-streptomycintylocine) also were purchased from Grand Island. Disposable plastic cultureware were obtained from Falcon Plastics. Radioactively labeled nucleic acid bases, nucleosides, nucleoside 5’-mono-, di-, and triphosphates and Liquifluor scintillation concentrate were purchased from New England Nuclear Corp. Nonlabeled deoxynucleoside triphosphates, dithiothreitol, fibrous DEAE-cellulose, salmon sperm DNA, bovine serum albumin (fraction V), and Tris base were purchased from Sigma Chemical Company. ATP and thymidine were obtained from CalBiochem. Sephadex G-206 was a product of Pharmacia; and glass fiber filter discs (GF/A) were obtained from ‘Abbreviations used: DMEM-HG, Dulbecco’s modification of Eagle’s minimal medium; NEM, N-ethylmaleimide; RSB, hypotonic swelling buffer; and PBS, phosphate buffered saline; BSA, bovine serum albumin.

AND REED Whatman. N-Ethylmaleimide (NEM) was purchased from Pierce Chemical Co. Escherichia coli DNA polymerase I was purified through DEAE-cellulose chromatography (15), and poly[d(A-T)] was prepared by the method of Schachman et al. (16). Electrophoretically pure bovine pancreatic DNase I was purchased from Worthington Biochemical Corp. Triton X-100 was purchased from Rohm and Haas Company. Porcine microsomal carboxyesterase and soybean trypsin inhibitor were kindly given to us by Dr. Kirk C. Aune. [‘C]Bovine serum albumin (2.5 x lo8 cpm/mg protein) was the generous gift of Dr. W. P. Tate. Tissue Culture

Methods

Conditions for culturing the Chinese hamster cell line in monolayers have been described before (14). Large scale growth of VT9 for preparation of DNA polymerases, however, was carried out more efficiently in suspension culture. Chinese hamster cells adapt rapidly to spinner suspension cultures, where they grow with a doubling time of approximately 20 h. Spinner cultures (500 ml) were inoculated with cells grown in monolayer at a density of l-2 x 10’ cells/ml, and grown logarithmically at 35°C to a cell density of 8-10 x 10’. Spinner culture medium contained 7.5% fetal calf serum. For preparation of large quantities of Chinese hamster DNA polymerase, 500 ml spinner cultures were used to inoculate three liter spinner vessels containing 2500 ml of the same medium with antibiotics. In approximately three days the 3000 ml cultures attained densities of 4-6 x lo6 cells/ml, and were harvested for preparation of cell-free extracts. Preparation

of Cell-Free

Extracts

Cells grown in suspension cultures were harvested from the medium by centrifugation at 2000 rpm for 15 min in the JA-10 rotor of a Beckman J-21B preparative centrifuge at 4°C. The cells were resuspended in 200 ml of phosphate buffered saline (PBS) and collected by centrifugation in the JA-10 rotor. Washed cell pellets were finally resuspended at a concentration of 3 x 10’ cells/ml in 50 ml of iced hypotonic swelling buffer (RSB) containing 0.01 M Tris-HCI, pH 7.5, 0.01 M NaCl, 0.001 M MgCl,, and 0.005 M CaCl, (17), where they were allowed to swell for 30 min at 4°C. Cells grown in 150 mm monolayer dishes for the production of extracts were washed one time with 10 ml cold PBS after aspirating the culture medium. The monolayers were scraped from the plates in 2 x 5 ml aliquots of PBS. Cell clumps were triturated with a 10 ml large bore pipette, and centrifuged at 4°C at 2000 rpm for 15 min. The washed cell pellets were resuspended in 2 ml per dish (approximately l-2 x 10’ cells/ml) of RSB and allowed to swell for 30 min on ice. Swelling of the cells was monitored at 100x in a

CHINESE

HAMSTER

phase contrast microscope. When all cells were swollen (approximately 30 min), the suspensions were transferred to an iced Dounce homogenizer and ruptured with 10 to 20 strokes of the tight-fitting (A) glass plunger. Cell disruption also was monitored by phase contrast microscopy. Homogenization was ended when >95% of the cells had been ruptured. The cell homogenates were fractionated immediately as follows: unbroken cells, nuclei, and membranes were sedimented in the cold by centrifugation in the International P.R-J centrifuge at 1500 rpm for 15 min, yielding a turbid cytoplasmic supernatant fraction described in Table I. The heterogeneous pellets were suspended in 101ml of RSB, resedimented to wash the fraction free of cytoplasmic elements, and again suspended in 10 ml of RSB. This final suspension was layered over a 30-ml cushion of RSB made 0.88 M sucrose in a 50 ml conical centrifuge tube. Nuclei sedimented through the cushion (2000 rpm for 15 min), while membranes remained above at the sucrose interface. All subcellular fractions were adjusted immediately to 1 mM dithiothreitol. Cytoplasmic factions (2000 rpm supernatants) were subjected to high speed centrifugation in the Beckman JA-20 rotor (15,000 rpm for 30 min). Routinely, cytoplasmic DNA polymerase is purified from the final clarified cytosol supernatant. Protein concentrations within this fraction have ranged from 1.2 to 2 mg/ml.

Enzyme Assays Chinese hamster DNA polymerase activities were measured under optimized conditions in 50 ~1 reaction mixtures containing up to 20 ~1 of crude extract or purified enzyme fractions, 0.2 A*” units of salmon sperm DNA activated with pancreatic DNase by the procedure of Aposhian (18), 30 PM each of 5 -dATP, dGTP, dCTP, and dlTP (methyl-labeled with ‘H to a specific activity of 70 cpm/pmol), 10 mM MgCl, and 50 mM Tris-HCl., pH 7.5. Unless otherwise specified, reaction mixtures were incubated for 30 min (cytoplasmic DNA polymerase) or for 60 min (nuclear polymerase) at 3’7”C, and terminated by the addition of I ml iced 10% trichloroacetic acid containing 0.1 M sodium pyrophosphate. Mixtures were kept on ice for 5 min, and then filtered through 2.4~cm glass fiber filters with four 2-ml washed of cold 5% Cl,CCOOH followed by one 2-ml wash of cold .Ol M HCI. The washed filters were dried in an oven at 120°C and counted in liquid scintillation vials containing 10 ml of Liquifluor diluted in toluene according to the manufacturer. One unit of DNA polymerase activity has been defined as the amount of enzyme that catalyzes polymerization of one nmole of deoxynucleoside 5’triphosphate [8H]dTI’P at 37°C in 60 min. Protein concentrations in all enzyme preparations were determined relative to bovine serum albumin by the method of Lowry et al. (20), including in the

549

DNA POLYMERASES determinations blanks containing appropriate centrations of dithiothreitol buffers.

con-

Sucrose Density Gradients Sucrose gradients (5-20% w/v) in .05 M KPO,, 601 M dithiothreitol, pH 7.5, were generated in the 5 ml tubes of a Beckman SW50.1 rotor. The gradients were stabilized at 4°C for 6 h. Protein samples were layered on top of the gradients and the samples were centrifuged at 30,000 rpm for 15 h in an L3-50 preparative centrifuge at 4°C. The rotor was allowed to coast to a stop and the contents of each tube were harvested from the bottom with a Beckman gradient fractionator. Eight-drop fractions were collected and assayed for DNA polymerase and [I’C]BSA as described in the legend to Fig. 7. RESULTS

Subcellular Distribution of DNA Polymerase in Chinese Hamster Cells Since a number of investigators have demonstrated that mammalian cells contain several DNA polymerases (3-10, 21, 22), we felt it necessary to examine their subcellular distribution in freshly harvested Chinese hamster cells by well established techniques (12). Although the buffer used to rupture cells in these studies was hypotonic, and perhaps might have caused artifactual redistribution of the cells’ DNA polymerase, that buffer (RSB) has been designed by others (17) to maintain integrity of subcellular structures. Attempts were made to rupture cells under isotonic conditions, but they proved either highly inefficient with respect to cell breakage (Dounce homogenization, freeze-thaw and rapid ejection through a syringe orifice) or deleterious to the recovery of enzyme activity (sonication). Eighty-two percent of the DNA polymerase activity found in crude cell-free homogenates prepared in RSB remains in the supernatant fraction after low-speed centrifugation (Table I). The remaining 18% of the activity is associated with the sucrose-washed nuclei. The DNAdependent DNA polymerase assayed in the crude cytoplasm appeared almost exclusively (96%) in the clarified cytoplasmic supernatant. Recovery of DNA polymerase activity after high-speed centrifugation (step 3) was not quantitative (77.4%). At this stage of the fractionation dithiothreitol

550

ROUFA. TABLE

MOSES

I

DISTRIBUTION OF DNA POLYMERASE ACTIVITY AMONG CHINFSE HAMSTER SUBCELLULARFRACTIONZ Cell fraction

DNA polymerase (units)

1. Hypotonic homogenate 2. a. Sucrose-washed nuclei b. Cellmembranes c. Cytoplasm 3. a. Cytosol b. Particulates

(%)

39.2

(bcovery) 100%

7.0

(18)

0 32.1 23.8 1.0

( 0) (82) (96) ( 4)

Protein (mg) 15.7 5.6

99.8% 77.4%

4.0 7.0 4.9 1.7

n Cells (300 mg wet weight) were harvested from monolayer culture as described in Experimental Methods. After washing and swelling, the cells were homogenized in 4 ml of RSB and fractionated by differential centrifugation as described in the text. Each fraction was readjusted to the initial 4 ml volume and made 1 mM dithiothreitol when it was generated. DNA polymerase activities were determined in the standard assay immediately following the total fractionation procedure. All assays were performed in duplicate as a function of increasing fraction volume. For DNA polymerase assays a background of 1.5 pmol [3HJTTP/30 min (minus template) has been subtracted from observed values. Recovery indicates the total enzyme activity accounted for after each centrifugation, while the (9’0) column indicates the distribution of that activity in each preparative subfraction.

substantially stabilizes the enzyme activity. Thus, sulfhydryl lability may account for the lack of quantitative recovery during step 3. Addition of reducing agent before centrifugation, however, has not improved yields of soluble cytoplasmic DNA polymerase. Purification of DNA Polymerase from the Soluble Cytoplasmic Fraction

In order to characterize the DNA polymerase(s) localized in the soluble cytoplasmic fraction, we have purified that activity further. The procedures used are summarized in Table II. A soluble cytoplasmic fraction (cytosol) derived from cells grown in a large suspension culture contained 184 units of DNA polymerase activity and 78 mg protein after storage at -96°C. Adjusting the pH of the cytosol

AND REED

fluid to pH 5.1 with 1 M acetic acid yielded a precipitate that contained all of the DNA polymerase activity and about half of the fraction’s protein. The precipitate was resuspended in .02 M Tris-HCl buffer pH 7.5 containing 1 mM dithiotreitol (5 ml), and was dialyzed overnight at 4% against 400 ml of the same buffer. The resulting turbid suspension was clarified by centrifugation (10,000 rpm for 10 min in the JA-20 rotor) to give fraction 2 enzyme shown in the table. Fraction 2 enzyme was loaded directly onto a 0.9 x 30 cm column of DEAE-cellulose, equilibrated with the same buffer. After washing the column with two volumes (40 ml) of application buffer, the DNA polymerase was eluted as a single peak of activity with a linear salt gradient to 0.3 M NId,Cl (Fig. 1). The fractions eluting around 0.15 M NH&l contained the DNA polymerase activity and were pooled to yield fraction 3 enzyme. At this stage the enzyme is unstable, having a half-life of approximately 24 h at 4”C, even in the presence of 1 mM dithiothreitol. Fraction 3 enzyme also is labile to freezing, losing about half its activity with each freeze-thaw. The effect of freeze-thaw is responsible for differences in specific activities of fresh (Table I) and frozen (Table II) cytosol fractions. We have found, however, that fraction 3 can be concentrated rapidly by ultrafiltration through an Amicon PM-10 filter at 70 psi. In a 10x concentrated form cytoplasmic DNA polymerase has been stored on ice for TABLE

II

PURIFICATION OF DNA POLYMERASE FROM THE SOLUBLE CYTOPLASM OF CHINESE HAMSTER CELLS u Fraction

1. Cytosol 2. pH 5 Precipitate 3. DEAE-cellulose

Volume DNA Polymerase (ml)

50 10.5 35.2

(units)

(yield)

184 198 96.3

100% 108 52

F’rotein (ma) 78 41.3 5.6

n The purification of cytoplasmic DNA polymerase from cells grown in one 31 suspension culture (2 g) was carried out as described in the text. Assays were performed in duplicate according to procedures outlined in Experimental Methods.

CHINESE

HAMSTER F

,,“J--,CY" 1000-

02; 01

soo-

1* L

600-

551

thermore, preincubation of native DNA templates with fraction 3 polymerase does not alleviate the requirement for endonucleolytic activation of the DNA template. Properties of the Cytosol DNA Polymerase

400 -

0

DNA POLYMERASES

10 20 30 40 50 60 10 80 90 100 110 0 Frac+lons

FIG. 1. Elution of cytoplasmic DNA polymerase from a DEAEcellulose column. Fraction 1 DNA polymerase (80 ml) was dialyzed against two liters of 0.02 M Tris-HCl, 0.01 M dithiothreitol, pH 7.5, and absorbed to a DEAE-cellulose column (0.9 x 30 cm) equilibrated with the same buffer. The column was washed with two volumes (40 ml) of that buffer, and then a 200-ml linear gradient to 0.3 M NH&l in Tris-dithiothreitol was begun. Fractions (2.5 ml) were collected through the gradient, and the column was finally washed extensively with limit buffer. Flow rates of 0.5 mf/min were maintained by gravity. Aliquots (0.01 ml) of each fraction were assayed for DNA polymerase activity in standard 0.05 ml reaction mixtures for 30 min at 37°C. The protein profile was determined according to Lowry (20), where 0.025-ml aliquots of each fraction were included in standard l-ml assays. That profile is indicated at A’5o (. .). The elution gradient was monitored with a YSI conductivity bridge.

The enzymatic activity of cytosol DNA polymerase depends upon the addition of DNA template-primer, all four deoxynucleoside 5’-triphosphates and 10 mM MgCl, in the reaction mixtures (Table III). The data indicate that cytoplasmic DNA polymerase is strongly inhibited by millimolar concentrations of pyrophosphate and is sensitive to N-ethylmaleimide. Chinese hamster DNA polymerase activity was not enhanced by the inclusion of ribose ATP, in contrast to the analogous rabbit cytoplasmic polymerase (23). The experiment summarized by Table IV indicates that optimal initial rate of DNA polymerase activity is achieved with template activated by pancreatic DNase I. A small amount of polymerase activity (2.4%) was observed with native and heat denatured (15%) salmon sperm DNA. The structural integrity of our native, commercially prepared salmon sperm DNA is susTABLE

periods up to 1 month with only moderate losses of enzyme activity. Although fraction 3 enzyme is only l&fold purified over the original cell lysate (on the basis of recovered protein), substantial losses in catalytic activity have occurred when that fraction was subjected to further chromatographic steps (phosphocellulose or Sephadex G-200). We have attempted to stabilize the enzyme by addition of higher concentrations of sulfhydryl reducing agent and glycerol (10 and 20%) to all buffer without success. Furthermore, mixing the various fractions generated by chromatography procedures has not indicated that the polymerase is split into multiple complementing fractions. For these reasons we have elected to characterize fraction 3 enzyme. That fraction of cytoplasmic DNA polymerase does not degrade deoxynucleoside 5’-triphosphate substrates or the product [3H]DNA to acid soluble nucleotides during six hour incubations at 37°C. Fur-

III

REACTIONREQUIREMENTS OF PLASMIC DNA

CHINESE HAMSTER POLYMERASEO

Reaction components

A. Complete Minus activated DNA Minus dATP, dCTP, dGTP Minus MgCl, B. Complete Plus 7.2 mM pyrophosphate Plus 5 mM NEM Plus 5 mM ATP

CYTO-

DNA polymerase Activity [3H]‘ITP polymerized (pmol/30 min) 11.6 0.1 1.9 0.2 29.6 3.3 2.8 26.8

“In experiment A the complete reaction contained 0.05 units of fraction 3 cytoplasmic DNA polymerase.In experiment B the complete reaction contained 0.12 units of polymerase. Other components were as listed in Experimental Methods. Additions in experiment B were in the form of neutralized sodium salts in water. A minus enzyme assay background of 1.1 pmol [3H]‘R’P was subtracted from all values in the table.

552

ROUFA,

MOSES

pect, since E. coli DNA polymerase I, which is known to require 3’-OH primers produced by activation (24), employed native DNA as an excellent template. Heat denaturation might have unmasked 3’-OH’s buried within the duplex structure of the high molecular weight salmon sperm DNA. Whereas the E. coli DNA polymerase I efficiently utilized polyd(A-T) to prime synthesis of DNA, the Chinese hamster cytoplasmic polymerase was unable to synthesize DNA on that template. The data in Table IV suggest that this polymerase functions optimally with a duplex template containing 3’-OH primers. Chinese hamster cytoplasmic DNA polymerase catalyzes biosynthesis of DNA in a linear fashion for approximately 2 h at 37°C (data not shown), and the initial rate TABLE IV TEMPLATE SPECIFICITY OF CHINESE HAMSTER CYTOPLASMIC DNA POLYMERASEo Template

DNA polymerase activity (pmol ISH]TTP polymerized/30 mini Cytoplasmic polymerase

None Native DNA, units Activated DNA, 0.2 Also units Denatured DNA, 0.17 Azeounits Poly[d(A-Tl], 0.02 Az6"units

E. coli PO1 1

1.8

0

4.1

190

96.0

189.1

15.9

1.6

247.0

0 Reaction conditions for both the Chinese hamster and E. coli polymerases were described in Experimental Methods. Fraction 3 cytoplasmic DNA polymerase (0.2 units) and an equivalent DEAE-cellulose fraction of E. coli polymerase 1 (0.38 units) were added to 0.05 ml reaction mixtures as indicated in the table. Native salmon sperm DNA was dissolved in water at a concentration of 34 AYml. Activated DNA was prepared from that stock by treatment with pancreatic DNase 1, as described by Aposhian (18). Denatured DNA was prepared from the native DNA stock by heating it for 10 min (100°C) and then quickly cooled in iced water immediately prior to use in the reactions, All assays were performed with saturating amounts of each template; a minus enzyme reaction background of 0.9 pmol 13HJTTP has been subtracted from the values reported

AND REED

of reaction is affected by the extent to which the DNA template is activated. Since the time course of DNA biosynthesis also might have been affected by degradation of template-primer, by degradation of nucleotide substrates or by accumulation of inhibitory reaction products, we have examined the requirements for reinitiating DNA biosynthesis in reaction mixtures preincubated for 3 h at 37°C. Only when both DNA polymerase and templateprimer were added to preincubated reaction mixtures was the initial rate of synthesis restored (data not shown). Those results indicate that reaction by-products, such as pyrophosphate, have not accumulated to inhibitory levels and that nucleoside triphosphate substrates have been neither depleted nor destroyed during the 3-h preincubation. Others have reported that cytoplasmic DNA polymerases isolated from mammalian tissues and cells growing in tissue culture are heterogeneous in sedimentation properties (3, 26). We have observed that when the DEAE-cellulose column described in Fig. 1 is developed in 0.02 M phosphate with a KC1 gradient, cytoplasmic Chinese hamster DNA polymerase elutes as two peaks of equal specific enzyme activity. Such an elution is summarized in Fig. 2. To be certain that neither enzyme inhibitors nor activities that destroy the tsH]DNA product eluted between the two peaks of polymerase activity, we have tested effects of fractions numbered 40-61 on E. coli polymerase I-catalyzed DNA biosynthesis. No inhibitory activities were demonstrated in that region of the DEAE-cellulose column. Peaks A and B were pooled as indicated on the figure and were examined separately by the parameters discussed in Tables III and IV. Both enzymes (or forms of the same enzyme) are very similar in all catalytic properties studied. One difference between Peaks A and B, however, has become apparent. We have chromatographed Peak A and Peak B on gel filtration columns, and have observed small differences in their elution from Sephadex G-200. As illustrated in Fig. 3, DNA polymerase B elutes a few fractions ahead of Peak A. Both forms of the DNA

CHINESE HAMSTER DNA POLYMERASES

-’

u Y

-7 1200

-

0

IO

20

30

40

50

60

70

80

90

100

FIG. 2. Elution of cytoplasmic DNA polymerase from a DEAE-cellulose column in phosphate buffer. Fraction 2 enzyme (5 ml) was dialyzed against 500 ml of 0.02 M potassium phosphate, 0.001 M dithiothreitol, pH 7.5, and loaded onto a 0.9 x 30 cm column of DEAE-cellulose equilibrated with the same buffer. The column was washed with 2.5 volumes (0.50 ml) of potassium phosphate-dithiothreitol as 5-ml fractions were collected. A salt gradient to 0.4 M KC1 in phosphate buffer was begun, and 2.5 ml fractions were collected to the end of the gradient. DNA polymerase, protein concernrations and the salt gradient were assayed as described in Fig. 1.

600

0

13

14

IS

22

26

M

34

38

42

46

polymerase eluted from the column well ahead of the largest standard protein examined. These observations indicate that the cytoplasmic DNA polymerase of Chinese hamster cells is large (> 180,000 daltons) and consists of at least two molecular species. DNA Polymerase from the Nuclei nese Hamster Cells

0

50

FIG. 3. Sephadex G-200 elution of cytoplasmic DNA polymerases A and B. A column 0.9 x 60 cm was packed with Sephadex G-200 equilibrated with 0.05 M potassium phosphate, 0.001 M dithiothreitol, pH 7.5. A flow rate of 0.1 ml/min was established with a pressure head of 15 cm. Samples (1 ml) of peaks A and B concentrated 10x by ultrafiltration from the column summarized in Fig. 3, or a mixture of five standards (blue dextran, porcine microsomal carboxyesterase, bovine serum albumin, soybean trypsin inhibitor, and DNP-glycine with protein molecular weights of 180,090 (251, 68,090, 14,300, and 260, respectively, were chromatographed on the column. The standards were chromatographed both before and after enzyme samples and were assayed by optical density. Throughout the series of elutions, standards reproducibly eluted in the peak positions indicated by arrows. DNA polymerase assays were carried out on O.Ol-ml aliquots of each l-ml fraction and were incubated for 60 min at 37% as described in Experimental Methods.

553

of Chi-

Eighteen to twenty percent of the NEMsensitive (cytoplasmic) DNA polymerase activity in extracts of Chinese hamster cells was found in the pellet of sucrosewashed nuclei (Table I). When sucrosewashed nuclei were treated with hypotonic extraction buffer made 1% in Triton X-100 (Table V), virtually all of the DNA polymerase activity was solubilized, leaving a pellet of intact nuclei which did not by itself catalyze DNA synthesis from exogenous substrates. Others have demonstrated that nonionic detergents (such as Triton X-100) strip nuclei of their outer membranes, leaving behind nuclei bounded by only the inner membrane (12). After Triton X-100 treatment Chinese hamster nuclei appeared greatly expanded and much less dense under the phase contrast microscope (100x). The Chinese hamster DNA polymerase contained within the Triton wash has been characterized by several procedures, and appears to be identical to cytoplasmic DNA polymerase. In order to determine whether additional DNA polymerases could be isolated from nuclei after they were washed sequentially in sucrose and Triton X-100, we have extracted nuclear preparations with 0.5 M potassium phosphate (pH 7.5) containing 0.001 M dithiothreitol and 1% Triton X-100 (high salt-detergent extract). This procedure is similar to the method for obtaining nuclear DNA polymerases developed by others (36). As shown by data in Table V, high salt-detergent extracts contained substantial amounts of additional DNA polymerase activity. One high salt-detergent extract of nuclei prepared from a homogenate (310 mg of cellular protein) contained 46 units of DNA polymerase activity and 7 mg of protein (Table VI). Approximately

554

ROUFA. TABLE

MOSES

TABLE

V

EXTRACTION OF DNA POLYMERASE FROM NUCLEI OF CHINESE HAMSTER CELLS a Subcellular

AND REED

Fraction

A. Sucrose-washed nuclei B. Low-salt-Triton-washed nuclei 1. Triton wash C. High-salt-Triton extract of washed nuclei

DNA polymerase activity (units) Complete

% Recovery

64

The DNA polymerases of Chinese hamster cells. Subcellular distribution and properties of two DNA polymerases.

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 167, 547-559 (197% The DNA Polymerases Subcellular Distribution DONALD Maws McLean Department of Chin...
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