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 Characterizationof DNA Polymerase cy from

Spinach"
(Received for publication, July 23, 1981)

Masarou Misumi and ArthurWeissbach

From theDepartment of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110

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 relationship 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 polymerases has received support from the work of Sala et al.
(8) who have isolated a y-like DNA polymerase from spinach
chloroplasts. In addition, theseworkers have partiallypurified
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
thusto extend, in part, the nomenclature established for
animal DNA polymerases (7) to the plantkingdom.
MATERIALSANDMETHODS

Field grown Spznacza oleracea I. was purchased from local markets. [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 dideoxyTTP was from P-L Biochemicals, deoxyribonuclease I was purchased
from Worthington and poly(A)-oligo(dT)12-,8 and poly(dC)oligo(dG),n.lawere from Miles Laboratories, Inc. The plasmid pZE4C100 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 asdescribed 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 fluoride was purchased fromAldrich. Deoxynucleotides were from Sigma
and agarose was obtained from Sea Kem. DEAE-cellulose (DE-52)
and phosphocellulose (P-11) were supplied by Whatman. PhenylSepharose 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 20mM 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 p1of a reaction mixture. One unit of enzyme activity
is defmed as 1 nmol of deoxyribonucleotide incorporated into an acidinsoluble form in 1h 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 mMMgC12. 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 is0.1 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 p g / m l of PMSF and p-toluenesulfonyl fluoride.
Preparation of Cell Extracts-Five hundred g of fresh commercially purchased spinach were washed 3 times with Buffer A, suspended 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 homogenization was repeated for another 5 s at low speed. The homogenate
was filtered through 8layers 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 chromatography were pooled and phosphocellulose (70ml), previously equilibrated
in 0.1 M potassium phosphat,e (pH 7.5), was added. The mixture was
then further diluted by addition of 600 ml of Buffer E (pH 7.5). After
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-mlfractions with 8 column volumes of a linear gradient from 0.1-

' The abbreviations used are: PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate.

2323

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A DNA polymerase with the characteristics of the a

class of eukaryotic DNA polymeraseshas been purified
1000-fold from spinach leaves. The enzyme has a molecular weight of 160,000 A 10,000 in its native form
and is markedly inhibited by aphidicolin andN-ethylmaleimide,but
not bydideoxynucleosidetriphosphates. As isolated, the enzyme containsno detectable
deoxyribonuclease activity. A catalytically active 12kilodalton fragment of the DNA polymerase, apparently generated by endogenous proteolytic action,has
also been purified. Thenative enzyme is found predominantly in the cytoplasmic fraction of broken leaf cell
preparations and less than 10%
is found associated with
the nuclei.

2324

Spinach DNA 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 10,000X 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 procedure of Kolodner and Tewari (20).

RESULTS

Purification of Spinach DNA Polymerase a

This enzyme was purified by sequential column chromatography on DEAE-cellulose, phosphocellulose, and phenylSepharosefollowedbya
velocity sedimentation step ina
glycerol gradient. Fig.1, A and B, shows the elution profile of
the DNA polymerase on DEAE-cellulose and phosphocellulose, respectively.As described in under "Materials Methand
ods,'' the major peak of
DNA polymerase elutingfrom DEAEcellulose at 0.13 M KH2P04(fractions40-60) were pooled, and
adsorbed to phosphocellulose. The spinach DNA polymerase
was then eluted from phosphocelluloseat 0.2 M KH2PO4.This
peak of enzymatic activity wasdialyzed and applied to a
phenyl-Sepharose column in 1 M (NH4)~S04. The
chromatographic patternon the hydrophobicmatrix, phenyl-Sepharose, is shown in Fig, 2A and the sedimentation patternin the
finalglycerolgradient step inFig. 2B. Asummary of the
purification of the enzyme is shown in Table I. The enzyme
has beenpurified some 1000-foldand is stable for months
whenstoredin the presenceof1 m g / d ofbovineserum
albumin in liquid Nz.
As will be illustrated later, it is important that protease
inhibitors, such as PMSF and p-toluenesulfonyl fluoride be
present throughoutthe early steps of the purificationto avoid
degradation of the enzyme.
During the first steps of the purifkation, including the
phosphocellulose fractionation, thereis a significantcontamination of the DNA polymerase fractions with a deoxyribonuclease(data not shown). This nuclease activity can be
separated from the DNA polymeraseby sedimentation in

io4

I
#
80
40
60
FRACTION NUMBER
~~

20

I
I00

FIG. 1. Separation of spinach DNA polymerase a on DEAEcellulose (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~
concentration.

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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 m l )
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 m l ) 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 1ml phosphocellulose column. The enzyme waseluted 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 thesame
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 mixedwith 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-40and 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-labeledpCRl DNA (18) (2000 cpm/pmol) was used as
substrate a t a concentration of 10 pg/ml. After incubation, the reaction was terminated by addition of100 pl of carrier salmon sperm
DNA (2.5 mg/ml) followed by100 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 p1of 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 32Plabeled 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 1layer of sterile cheesecloth 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 1M 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 thesucrose 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 centrifugation a t 1000 X g for 10 min.
Purified nuclei were obtained by repeating the sucrose gradient
centrifugation twice. To the purified nuclear suspension in 20mM
Tris-HCl, 20 mM EDTA (pH 7.5), proteinase K and sodium dodecyl
sulfate were added to a final concentration of100 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 separatedinto 2 layers by centrifugation
a t 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

2325

Spinach DNA Polymerase a

-I

0
IiM

O?fM

02SM

O l S M OM

FRACTIONNUMBER

1 1

a
I-

II

I
top

FRACTION NUMBER

bottom

TABLE
I
Purification of spinach DNA polymerase a
SWP

I. Crude extract
11. DEAE-cellulose
111. Phosphocellulose
IV. Phenyl-Sepharose
V. Glycerol gradient

Total ac-

Total pro-

tivities
units

tein
mg

187
125
31
17

4530
38067
11
1.3
0.1

Specific
activity

0.04

0.33
2.82
13.0

Recovery
%

100

FIG. 3. Detection of endonuclease activity in the

17
9
2

glycerol

gradient sedimentation of spinach DNA polymerase

a. The
endonuclease assay was carried out as described by Hoffman et al.
40
4
(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
glycerol gradients (Fig. 2B) so that thefinal DNA polymerase indicated fraction from the glycerol gradient (Fig. 2 8 ) . Incubation
fractions are free of detectable nuclease activity. Examination was a t 37 C for 60 min, and the reaction was terminated by addition
of the nuclease activity separatedin the glycerol gradient step of 5 pl of a solution containing 5% SDS, 0.1 M EDTA, 0.1% bromphenol
(Fractions 17-19) show it to contain an endonuclease as evi- blue, and 70% glycerol. Two pl of the mixture were placed on a 1%
denced by its ability to degrade the closed circular DNA of agarose gel and electrophoresis was conducted at 45 mA for 1 h a t
plasmid pBR322 (Fig. 3). Neitherdegradation of pBR322 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
DNA nor degradation of H-labeled HeLa DNA is observed run, the gel was stained in a solution containing 2 pg/ml of ethidium
upon incubations with the DNA polymerase fractions (Frac- bromide for 20 min. I and II refer to the supercoiled and relaxed
tions 21-33) from the glycerol gradient (Figs. 2B and 3). T o circular DNA forms of pBR322 DNA, respectively.

examine the DNA polymerase activity for possible exonuclease contamination stringently, the peak
polymerase, Fraction
26of 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 absenceof deoxynucleoside triphosphates. The amountof
DNA solubilized under these conditions
was ~ 3 of%the DNA
synthesizing capacity of the enzymewhen either doublestranded orsingle-stranded [PIDNA was the substrate (data
not shown).
Molecular Weight

Velocity sedimentation centrifugation (Fig. 2B) indicated

that theisolated purified DNA polymerase has an S value of

approximately 7 in potassium phosphate solutions. T o examine this moreclosely, the sedimentationvalues of the enzyme
in thecrudeextract
were measured in glycerol gradients
containing high salt (0.25 M potassium phosphate) orlow salt
(0.05 M potassium phosphate). In0.25 M phosphate, theDNA
polymerase sedimented at about 7.5 S whereas in 0.05 M PO4
an S value of 10.3 was observed (Fig. 4). Reversible aggregations 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 alsoexamined on 6, 7, and 8% polyacrylamide gels
using nondenaturing conditions. Fig. 5 B shows the results

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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 3546 of Fig. 1B werepooled 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 phosphocellulose 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 stepelutions. 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 underMaterialsandMethods.
W,DNA polymerase activity; 0- - -0, deoxyribonuclease activity.

Spinach DNA Polymerase a

2326

185,000. Other protein(s) which remained at the origin accounted for the remaining 20% of the protein. On SDS-poly1
1
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
Q
0
in its native form is composed of these two subunits.
2 3000
The molecular weight of the enzyme can also be markedly
V
5 200influenced by the conditions under which the enzyme is isoa
lated from the crude extract.
If the proteaseinhibitors, PMSF
andp-toluenesulfonyl fluoride, are omittedduring the DEAEcellulose and phosphocellulose steps, glycerol gradient analysis of the DNApolymeraseactivityisolated
after DEAE0
10
20
30
40
cellulose and phosphocellulose chromatography revealed the
top
FRACTION NUMBER
bottom
FIG. 4. Effect of salt concentration upon the sedimentation catalytic activity migrated a t 1.6 S in glycerol gradients (Fig.
15% polyacrylamide gel
velocity of spinach DNA polymerase a. A high salt S-100 prepa- 7A) and showed a high mobility in
ration containing 50 mM Tris-HCI, 250 mM potassium phosphate (pH electrophoresis (Fig. 7B). From these data we estimate the
7.5) was prepared as described under "Materials and Methods" with
be 13,000.
molecular weight of this small catalytic fragment to
the following modification. The S-20 fractionwascentrifuged
a t It should be emphasizedthat formationof this small catalytic
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 fragment is not observed when protease inhibitors are present
throughout the purification procedure.
concentrated S-100 fraction containing 1-2 units of DNA polymerase
3 65

70s

1001
I

POI"

Properties of the Purified SpinachDNA Polymerase a

The following studies were performedon 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 between pH 7.0-8.5 was observed. A t pH 7.0 the activities in
Tris-HC1 and potassium phosphate buffers were about the
same; a t p H7.5 the activity in potassium phosphate was 10%
higher than in Tris-HC1. The enzymatic activitydrops 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 temperature of between 37-45 "C, although detectableactivity can be
seen as low as 10 "C or as high as 52 "C.
Salt Effect-The spinach DNA polymerasea is significantly
inhibited by either NaCl or KH2P04. A slight inhibition can

il L C

L ! L

10

top

20
~~

30

40

50

I I I

FRACTION
NUMBER
bottom

14

FIG. 5. Polyacrylamide gel electrophoresis of DNA polymerase a under nondenaturingconditions. 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 pretreatmentby DNase L A , DNA polymerase 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 forDNA polymerase a activity as described under "Materials
and Methods." B, Coomassie blue stained gel of spinach DNA polymerase a (n and the protein standards, catalase, fumarase, and lactic
dehydrogenase (10.

obtained with the7% polyacrylamide gel in which one major

band containing the DNA polymerase activity (Fig. 5A) was
noted. Using themethod of Hedrick andSmith (21) the
migration data obtained with the DNA polymerases in the 3
polyacrylamide gels shows the
molecular weightof 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 notshown here,
a densitometer tracing of the 7% gel revealed that 70% of the
protein was in this major band, andrevealed a minor contaminant (approximately 10%) with a molecular weight of about

-0.

-8

0.4
0.6
0.0
RELATIVE MOBILITY

0.2

10

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. Aftermigration, the gel was removed andstained with
Coomassie blue (R-250) ( B ) .Protein standards used in Lane I were
phosphorylaseA (100.000), bovine serum albumin @SA) (68,000).
ovalbumin (46,000). carbonic anhydrase (29,000). soybeantrypsin
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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activity was layered onto a 10-308 glycerol gradient containing 250

mM potassium phosphate buffer (pH 7.5). 0.5 mM dithiothreitol and
the gradientswere 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 theabsence of potassium
phosphate. -,
DNA polymerase a activity of the low salt S-100
preparation (no potassium phosphate); - - -, high salt S-100 preparation. The arrows show the positions of the marker proteins, ovalbumin (3.6 S ) and human y-globulin (7.0 S ) in this gradientrun. The
recovery of enzyme was about 558in both cases.

2327

Spinach DNA Polymerase a

1.6s

7.0s

0
Mg2

OR Mn

CONCENTRATION ImM)

FIG. 8. Effect of divalent ions on purified spinach DNA polymerase a. The DNA polymerase a activity was assayed as described under Materials and Methods, except that the concentrations ofMgCI, and MnC12 were varied as shown. The reaction was
carried out with 0.064 unit of Fraction V DNA polymerase a. [HI
T T P incorporation into an acid-insoluble form is shown.

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 phosphocellulose chromatography as described under Materials and Methods. However, the buffer solutions used in the gradientelutions
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. Centrifugation 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 polymerase 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 phosphate 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, trypsininhibitor (22,700); and 3, lysozyme
(17,200).

be observed even a t 20 mM and the inhibition is essentially

complete at 100 mM (Fig. 10). The DNA polymerase assays in
the studies presented inthis paperwere unifonnly carriedout
in 20 mM potassium phosphate (pH 7.5).
Template Studies-As found with most other purified DNA
polymerases from eukaryoticsources, the spinach enzyme
efficiently utilizes activated DNA,but shows diminished or no
activity on duplex linear DNA,be it salmon spermor 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

T (C)

FIG. 9. Temperature dependency of spinach DNA polymerase 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 syntheticDNA, poly(dC)-oligo(dG)lz.la.
Inhibitor Studies-Inhibitors such asaphidicolin, N-ethylmaleimide, 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 yarevery sensitive to ddTTP/dTTPratios
as low as 1:l (23) and the sameinhibitory effects on the ,f3 and
y polymerases have been reported for the other dideoxynucleoside triphosphate inhibitors (24).
Although insensitive to ddTTPinhibition, 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 represents 1930 cprn (2.4 pmol) incorporated into acid-insolublematerial.
The preparation or source of the DNA is also listed under Materials
and Methods.
Primer template
[HJDeoxyribonucleoErnwe
activity
side triphosphate
%

Activated DNA
Spinach nuclear DNA
Spinach chloroplast DNA
Salmon sperm duplex DNA
Salmon
sperm
singlestranded DNA
Poly(A)-oligo(dT)l~-la
Circular plasmid DNA of
pZMC- IO0
Poly(dC)-oligo(dG)12-,~

TTP
TTP

100

TTP

1
2
3

TTP

TTP
TTP

<l

dGTP

25

Cytoplasm

Nuclear salt extract*

Salt-extracted nuclei

<1

*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 a t 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);Vmaxwas 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 specificinhibitor of the a class of animal
cell DNA polymerases, also
is 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.

AphidiColin

-91.5

Radioactivity

Total
tion
acby
tivity

wm

Units

1826
14.2
+
324
1351
12.8
+
299
991
+
282
1.9
606
+
182
- 2.7 862
+
26 1 75
- 0.19 102
+
71

% inhibi-

aphidiColin

84
66.4

81
8.3
2.4

72

0.45

76

0.72
0.15

21

The preparation of cell fractions and the DNA polymerase assays

using C3H]TTPare 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
was 100 pg/ml.
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 l o 0 0 X g
to yield the nuclear saltextract(supernatant)and
self-extracted
nuclei (pellet).

The animal fl and y polymerases are unaffected at these

concentrations of aphidicolin (25). A plot of the data shows
the K, for aphidicolin to be 1.7p~ (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 polymerase 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 fromDNApolymerase
a by a
putative endogenous proteolytic cleavage as discussed previously 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 dideoxyTTP. 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.
DISCUSSION

The DNApolymerase 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 weightforms in solutions of low ionic strength. It is
strongly inhibited by N-ethylmaleimide but not by dideoxynucleoside triphosphates. Aphidicolin is also as potent an
inhibitor of the plant a polymerase as itis for the animal cell
DNA polymerase a.Thus, by the usual parameters, the plant
enzyme should be consideredan a polymerase. In this respect,
the spinach enzyme also resembles the a polymerase-like
activity partially purified fromcultured rice cells (9,101.Both

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Crude extract

Washed nuclei

Fraction

Crude nuclei

TTP

TABLE
111
DNA polymerase activity of spinach cell fractions

Spinach DNA Polymerase a

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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Lett. 59, 125-130

2. Castroviejo, M., Tarrago-Litvak, L., and Litvak, S. (1975) Nucleic

Acids Res. 2, 2077-2090
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5. Gardner, J. M., and Kado, C. I. (1976) Biochemistry 15,688-696
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521-527
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Res. Commun. 82,1984-1090
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Arch. Biochem. Biophys. 155,213-220
16. Knopf, K. W., Yamada, M., and Weissbach, A. (1976) Biochemi s t v 15, 4540-4548
17. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412
18. Van den Berg, J., Ooyen, A., Mantei, N., Schambock, A,, Grosveld,
G . , Falvell, R. A., and Weissmann, C. (1978) Nature 2 7 6 , 3 7 4 4
19. Jeffreys, A. J., and Flavell, R. A. (1977) Cell 12,429-439
20. Kolodner, R., and Tweari, K. K. (1975) Biochim. Biophys. Acta
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21. HedriLk, J. L.,~
and Smith, A. J. (1968) Arch. Biochem. Biophys.
126, 155-164
22. Bedbrook, J. R., Kolodner, R., and Bogarad, L. (1977) Cell 11,
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23. Edenberg, H. J., Anderson, S., and DePamphilis, M. L. (1978) J.
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24. Zimmermann, W., Chen, S. M., Bolden, A,, and Weissbach, A.
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the rice and spinach enzymes sediment around 7 S , are sensitive to N-ethylmaleimide and aphidicolin, and are inhibited
by salt. However, there arealso important differencesbetween
the rice a polymerase and the spinach a polymerase characterized 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. Amileniet al. (8) were unable to purify
the rice polymerase beyond DEAE-celluloseand phosphocellulose 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-kilodaltonfragment
which is catalytically active and which has properties simiiar
to thenative enzyme. This represents the smallest polypeptide
with DNA polymerase activity that has been reported and
further characterization of this fragment is underway. Characterization 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 synthesize 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 couldalso 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 thisrespect it is also possible that thetwo
putative subunits we observe on SDS-polyacrylamidegel electrophoresis are themselves, proteolytic cleavage products of
the native 160-kilodalton polypeptide.
Although our data do not delineate the cellular compartment(s) in whichthe 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 thed 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

2329

:
The isolation and characterization of DNA
polymerase alpha from spinach.
M Misumi and A Weissbach
J. Biol. Chem. 1982, 257:2323-2329.

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