Txe JOURNAL OB BIOLOGICAI. CHEMISTRY Vol 257. Nu. 5, Issue of March IO, pp 2323-2329. 198182 Prmted m U S A.

The Isolation and Characterization of DNA Polymerase cy from Spinach"

(Received for publication, July 23, 1981)

Masarou Misumi and Arthur Weissbach From the Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 071 10

A DNA polymerase with the characteristics of the a class of eukaryotic DNA polymerases has been purified 1000-fold from spinach leaves. The enzyme has a mo- lecular weight of 160,000 A 10,000 in its native form and is markedly inhibited by aphidicolin and N-ethyl- maleimide, but not by dideoxynucleoside triphos- phates. As isolated, the enzyme contains no detectable deoxyribonuclease activity. A catalytically active 12- kilodalton fragment of the DNA polymerase, appar- ently generated by endogenous proteolytic action, has also been purified. The native enzyme is found predom- inantly in the cytoplasmic fraction of broken leaf cell preparations and less than 10% is found associated with the nuclei.

The characterization of the DNA polymerases of higher plants is still in an early stage. Partial purification of DNA polymerases has been accomplished from wheat germ (1-4), crown gall tumors (5), and Pisum satiuum (6) and a relation- ship of a wheat germ enzyme to the DNA polymerase y class of animal DNA polymerases ( 7 ) has been suggested (1, 2). This putative similarity between plant and animal DNA po- lymerases has received support from the work of Sala et al. (8) who have isolated a y-like DNA polymerase from spinach chloroplasts. In addition, these workers have partially purified a DNA polymerase a-like activity from cultured rice and other plant cells (9). This enzyme is inhibited by aphidicolin (lo), a known specific inhibitor of animal cell DNA polymerase a (11).

In this paper we report the preparation of highly purified DNA polymerase from spinach leaves which has properties remarkably similar to those ascribed to mammalian DNA polymerase a. We, t,herefore, propose that this enzyme be designated a member of the a class of DNA polymerases and thus to extend, in part, the nomenclature established for animal DNA polymerases (7) to the plant kingdom.

MATERIALS AND METHODS

Field grown Spznacza oleracea I. was purchased from local mar- kets. [3H]Deoxyguanosine triphosphate and [methyl-'H]thymidine were purchased from Amersham Corp. Aphidicolin was obtained from Dr. David Abraham of the NationaI Cancer Institute and dideoxy- TTP was from P-L Biochemicals, deoxyribonuclease I was purchased from Worthington and poly(A)-oligo(dT)12-,8 and poly(dC)- oligo(dG),n.la were from Miles Laboratories, Inc. The plasmid pZE4C- 100 DNA was the kind gift of Dr. W. Zimmerman, Roche Institute of Molecular Biology. Salmon sperm DNA was purchased from Sigma and activated by nuclease treatment as described by Pedrali-Noy and

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduerfisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact,

-

Weissbach (12). PMSF' was from Sigma andp-toluenesulfonyl fluo- ride was purchased from Aldrich. Deoxynucleotides were from Sigma and agarose was obtained from Sea Kem. DEAE-cellulose (DE-52) and phosphocellulose (P-11) were supplied by Whatman. Phenyl- Sepharose was obtained from Pharmacia Fine Chemicals. Lysozyme, ovalbumin, and trypsin inhibitor were obtained from Bio-Rad and bovine serum albumin was from Miles Laboratories, Inc.

Assay for DNA Polymerase a-DNA polymerase activity was assayed in 0.05 ml of the following reaction mixture unless otherwise stated 20 mM potassium phosphate. (pH 7.5), 10 mM MgCL, 0.5 mM dithiothreitol, 0.05 mM each of dATP, dCTP, dGTP, and [methyl-"HI TTP with a specific activity of 800 cpm/pmol, 250 pg/ml of bovine serum albumin, and 200 p g / d of activated salmon sperm DNA. The incubation was carried out at 37 "C for 30 min. The radioactivity incorporated into an acid-insoluble form was assayed as previously described (13). For assays of the column fractions, 5 p1 of each fraction were added to 45 p1 of a reaction mixture. One unit of enzyme activity is defmed as 1 nmol of deoxyribonucleotide incorporated into an acid- insoluble form in 1 h at 37 "C.

Purification of DNA Polymerase a-Unless otherwise stated, all purification procedures were conducted at 0-4 "C.

Buffer A is 10 m~ Tris-HC1 (pH 7.5 at 25 "C), 20 mM NH,Cl, and 1 mM MgC12. Buffer B is 50 mM Tris-HC1 (pH 7.5 at 25 "C), 5 mM MgC12, 0.5 m~ dithiothreitol, and 0.5% Triton X-LOO. Buffer C is 20 mM potassium phosphate (pH 7.5 at 25 "C), 0.5 mM dithiothreitol, and 20% glycerol. Buffer D is 0.4 M potassium phosphate (pH 7.5 at 25 "C), 0.5 mM dithiothreitol, and 20% glycerol. Buffer E is 0.1 M potassium phosphate (pH 7.5 at 25 "C), 0.5 mM dithiothreitol, and 20% glycerol. Buffer F is 0.4 M potassium phosphate (pH 7.5 at 25 "C), 0.5 mM dithiothreitol, and 20% glycerol. All buffers except A contained 50 pg/ml of PMSF and p-toluenesulfonyl fluoride.

Preparation of Cell Extracts-Five hundred g of fresh commer- cially purchased spinach were washed 3 times with Buffer A, sus- pended in 250 ml of Buffer B, and homogenized with a Waring blender for 5 s at low speed. Two M potassium phosphate buffer (pH 7.5 at 25 "C) was added to a final concentration of 0.25 M, and the homogeni- zation was repeated for another 5 s at low speed. The homogenate was filtered through 8 layers of sterile cheesecloth and centrifuged at 20,000 X g for 30 min, and the clear supernatant fluid was used as the 5-20 fraction. The S-20 fraction was dialyzed against 12.5 volumes of Buffer C overnight and the precipitate formed during dialysis was removed by centrifugation at 20,000 X g for 30 min.

DEAE-cellulose Chromatography-The 20,000 X g supernatant (320 ml) was loaded onto a 600-ml column of DEAE-cellulose which was previously equilibrated with Buffer C. After a wash with 1.5 column volumes of the same buffer, the column was eluted in 25-ml fractions with 6.5 column volumes of a linear gradient from 0.02-0.4 M potassium phosphate (pH 7.5) using Buffer C and Buffer D. DNA polymerase a activity was detected as a peak eluting at 0.13 M potassium phosphate (Fig. I).

Phosphocellulose Chromatography-The peak fractions (530 ml) of DNA polymerase a obtained from DEAE-cellulose chromatogra- phy were pooled and phosphocellulose (70 ml), previously equilibrated

then further diluted by addition of 600 ml of Buffer E (pH 7.5). After in 0.1 M potassium phosphat,e (pH 7.5), was added. The mixture was

4 h of stirring, the slurry was placed in a column and the resin was washed with 2 column volumes of Buffer E. The column was eluted in 7-ml fractions with 8 column volumes of a linear gradient from 0.1-

' The abbreviations used are: PMSF, phenylmethylsulfonyl fluo- ride; SDS, sodium dodecyl sulfate.

2323

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2324 Spinach DNA

0.4 M potassium phosphate (pH 7.5) using Buffer E and Buffer F. DNA polymerase a activity appeared as a single peak eluting at 0.2 M potassium phosphate (Fig. 1B).

Phenyl-Sepharose Chromatography-The peak fractions ( 8 4 ml) of DNA polymerase a from phosphocellulose chromatography were pooled and dialyzed against Buffer D containing 1 M ammonium sulfate. The dialyzed solution was applied to a phenyl-Sepharose column (6.8 ml) equilibrated with the same buffer. The column was eluted in stepwise fashion with Buffer C containing 0.35, 0.25, 0.15, and 0 M ammonium sulfate, respectively. DNA polymerase activity was recovered during the last elution step.

To concentrate the enzyme, the peak fractions of DNA polymerase a activity from phenyl-Sepharose were pooled and applied onto a 1- ml phosphocellulose column. The enzyme was eluted in one step with 0.4 M potassium phosphate (pH 7.5) containing 20% glycerol and 0.5 mM dithiothreitol.

Glycerol Gradient Fraction-The concentrated enzyme in 0.6 ml was layered onto a gradient consisting of 10-30% glycerol in 0.25 M potassium phosphate buffer (pH 7.5) containing 0.5 mM dithiothreitol and centrifuged at 36,000 rpm for 16.5 h at 4 "C in a SW 40.1 rotor. After centrifugation, 11-drop fractions (0.33 ml) were collected to yield 41 fractions.

Identical control gradients which contained ovalbumin and human a-globulin as standard proteins were run simultaneously in the same rotor. The molecular weight was estimated from the sedimentation data by the method of Martin and Ames (14).

Protein Determination-Protein was determined by the method of Bohlen et al. (15), with bovine serum albumin as a standard.

Gel Electrophoresis-Polyacrylamide gel electrophoresis under nondenaturing conditions was performed according to the procedure described by Knopf et al. (16). For the determination of the DNA polymerase a activity, the gel was cut into 2-mm portions. Each gel fraction was mixed with 0.2 ml of 0.2 M potassium phosphate (pH 7.5) containing 1 m~ dithiothreitol, 20% (v/v) glycerol, and 0.1% Nonidet P-40 and incubated overnight with shaking at 4 "C. The total recovery of the enzyme was 19%. SDS-polyacrylamide gel electrophoresis was performed as previously described (17). Visualization of protein bands was accomplished with Coomassie blue (R-250) and denistometer tracings were performed on the Ortex model 4310 densitometer.

Nuclease Assay-The incubation was carried out under standard DNA polymerization conditions (cf above), except that native and denatured 32P-labeled pCRl DNA (18) (2000 cpm/pmol) was used as substrate a t a concentration of 10 pg/ml. After incubation, the reac- tion was terminated by addition of 100 pl of carrier salmon sperm DNA (2.5 mg/ml) followed by 100 pl of 6% perchloric acid. The mixture was placed on ice for 10 min, and then centrifuged at 10,000 rpm for 10 min. Two-hundred p1 of the supernatant were placed in a scintillation vial with 10 ml of Aquasol (New England Nuclear) and the radioactivity was determined in a scintillation counter. The 32P- labeled pCRl DNA was prepared by nick translation (19) and was the kind gift of Dr. Bennett Cohen of the Roche Institute of Molecular - Biology.

PreDaration of Spinach Nuclear and Chloroplast DNA-Fresh spinaih leaves (750 g) were sliced into small pieces, suspended in 500 ml of a buffer containing 0.32 M sucrose, 20 mM Tris-HC1 (pH 8.0), 3 mM CaC12, and homogenized with a Waring blender for 5 s at low speed. The homogenate was filtered through 1 layer of sterile cheese- cloth and centrifuged at 1000 X g for 5 min and the resulting pellets were suspended in 60 ml of the above extraction buffer. Five ml of this suspension were layered onto a discontinuous sucrose gradient consisting of 15 ml of 1 M sucrose and 10 ml of 2 M sucrose containing 20 mM Tris-HC1 (pH 8.0), 3 mM CaClz and centrifuged at 1000 X g for 15 min.

After removal of the resulting nuclei band at the sucrose junction, the nuclear suspension was diluted 2-fold with a buffer containing 20 mM Tris-HC1 (pH 8.0), 3 mM CaC12, and concentrated by centrifuga- tion at 1000 X g for 10 min.

Purified nuclei were obtained by repeating the sucrose gradient centrifugation twice. To the purified nuclear suspension in 20 mM Tris-HCl, 20 mM EDTA (pH 7.5), proteinase K and sodium dodecyl sulfate were added to a final concentration of 100 pg/ml and 0.1%, respectively. After incubation at 37 "C for 1 h, an equal volume of buffer-saturated phenol was mixed with this suspension, which was shaken gently for 30 min and separated into 2 layers by centrifugation at 4000 X g for 15 min. The aqueous phase was re-extracted one more time with phenol. The crude nucleic acid solution was precipitated by addition of 1/10 volume of 3 M NaOAc, pH 5.5, and 2 volumes of cold ethanol at -20 "C for 18 h. The precipitate was collected at 10,000

Polymerase a

X g for 20 min, washed with 70% ethanol, and dissolved in 1 ml of 10 mM Tris-HC1 (pH 7.6), 10 n" EDTA. This DNA solution was purified by isopycnic centrifugation in a CsCl solution (density = 1.70 g/cm3) at 45,000 rpm for 20 h at 20 "C in a Spinco 50 Ti rotor. After combining the peak fractions of the DNA, as measured at 260 nm, the DNA solution was diluted 2-fold with Hz0 and precipitated with 2 volumes of cold ethanol a t 4 "C for 2 h. After the DNA precipitate was collected by centrifugation at 10,000 X g for 20 rnin, it was washed with 70% ethanol and dissolved in 10 n" Tris-HC1 (pH 7.8), 1 mM EDTA.

Chloroplast DNA from spinach leaves was prepared by the proce- dure of Kolodner and Tewari (20).

RESULTS

Purification of Spinach DNA Polymerase a This enzyme was purified by sequential column chromatog-

raphy on DEAE-cellulose, phosphocellulose, and phenyl- Sepharose followed by a velocity sedimentation step in a glycerol gradient. Fig. 1, A and B, shows the elution profile of the DNA polymerase on DEAE-cellulose and phosphocellu- lose, respectively. As described in under "Materials and Meth- ods,'' the major peak of DNA polymerase eluting from DEAE- cellulose at 0.13 M KH2P04 (fractions 40-60) were pooled, and adsorbed to phosphocellulose. The spinach DNA polymerase was then eluted from phosphocellulose at 0.2 M KH2PO4. This peak of enzymatic activity was dialyzed and applied to a phenyl-Sepharose column in 1 M (NH4)~S04. The chromato- graphic pattern on the hydrophobic matrix, phenyl-Sepha- rose, is shown in Fig, 2A and the sedimentation pattern in the final glycerol gradient step in Fig. 2B. A summary of the purification of the enzyme is shown in Table I. The enzyme has been purified some 1000-fold and is stable for months when stored in the presence of 1 m g / d of bovine serum albumin in liquid Nz.

As will be illustrated later, it is important that protease inhibitors, such as PMSF and p-toluenesulfonyl fluoride be present throughout the early steps of the purification to avoid degradation of the enzyme.

During the first steps of the purifkation, including the phosphocellulose fractionation, there is a significant contam- ination of the DNA polymerase fractions with a deoxyribo- nuclease (data not shown). This nuclease activity can be separated from the DNA polymerase by sedimentation in

h io4

I # I 20 40 60

~~

80 I00 FRACTION NUMBER

FIG. 1. Separation of spinach DNA polymerase a on DEAE- cellulose (A) and phosphocellulose (B). Fractions 40-62 obtained in the DEAE-cellulose chromatography were pooled and applied to phosphocellulose. Five-p1 aliquots were assayed in both A and B. The experimental details are given under "Materials and Methods." W, DNA polymerase activity; - - -, K2HP04 + KHzPO~ concen- tration.

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Spinach DNA Polymerase a

I IiM O?fM 02SM OlSM OM

1 1 1

a I-

top FRACTION NUMBER bottom

FIG. 2. Elution pattern of spinach DNA polymerase a on phenyl-Sepharose ( A ) and velocity sedimentation of the phenyl-Sepharose peak in a glycerol gradient (B). Fractions 35- 46 of Fig. 1B were pooled for phenyl-Sepharose chromatography. Fractions 26-31 derived from the phenyl-Sepharose chromatography (Fig. 2.4) were pooled, concentrated on a 1-ml column of phosphocel- lulose and layered onto a 10-30% glycerol gradient in 0.25 M potassium phosphate (pH 7.5). The procedures are described under “Materials and Methods.” The molarities in A refer to the concentration of (NH&S04 used in the step elutions. Five pl of each fraction shown in A and B were assayed. In B, for enzymatic activity, [’HH]-labeled HeLa DNA (3.7 X lo4 cpm/pg) was used as a substrate in the nuclease assays. All assays are described under “Materials and Methods.” W, DNA polymerase activity; 0- - -0, deoxyribonuclease activ- ity.

TABLE I Purification of spinach DNA polymerase a

Total ac- Total pro- Specific SWP tivities tein activity Recovery

units mg %

I. Crude extract 187 4530 0.04 100 11. DEAE-cellulose 125 380 0.33 67

111. Phosphocellulose 31 11 2.82 17 IV. Phenyl-Sepharose 17 1.3 13.0 9 V. Glycerol gradient 4 0.1 40 2

glycerol gradients (Fig. 2B) so that the final DNA polymerase fractions are free of detectable nuclease activity. Examination of the nuclease activity separated in the glycerol gradient step (Fractions 17-19) show it to contain an endonuclease as evi- denced by its ability to degrade the closed circular DNA of plasmid pBR322 (Fig. 3). Neither degradation of pBR322 DNA nor degradation of ‘H-labeled HeLa DNA is observed upon incubations with the DNA polymerase fractions (Frac- tions 21-33) from the glycerol gradient (Figs. 2B and 3). To examine the DNA polymerase activity for possible exonucle- ase contamination stringently, the peak polymerase, Fraction 26 of Fig. 2B, was incubated with a nicked translated [”PI DNA (plasmid pCR1, specific activity 2000 cpm/pmol) under the standard conditions of the DNA polymerase assay but in the absence of deoxynucleoside triphosphates. The amount of DNA solubilized under these conditions was ~ 3 % of the DNA synthesizing capacity of the enzyme when either double- stranded or single-stranded [“’PIDNA was the substrate (data not shown).

Molecular Weight

Velocity sedimentation centrifugation (Fig. 2B) indicated that the isolated purified DNA polymerase has an S value of

0 -I

FRACTION NUMBER

1

2325

II

I

FIG. 3. Detection of endonuclease activity in the glycerol gradient sedimentation of spinach DNA polymerase a. The endonuclease assay was carried out as described by Hoffman et al. (29) with the following modification: the reaction mixtures contained, in 25 pl, 50 mM Tris-HCI (pH 8.0), 5 mM mercaptoethanol, 10 mM MgClz, 20 p g / d of pBR322 (supercoiled DNA), and 2.5 pl of the indicated fraction from the glycerol gradient (Fig. 2 8 ) . Incubation was a t 37 “C for 60 min, and the reaction was terminated by addition of 5 pl of a solution containing 5% SDS, 0.1 M EDTA, 0.1% bromphenol blue, and 70% glycerol. Two pl of the mixture were placed on a 1% agarose gel and electrophoresis was conducted at 45 mA for 1 h a t room temperature on a minigel apparatus (30). The running buffer was 40 m~ Tris-acetate (pH 7.8) containing 0.01 M EDTA. After the run, the gel was stained in a solution containing 2 pg/ml of ethidium bromide for 20 min. I and II refer to the supercoiled and relaxed circular DNA forms of pBR322 DNA, respectively.

approximately 7 in potassium phosphate solutions. To exam- ine this more closely, the sedimentation values of the enzyme in the crude extract were measured in glycerol gradients containing high salt (0.25 M potassium phosphate) or low salt (0.05 M potassium phosphate). In 0.25 M phosphate, the DNA polymerase sedimented at about 7.5 S whereas in 0.05 M PO4 an S value of 10.3 was observed (Fig. 4). Reversible aggrega- tions induced by lowering of raising the salt concentration are characteristic of the a class of animal cell DNA polymerases (15).

The purified spinach DNA polymerase a (Fraction V, Table I) was also examined on 6, 7, and 8% polyacrylamide gels using nondenaturing conditions. Fig. 5B shows the results

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2326 Spinach DNA

3 65 70s 1 1

Q 0

0 V

2 300-

5 2 0 0 - a

0 10 2 0 30 40

top FRACTION NUMBER bottom FIG. 4. Effect of salt concentration upon the sedimentation

velocity of spinach DNA polymerase a. A high salt S-100 prepa- ration containing 50 mM Tris-HCI, 250 mM potassium phosphate (pH 7.5) was prepared as described under "Materials and Methods" with the following modification. The S-20 fraction was centrifuged a t 45,000 rpm for 120 min a t 4 "C in a Ti-50 rotor. The supernatant was concentrated 6- to 7-fold by ultrafiltration for 6 h a t 4 "C. The concentrated S-100 fraction containing 1-2 units of DNA polymerase activity was layered onto a 10-308 glycerol gradient containing 250 mM potassium phosphate buffer (pH 7.5). 0.5 mM dithiothreitol and the gradients were centrifuged a t 45.000 rpm for 18 h a t 4 "C in a SW 50.1 rotor. After centrifugation, 5 pl of each fraction were assayed for DNA polymerase a as described under "Materials and Methods." A low salt S-100 fraction in just 50 mM Tris-HCI with 1.1 units of DNA polymerase was prepared as above in the absence of potassium phosphate. -, DNA polymerase a activity of the low salt S-100 preparation (no potassium phosphate); - - -, high salt S-100 prepa- ration. The arrows show the positions of the marker proteins, oval- bumin (3.6 S) and human y-globulin (7.0 S) in this gradient run. The recovery of enzyme was about 558 in both cases.

A B

1001 n POI" ,

L I I - L ! L

X

0 10 20 30 40 50

i l L C

top FRACTION NUMBER bottom ~~

FIG. 5. Polyacrylamide gel electrophoresis of DNA polym- erase a under nondenaturing conditions. The polyacrylamide gel electrophoresis was done in a disc gel (7% polyacrylamide gel) a t 2.5 mA/gel for 8 h a t 4 "C as described by Knopf et al. (16) except that the purified enzyme (4.5 pg: Fraction 27 in Fig. 2B) was subjected to the electrophoresis without pretreatment by DNase L A , DNA polym- erase a assay of polyacrylamide gel fraction after electrophoresis; the gel was sliced into 2-mm portions, and each gel fraction (0.2 ml) was assayed for DNA polymerase a activity as described under "Materials and Methods." B, Coomassie blue stained gel of spinach DNA polym- erase a (n and the protein standards, catalase, fumarase, and lactic dehydrogenase (10.

obtained with the 7% polyacrylamide gel in which one major band containing the DNA polymerase activity (Fig. 5A) was noted. Using the method of Hedrick and Smith (21) the migration data obtained with the DNA polymerases in the 3 polyacrylamide gels shows the molecular weight of the spinach a polymerase to be 154,000. This molecular weight would be consistent with the sedimentations velocity (7.5 S ) observed in glycerol gradients. Although the data are not shown here, a densitometer tracing of the 7% gel revealed that 70% of the protein was in this major band, and revealed a minor contam- inant (approximately 10%) with a molecular weight of about

Polymerase a

185,000. Other protein(s) which remained at the origin ac- counted for the remaining 20% of the protein. On SDS-poly- acrylamide gels, the enzyme (Fraction V, Table I) shows two major polypeptides of 78,000 and 72,000 daltons (Fig. 6, A and B ) . We tentatively conclude the spinach DNA polymerase a in its native form is composed of these two subunits.

The molecular weight of the enzyme can also be markedly influenced by the conditions under which the enzyme is iso- lated from the crude extract. If the protease inhibitors, PMSF andp-toluenesulfonyl fluoride, are omitted during the DEAE- cellulose and phosphocellulose steps, glycerol gradient analy- sis of the DNA polymerase activity isolated after DEAE- cellulose and phosphocellulose chromatography revealed the catalytic activity migrated a t 1.6 S in glycerol gradients (Fig. 7A) and showed a high mobility in 15% polyacrylamide gel electrophoresis (Fig. 7B). From these data we estimate the molecular weight of this small catalytic fragment to be 13,000. It should be emphasized that formation of this small catalytic fragment is not observed when protease inhibitors are present throughout the purification procedure.

Properties of the Purified Spinach DNA Polymerase a The following studies were performed on the Step 5 fraction

(Table I) of the enzyme isolated in the presence of protease inhibitors.

pH Optimum-In Tris-HC1 buffers, a broad optimum be- tween pH 7.0-8.5 was observed. At pH 7.0 the activities in Tris-HC1 and potassium phosphate buffers were about the same; a t pH 7.5 the activity in potassium phosphate was 10% higher than in Tris-HC1. The enzymatic activity drops sharply below pH 7 in phosphate buffers.

Cation Requirements-The Mg'+ and Mn2+ requirements are shown in Fig. 8. The Mg" is optimum a t about 10 mM and Mn" a t about 2 mM.

Temperature Effect-Fig. 9 reveals an optimum tempera- ture of between 37-45 "C, although detectable activity can be seen as low as 10 "C or as high as 52 "C.

Salt Effect-The spinach DNA polymerase a is significantly inhibited by either NaCl or KH2P04. A slight inhibition can

A B

I I I - 1 4

, - -0. -8

I

1- 0 0.2 0.4 0.6 0.0 10

RELATIVE MOBILITY

FIG. 6. SDS-polyacrylamide gel electrophoresis of spinach DNA polymerase a. Electrophoresis was conducted in a 10% gel at 7 mA/tube for 7 h as described by Weber and Osborn (17) with 6.5 pg of DNA polymerase a. Bromphenol blue was used as a tracking marker. After migration, the gel was removed and stained with Coomassie blue (R-250) ( B ) . Protein standards used in Lane I were phosphorylase A (100.000), bovine serum albumin @SA) (68,000). ovalbumin (46,000). carbonic anhydrase (29,000). soybean trypsin inhibitor (22,700). and lysozyme (14,300). The molecular weight of the dissociated subunits (Lane In were 78,000 and 72,000, respectively, as shown in A.

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Spinach DNA Polymerase a 2327

A 1.6s 7.0s - 4 4

FRACTION NUMBER FIG. 7. Glycerol gradient centrifugation of DNA polymerase

a prepared in the absence of protease inhibitors (A) and Poly- acrylamide gel electrophoresis of DNA polymerase a under nondenaturing condition (B). A, a crude extract (S-20 fraction) was prepared as described under “Materials and Methods,” except that the buffers used to make the crude extract contained 50 pgjml of PMSF as the sole protease inhibitor. The DNA polymerase activity in the crude extract was purified by DEAE-cellulose and phospho- cellulose chromatography as described under “Materials and Meth- ods.” However, the buffer solutions used in the gradient elutions contained no protease inhibitors. The polymerase a peak obtained by the fxst two steps of column chromatography (Fraction 111) (16.4 mg) was layered onto a gradient consisting of 10-30% glycerol in 0.25 M potassium phosphate buffer containing 0.5 M dithiothreitol. Centrif- ugation was carried out at 20,000 rpm for 36 h at 4 “C in a SW 25.1 rotor. After centrifugation, 22-drop fractions (1.0 ml) were collected into 34 tubes. Five p l of each fraction were assayed for DNA polym- erase a activity as described under “Materials and Methods.” B, The procedure for polyacrylamide gel electrophoresis as described by Knopf et al. (16) was performed using a slab gel (15% polyacrylamide) with some modifications. The enzyme peak (4 p g ; Fraction 5 of A ) with pretreated was pretreated with 25 p g j m l of pancreatic DNase I in a reaction mixture containing, in 0.1 ml, 50 mM potassium phos- phate buffer (pH 7.5), 10 mM 2-mercaptoethanol, 10% glycerol, and 5 mM MgCI2. After incubation at 37 “C for 5 min, the reaction mixture was chilled in ice and then electrophoresed at 4.1 V/cm for 8 h. At the same time protein standards were run from left to right using 1 , ovalbumin (46,000); 2, trypsin inhibitor (22,700); and 3, lysozyme (17,200).

be observed even at 20 mM and the inhibition is essentially complete at 100 mM (Fig. 10). The DNA polymerase assays in the studies presented in this paper were unifonnly carried out in 20 mM potassium phosphate (pH 7.5).

Template Studies-As found with most other purified DNA polymerases from eukaryotic sources, the spinach enzyme efficiently utilizes activated DNA, but shows diminished or no activity on duplex linear DNA, be it salmon sperm or spinach DNA, single-stranded linear DNA, or circular duplex DNA derived from the plasmid pZMC-100 ( 2 2 ) or from spinach chloroplasts (Table 11). The enzyme cannot copy a synthetic polynucleotide such as poly(A) (by contrast the y class of

0

Mg2’ OR Mn“ CONCENTRATION ImM)

FIG. 8. Effect of divalent ions on purified spinach DNA po- lymerase a. The DNA polymerase a activity was assayed as de- scribed under “Materials and Methods,” except that the concentra- tions of MgCI, and MnC12 were varied as shown. The reaction was carried out with 0.064 unit of Fraction V DNA polymerase a. [’HI TTP incorporation into an acid-insoluble form is shown.

P

T (“C)

FIG. 9. Temperature dependency of spinach DNA polymer- ase a. The reaction conditions were described under “Materials and Methods” and contained 0.005 unit of Fraction V DNA polymerase a. The assays were carried out for 30 min.

FIG. IO. Effect of salts on spinach DNA polymerase a. The standard reaction conditions for DNA polymerase a (cf. “Materials and Methods”) were used in the assay with 0.016 unit of Fraction V DNA polymerase a. 100% activity was 1850 cpm. t“., NaC1; 0- - -0, KPO,.

DNA polymerases are known to copy poly(A) (lo)), but can copy the synthetic DNA, poly(dC)-oligo(dG)lz.la.

Inhibitor Studies-Inhibitors such as aphidicolin, N-ethyl- maleimide, and dideoxynucleoside triphosphates are valuable tools when used to define and identify the various eukaryotic DNA polymerases (7,lO). In our hands the spinach enzyme is insensitive to dideoxy-TTP at concentrations up to 1 mM in the presence of 50 p~ dTTP (molar ratio 20:l). Animal DNA polymerases /3 and yare very sensitive to ddTTP/dTTP ratios as low as 1:l (23) and the same inhibitory effects on the ,f3 and y polymerases have been reported for the other dideoxynu- cleoside triphosphate inhibitors (24).

Although insensitive to ddTTP inhibition, the spinach DNA polymerase is exquisitely sensitive to N-ethylmaleimide, being

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2328 Spinach DNA Polymerase a

TABLE 11 Primer template studies with DNA polymerase a

DNA polymerase a assays with 0.015 unit of enzyme were carried out as described under “Materials and Methods,” except that the appropriate templates were used as substrate. The 100% value rep- resents 1930 cprn (2.4 pmol) incorporated into acid-insoluble material. The preparation or source of the DNA is also listed under “Materials and Methods.”

Primer template [”HJDeoxyribonucleo- Ernwe activity -

side triphosphate %

Activated DNA TTP 100 Spinach nuclear DNA TTP 5 Spinach chloroplast DNA TTP 1 Salmon sperm duplex DNA TTP 2 Salmon sperm single- TTP 3

Poly(A)-oligo(dT)l~-la TTP <l Circular plasmid DNA of TTP <1

Poly(dC)-oligo(dG)12-,~ dGTP 25

stranded DNA

pZMC- IO0 -

A

0 ” “ “ ’ “ ‘ 2 4 6 8

’*I 8

APHlOlCOLlN CONCENTRATION oJg/ml) FIG. 11. Effect of aphidicolin on spinach DNA polymerase

a. A, DNA polymerase a assays were performed using the condition described under “Materials and Methods” for 30 min at 37 “C in the presence of different concentration of aphidicolin. The concentrations of MTP, dCTP, and dGTP were 50 PM each, and t3H]thymidine triphosphate (specific activity 800 cpm/pmol) was also at 50 p ~ . The incubations contained Fraction V DNA polymerase (0.013 unit) and 100% activity, in the absence of aphidicolin, equals 1649 cpm of [3H] TTP incorporated. B, Data in A was plotted as described by Sala et al. (10); Vmax was taken as 1649 cpm.

inhibited to 90% by 0.1 m~ N-ethylmaleimide and showing essentially complete inhibition at 1 mM N-ethylmaleimide. Aphidicolin, a known specific inhibitor of the a class of animal cell DNA polymerases, is also a strong inhibitor of the spinach DNA polymerase (Fig. 1lA). A 70% inhibition is observed at 1 p g / d of aphidicolin and over 90% inhibition by 10 pg/ml.

TABLE 111 DNA polymerase activity of spinach cell fractions

Aphidi- Radioac- Total ac- tion by Fraction” Colin tivity tivity aphidi- % inhibi-

Colin w m U n i t s

Crude extract - 1826 91.5 + 324 14.2 84

Cytoplasm - 1351 66.4 + 299 12.8 81

Crude nuclei - 99 1 8.3 + 282 2.4 72

Washed nuclei - 606 1.9 + 182 0.45 76

Nuclear salt extract* - 862 2.7 + 26 1 0.72 75

Salt-extracted nuclei - 102 0.19 + 71 0.15 21

The preparation of cell fractions and the DNA polymerase assays using C3H]TTP are described under “Materials and Methods.” The radioactivity is that incorporated into an acid-insoluble form in the DNA polymerase assay. When added, the aphidicolin concentration

Nuclei (8.8 X lo6) were extracted with 2 ml of 0.30 M KH2P04 (pH 7.5) at 0 “C and the resulting suspension was centrifuged at lo00 X g to yield the nuclear salt extract (supernatant) and self-extracted nuclei (pellet).

The animal f l and y polymerases are unaffected at these concentrations of aphidicolin (25). A plot of the data shows the K, for aphidicolin to be 1.7 p~ (Fig. 11B) with the purified spinach DNA polymerase a.

Intracellular Distribution of Spinach DNA Polymerase a-Although DNA polymerase a is known to be a replicative enzyme in animal cells, it is distributed both in the nucleus and cytoplasm with Iocalization centered in the perinuclear region (26). When spinach leaves are broken and examined for the distribution of the aphidicolin-sensitive DNA polym- erase a, most of the activity (70-9O%) is found in the cytoplasm with less than 10% associated with the nucleus (Table 111). The nuclear bound DNA polymerase a is extractable with 0.35 M KHzPO, as is known for the similar enzyme found in animal cell nuclei (12). The apparent intracellular distribution of spinach a is thus quite similar, at a gross level, to that of the animal cell a polymerase.

Properties of the Catalytic Small Fragment-The small catalytic fragment formed from DNA polymerase a by a putative endogenous proteolytic cleavage as discussed previ- ously showed the same Mg2‘ and Mn2+ requirements a~ the native enzyme. In addition, it showed a corresponding, similar inhibition by aphidicolin and was not affected by dideoxy- TTP. The catalytic fragment was, however, less sensitive to inhibition by NaCl, being inhibited 35% at 50 mM NaCl and 63% at 100 mM NaCl. This should be compared to the results of Fig. 10.

was 100 pg/ml.

DISCUSSION

The DNA polymerase isolated in these studies bears a striking similarity to the a polymerases found in the cells of higher animals (10, 20, 23). The plant enzyme is of high molecular weight (160,000) and reversibly aggregates to high molecular weight forms in solutions of low ionic strength. It is strongly inhibited by N-ethylmaleimide but not by dideox- ynucleoside triphosphates. Aphidicolin is also as potent an inhibitor of the plant a polymerase as it is for the animal cell DNA polymerase a. Thus, by the usual parameters, the plant enzyme should be considered an a polymerase. In this respect, the spinach enzyme also resembles the a polymerase-like activity partially purified from cultured rice cells (9,101. Both

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Spinach DNA

the rice and spinach enzymes sediment around 7 S , are sen- sitive to N-ethylmaleimide and aphidicolin, and are inhibited by salt. However, there are also important differences between the rice a polymerase and the spinach a polymerase charac- terized in this study. The rice enzyme retained an associated nuclease activity after a 300-fold purification whereas we find no detectable nuclease activity after a 1000-fold purification of the spinach enzyme. Amileni et al. (8) were unable to purify the rice polymerase beyond DEAE-cellulose and phosphocel- lulose steps because of enzyme instability; yet the spinach enzyme is much more amenable to handling and we have been able to obtain highly purified preparations.

In this respect, we note that the spinach a polymerase is highly sensitive to endogeneous proteolytic digestion in crude extracts unless protease inhibitors are present. The proteolysis is extraordinary in that it generates a 12-kilodalton fragment which is catalytically active and which has properties simiiar to the native enzyme. This represents the smallest polypeptide with DNA polymerase activity that has been reported and further characterization of this fragment is underway. Char- acterization of the properties of the high molecular weight form (160,000) and small fragment of DNA polymerase a (molecular weight 12,000) shows that there seem to be no major differences between them except for the molecular weight and subunit composition. Thus, the small fragment of spinach DNA polymerase a has retained the ability to syn- thesize DNA and shows the same Mn2+ and Mg2+ dependency and inhibition by aphidicolin as the putative native enzyme. However, it is not known whether this miniform of DNA polymerase a is ever normally present in the cell and whether it might play a significant role in DNA replication in uiuo. One could also speculate that there is a regulatory mechanism which governs the conversion of the high molecular weight and small molecular weight forms of DNA polymerase a in uiuo and therefore serves a role in DNA replication. The derivation of the small catalytically active polypeptide is unknown but it may be generated from either one or both of the putative subunits (78 and 72 kilodaltons) of the high molecular weight a polymerase or from the 160-kilodalton protein directly. In this respect it is also possible that the two putative subunits we observe on SDS-polyacrylamide gel elec- trophoresis are themselves, proteolytic cleavage products of the native 160-kilodalton polypeptide.

Although our data do not delineate the cellular compart- ment(s) in which the spinach DNA polymerase a functions or its role, our studies show it to distribute in broken cellular preparations in much the same way that the corresponding animal cell a polymerase does. Previous work by Sala et al. (27) suggests that the d i k e DNA polymerase of cultured rice cells plays an essential role in the replication of nuclear DNA but not in the replication of plastid or mitochondrial DNA. If

Polymerase a 2329

this is true of the spinach DNA polymerase a it provides a strong analogy to animal cells in which it is known that nuclear DNA replication requires the a polymerase (28).

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M Misumi and A Weissbach  polymerase alpha from spinach.The isolation and characterization of DNA:

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