1. science
2. medicine
3. genetics

A High Molecular
Weight
melanogaster
Embryos
PURIFICATION,
STRUCTURE,
AND
DNA
PARTIAL
Polymerase
from
Drosophila
-
CHARACTERIZATION*
(Received for publication,
Geoffrey
From
R. Banks,+
the Department
John
A. Boezi,#
of Biochemistry,
Stanford
April 27, 1979)
and I. R. Lehman
University
The DNA polymerase
of early embryos
of Drosophila
melanogaster
has been purified
to near-homogeneity.
The purified
enzyme
gave a single, catalytically
active
protein
band after polyacrylamide
gel electrophoresis,
under
nondenaturing
conditions.
Four
polypeptides
with
molecular
weights
43,000,
46,000,
58,000,
and
146,000 were resolved
when this band was electrophoresed
under
denaturing
conditions.
At high
ionic
strengths,
the DNA polymerase
had a sedimentation
coefficient
of 6.7 S, a Stokes radius of 78 A and frictional
ratio of 1.81, parameters
that yield a molecular
weight
of 280,000.
The purified
DNA polymerase
possessed
no detectable endo- or exodeoxyribonuclease,
ATPase,
or RNA
polymerase
activity.
Using an “activated”
DNA template-primer,
the enzyme
had a pH optimum
of 8.5. It
was stimulated
by (NH&SO+
KCl, and to a lesser extent, NaCl. A divalent
metal cation
was absolutely
required;
MgClz stimulating
activity
7-fold
more than
MnC12. It was inhibited
by low concentrations
of Nethylmaleimide
and Aphidicolin.
Thus the DNA polymerase of D. melanogaster
resembles
most closely the (YDNA polymerases
that have been purified
from mammalian
cells.
Considerable
progress has been made in establishing
the
function of the multiple DNA polymerases
in the replication
and repair of prokaryotic
chromosomes
(for recent reviews see
Iiefs. l-7). This progress has been achieved
largely by a
combined
biochemical
and genetic approach
that has been
aided greatly by the relatively large amounts of these enzymes
that can be obtained
from prokaryotes,
and the ease with
which DNA polymerase-deficient
mutants can be isolated. In
several instances the DNA polymerases,
both mutant and
wild type, have been obtained
in homogeneous
form and
detailed structural
and mechanistic
studies have been performed. Eukaryotes
are also known to contain multiple DNA
polymerases;
however, in contrast to prokaryotes
these enzymes are available in only limited quantities
(g-10). Eukary* This work was supported by grants from the National
Institutes
of Health (GM-06196) and the National Science Foundation (PCM74.00865). The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be
hereby marked “aduertisement”
in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
4 Supported on sabbatical leave by a fellowship from Smith Kline
and French Laboratories and a Senior fellowshio of the California
Division of the American Cancer Society. Permanent address, National Institute for Medical Research, Mill Hill, London, NW7 1AA.
§ Supported on a sabbatical leave by the Josiah Macy Foundation.
Permanent address, Department of Biochemistry, Michigan State
University, East Lansing, Mich. 48824.
9886
School
of Medicine,
Stanford,
California
94305
otes possess no easy method of genetic analysis, further limiting studies of their mechanisms
of DNA replication.
As a
result, the combined biochemical-genetic
approach that has
proved so powerful in the analysis of prokaryotic
DNA replication has thus far been limited to lower eukaryotes
or to
virus-infected
mammalian
cells (11-15).
Drosophila
melanogaster
possesses many of the attributes
of a prokaryote.
During
the fist hours of its embryonic
development,
the chromosomal
DNA is replicated
in about 4
min, the nuclei dividing
mitotically
every 10 min until a
syncytium
containing
some 6000 nuclei is formed (16). Such
rapid DNA replication
is achieved
by the use of a large
number
of chromosomal
replication
origins rather than a
rapid rate of fork movement (17,18), providing an explanation
for the high concentrations
of DNA polymerase
(19), DNA
ligase,’ DNA nicking-closing
enzyme and histones” found in
these embryos. Further, D. melanogaster
has been characterized genetically
and mutants with defects in DNA repair and
recombination
are known (20).
As part of a combined biochemical
and genetic analysis of
DNA replication
in D. melanogaster,
we have undertaken
the
purification
of the DNA polymerase
from early (2 to 16 h)
embryos. Previous attempts at purification
have been hampered by proteolysis
of the enzyme in the course of purification, which generated both size and charge heterogeneity
(21,
22). In this paper, we describe a relatively
simple purification
procedure
which minimizes
proteolysis,
giving a nearly homogeneous protein of high molecular
weight. This procedure
can be readily scaled up to yield quantities
of DNA polymerase suitable for structural
and mechanistic
studies. We also
present a possible subunit structure and partial characterization of the enzyme.
EXPERIMENTAL
PROCEDURES
Materials
Nucleotides
and Homopolymers-Unlabeled
deoxyribonucleoside
triphosphates,
poly- and oligonucleotides
were purchased
from P-L
Biochemicals;
[“H]dTTP
was from New England
Nuclear.
Nucleic
Acids-Calf
thymus
DNA,
Calbiochem
A grade,
was
treated
with pancreatic
DNase
until maximally
active
with Escherichia
coli DNA polymerase I (23). Poly(rA), of unspecified chain
length,
poly[d(A-T)],
(~A)Too, and (dT)I,
were purchased
from P-L
Biochemicals.
ColEl
DNA with a 17.5 kilobase
pair Drosophila
DNA
insert, and phage P22 [:‘H]DNA
and supercoiled
ColEl
DNA were
gifts from
Dr. Gregory
Guild
and Dr. George
Weinstock
of this
department.
Chromatography-Phosphocellulose
Pll,
hydroxyapatite
HTP,
and Sepharose
6B were purchased
from
Whatman,
Bio-Rad,
and
Pharmacia,
respectively.
Single-stranded
DNA-cellulose
and blue
dextran-Sepharose
’ Unpublished
’ D. Brutlag,
were gifts from Doctors David Bates and Ralph
observations.
personal
communication.
Drosophila
DNA
Meyer
of this department.
Collodion
membranes
were purchased
from Schleicher
and Schuell,
Keene, N. H.
Enzyme
and Protein
Standards-Bovine
pancreatic
DNase,
E.
coli alkaline
phosphatase,
P-galactosidase,
and rabbit
muscle
phosphorylase
a were obtained
from Worthington,
rabbit
muscle L-lactate
dehydrogenase,
bovine
liver catalase;
bovine thyroglobulin
and horse
spleen apoferritin
from Sigma and BSA3 from Pentex.
Myosin
from
Dictyostelium
discoideum
was a gift of Dr. James Spudich
(Stanford).
Chemicals-Polymin
P was obtained
from BASF Wyandotte
Corp.
A 10% (v/v)
stock solution
was prepared
as described
by Jendrisak
and Burgess
(24); the pH was adjusted
to 7.5 by addition
of concentrated HCl and clarified
by filtration.
PMSF,
purchased
from Sigma,
was dissolved
in isopropyl
alcohol
to make a stock 0.1 M solution.
Baker Analytical
Grade sodium
bisulfite
was dissolved
in water,
the
pH of the solution
adjusted
to 7.5 with solid NaOH
and made up to
a concentration
of 1 M. Acrylamide,
N,N’-methylenebisacrylamide,
N,N,N,‘N’-tetramethyleneethylenediamine,
dithiothreitol,
bromphenol blue, and Coomassie
blue were from Bio-Rad,
and thioglycolic
acid was from Sigma. Aphidicolin
was a gift of Dr. Giovanni
Ciarrocchi
(University
of California,
Berkeley).
A 1 mg/ml
of stock solution
was
prepared
in 60% dimethyl
sulfoxide.
Methods
Processing
of Drosophila
Embryos-D.
melanogaster
(Oregon
R)
embryos
of average age 9 h were collected,
washed,
and dechorionated
according
to the method
of Brake1 and Blumenthal
(21). They were
washed
with a solution
composed
of 30 mu Tris-HCI
(pH 8.0), 0.25
M sucrose,
10 mru EDTA,
and 2.5 mru CaCh. After removal
of excess
buffer,
they were stored
at -80°C.
DNA Polymerase
Assay-Reaction
mixtures
(0.1 ml) contained
50
mu Tris-HCl
(pH 8.5), 5 mM 2-mercaptoethanol,
20 mru (NH&S04,
10 mre MgCL,
200 pg of BSA, 25 pg of activated
calf thymus
DNA,
100 PM each of dATP,
dGTP,
dCTP,
and [“H]dTTP
(70 cpm/pmol),
and enzyme,
unless otherwise
indicated.
The enzyme
was diluted
in
50 mM Tris-HCl
(pH 8.5), 2 mg/ml
of BSA,
10% glycerol.
After
incubation
for 30 min at 37”C, reactions
were stopped
by the addition
of 2 ml of 10% trichloroacetic
acid containing
1% potassium
pyrophosphate.
The tubes
were kept in ice for 10 min, and the DNA
collected
by filtration
on Whatman
GF/C
glass fiber filter discs. The
filters were washed
with 10 ml of cold 5% trichloroacetic
acid containing 1% potassium
pyrophosphate,
followed
by 10 ml of cold ethanol.
After
drying,
the filter
discs were
counted
using a toluene-based
scintillant.
One unit of activity
is that amount
which
catalyzes
the
incorporation
of 1 nmol of dNTP
into acid-insoluble
material
in 60
min, at 37’C.
Under
these conditions
activity
is 0.7 of optimal
(see
“Results”).
N&ease
Assays-Reaction
mixtures
were identical
with those for
DNA
polymerase
assays except
that native
or heat-denatured
P22
[3H]DNA
(1.0 pg, 3 x IO4 cpm/pg)
was the substrate;
in some instances
unlabeled
dNTPs
(100 C(M) were added.
Reactions
were terminated
by addition
of 0.2 ml of cold 10% trichloroacetic
acid containing
1%
potassium
pyrophosphate.
The tubes were kept in ice for 10 min and
then centrifuged
at 7000 rpm for 10 min. Aliquots
(0.1 ml) of the
supematant
fraction
were counted
in a T&on-based
scintillant.
Protein
Determinations-Protein
was determined
by the method
of Lowry
et al. (25) after precipitation
with 7% trichloroacetic
acid;
BSA was the protein
standard.
Polyacrylamide
Gel Electrophoresis
in the Presence
of SDSPolyacrylamide
slab gels containing
7% SDS and a 3.3% stacking
gel
were run essentially
as described
by Laemmli
(26). The protein
samples
were first precipitated
with cold 7% trichloroacetic
acid. The
precipitates
were redissolved
in a solution
composed
of 0.2 M P-amino2-hydroxymethyl-1,3-propanediol
(Trizma)
base, 0.2 M dithiothreitol,
3% SDS, 30% glycerol,
and 0.04% bromphenol
blue, and the protein
denatured
by heating
at IOO’C for 3 min.
Polyac&amide
Gel Electrophoresis
under
Nondenaturing
Conditions-slab
gels containing
3.5% polyacrylamide
were prepared
in
375 mM Tris-HCl
(pH 8.9), 3.5% acrylamide,
0.2% N,N’-methylenebisacrylamide,
0.58 pi/ml of N,N,N,‘N’-tetramethyleneethylenediamine,
0.7 mg/ml
of ammonium
persulfate,
and 10% glycerol.
The concentration of bisacrylamide
was kept constant
for the 5 and 7.5% gels. After
polymerization,
the gels were prerun
at 4°C overnight
using 10 mM
Tris/glycine
(pH 8.3), 0.01% thioglycolic
acid, and 10% glycerol
for
’ The abbreviations
used are: BSA and BSA*, bovine
serum
albumm and its dimer; PMSF,
phenylmethylsulfonyl
fluoride;
RM, relative
mobility;
SDS, sodium
dodecyl
sulfate;
NEM,
N-ethylmaleimide.
9887
Polymerase
the electrode
buffers.
These were replenished
with fresh, cold buffer
immediately
before loading protein
samples
onto the gel. Electrophoresis was performed
at 4°C at a constant
current
of 15 mA until the
bromphenol
blue marker
dye reached
the bottom
of the gel and all
Rnr values
were determined
relative
to the position
of the dye. TO
recover
DNA polymersse
activity,
a sample
lane was cut into 2-mm
slices using a multirazor
blade gel slicer. Each slice was transferred
to
0.2 ml of a solution
composed
of 50 mu Tris-HCl
(pH 8.5), 0.2 M
(NH&S04,
2 mM 2-mercaptoethanol,
2 mg/ml
of BSA, and 10%
glycerol
and gently
agitated
overnight
at 4°C. An aliquot
of each
supernatant
was then assayed
for DNA polymerase
activity.
Thirty
to forty per cent of the activity
applied to the gel was recovered;
after
a further
15 h, recovery
increased
to 50 to 60%. To identify
activity
with a protein
band, the cuts resulting
from the slicing operation
were
extended
into a second,
adjacent,
and identical
sample
lane, which
was stained
with Coomassie
blue. The number
of slices from the gel
origin to the DNA
polymerase
activity
was then counted
along the
side of this second stained
lane. To transfer
the protein
band with
associated
DNA polymerase
activity
to an SDS-polyacrylamide
slab
gel, a third sample lane was run. A 5-cm segment
of the lane containing
the band (its position
having
been determined
from
the first two
sample
lanes) was excised
and the protein
within
the gel denatured
by incubation
of the segment
in 0.5 ml of the denaturing
buffer
at
100°C for 5 min. Excess buffer was removed
and the segment
carefully
placed in a wide slot of an SDS-polyacrylamide
slab gel so that it was
in contact
with the stacking
gel and the direction
of protein
migration
was perpendicular
to that in the original
native
gel. Lanes flanking
the segment
contained
marker
proteins.
Determination
of Sedimentation
Coefficients-Preformed
linear
10 to 30% glycerol
gradients
in 50 mru potassium
phosphate
(pH 7.5),
1 mM EDTA,
1 mM 2-mercaptoethanol,
and 1 mM PMSF
were
prepared
in nitrocellulose
tubes suitable
for centrifugation
in a Beckman SW 50.1 rotor.
In some instances,
200 mM (NH4)pS04
was also
present.
A 150~~1 aliquot of the enzyme,
in the presence
or absence
of
200 mu (NH&SO4
was layered
onto the gradient,
which
was centrifuged at 45,000 rpm for 15 h at 4°C. Nine-drop
fractions
were collected
through
a needle inserted
into the bottom
of the tube, and an aliquot
of each fraction
assayed for DNA polymerase
activity.
Both L-lactate
dehydrogenase
and catalase
were included
as internal
markers
and
were assayed
essentially
as described
in the Worthington
Enzyme
Manual.
Sedimentation
coefficients
were determined
by the procedure of Martin
and Ames (27).
Determination
of Stokes Radius-A
column
of Sepharose
6B (0.95
x 64.5 cm) was equilibrated
with 10 mM potassium
phosphate
(pH
7.5). 200 mM (NH&S04,
and 10% glycerol
at 4°C. Samples
(0.4 ml)
were applied to the column,
which was run at a flow rate of about 2.5
ml/h;
0.4-ml fractions
were collected.
The void volume
was determined using blue dextran
and the column
calibrated
with L-lactate
dehydrogenase,
catalase,
/3-galactosidase,
thyroglobulin,
BSA, and
apoferritin.
Buffers-All
potassium
phosphate
buffers
were at pH 7.5 and
contained
1 mM 2-mercaptoethanol,
1 mM EDTA,
1 mM PMSF,
10
mM sodium
bisulfite,
and 10% glycerol
unless
otherwise
indicated.
They were prepared
at room temperature
and cooled to 4°C so that
the PMSF
would not precipitate.
Sodium
bisulfite
and 2-mercaptoethanol
were added immediately
before
use. The ionic strength
of
buffers
were routinely
checked
with a Radiometer
conductivity
meter.
RESULTS
Purification
of D. melanogaster
DNA
Polymerase
All operations were performed
at 0-4°C. A summary of the
purification
is given in Table I.
Step 1. Preparation
of Post-mitochondrial
Supernatant
Fraction-Frozen
embryos (540 g) were suspended in 2.5 liters
of cold 30 mu Tris-HCl
(pH 8.0), 10 mM EDTA, 2.5 mM CaC12,
0.25 M sucrose, 1 mM PMSF, and 10 mM sodium bisulfite, and
homogenized
in 40-ml portions by three strokes of a 40-ml
stainless steel Teflon homogenizer.
The resulting suspension
was centrifuged
at 16,000 x g for 20 min and the supernatant
fluid filtered through
four layers of cheesecloth
to remove
lipid-like
material (Fraction I).
Step 2. Polyethyleneimine
and Ammonium
Sulfate Precipitations-Fraction
I was diluted 2.2-fold and a suspension
of cold 10% Polymin P (30 ml/liter
of diluted Fraction I) was
Drosophila
9888
DNA Polymerase
TABLE
Purification
Fraction
and step
I. Post-mitochondrial
supernatant
II. Polymin
P and (NH&S04
precipitations
III. Phosphocellulose
IV. DNA-cellulose
V. Hydroxyapatite
VI. Glycerol
gradient
sedimentation
VII. Blue dextran-Sepharose
of DNA
polymerase
from
I
D. melanogaster
Volume
Protein
ml
2,602
700
mg
25,760
4,040
510
650
50
8.3
3.4
added dropwise with stirring. The resulting suspension was
stirred for an additional
20 min and the precipitate
collected
by centrifugation
at 16,000 x g for 20 mm. The pellet was
extracted by suspension in 2.5 liters of 50 mM Tris-HCl
(pH
8.0), 250 IXIM (NH&S04,
1 mu 2-mercaptoethanol,
1 mu
EDTA, 1 mM PMSF, and 10 mu sodium bisulfite in a Waring
Blendor,
followed
by stirring for a further
20 min. After
centrifugation
at 16,000 x g for 20 min, solid (NH.&&&
was
added slowly to the supernatant
fraction to 40% saturation.
The resulting
suspension was centrifuged
and the pellets
dissolved in 560 ml of 60 mM potassium phosphate
buffer by
stirring in a Waring Blendor. The solution was dialyzed overnight against 30 liters of the 60 mu potassium
phosphate
buffer (Fraction II).
Step 3. Phosphocellulose Chromatography-Fraction
II
was centrifuged
at 16,000 x g for 20 min, the supernatant
fraction saved and the pellets extracted with I50 ml of 60 mM
potassium phosphate buffer by means of a glass-Teflon
homogenizer. After centrifugation,
the two supernatant
fractions
were combined and loaded onto a column (6.5 x 17 cm) of
phosphocellulose,
equilibrated
with 60 mu potassium phosphate, at a rate of 100 ml/h. The column was washed overnight
with 1.5 liters of this buffer and the DNA polymerase activity
eluted with 160 ITIM potassium
phosphate.
Active fractions
were pooled and dialyzed overnight against 8 liters of 10 mM
potassium phosphate (Fraction III).
Step 4. DNA-Cellulose Chromatography-Fraction
III was
loaded onto a column (4 x 20 cm) of single stranded DNAcellulose equilibrated
with 10 mu potassium phosphate buffer.
The column was washed with 500 ml of this buffer at 60 ml/
h and a l-liter linear gradient from 10 to 200 mu potassium
phosphate was applied. DNA polymerase activity was eluted
at 60 mM potassium
phosphate,
and active fractions were
pooled (Fraction IV).
Step 5. Hydroxyapatite
Chromatography-Fraction
IV
was dialyzed overnight
against 5 liters of 25 mM potassium
phosphate and loaded onto a column (2.0 X 5.5 cm) of hydroxyapatite
equilibrated
with the 25 mu potassium phosphate buffer at 65 ml/h. The column was washed with 50 ml
of this buffer and a 200-ml linear gradient from 25 to 400 mu
potassium phosphate
was applied. Polymerase
activity was
eluted at 140 mM potassium phosphate,
and active fractions
were pooled (Fraction V).
Step 6. Ammonium Sulfate Concentration and Glycerol
Gradient Sedimentation-Solid
(NH.+)&04 was added slowly
with stirring to Fraction V until 60% saturation
was reached,
and the suspension was stirred for an additional
60 min. The
precipitate
was collected by centrifugation
at 27,006 X g for
10 min and dissolved in 2.5 ml of 50 mu potassium phosphate
buffer that was 8% in glycerol but lacked sodium bisultite.
The resulting
solution was dialyzed against 1 liter of this
buffer for 6 h, centrifuged,
and the supernatant
fluid layered
onto five preformed
10 to 30% glycerol gradients containing
50 mM potassium phosphate, 1 mu 2-mercaptoethanol,
1 mM
EDTA,
and 1 mru PMSF, prepared
in polyallomer
tubes
480
94
33
3.9
0.23
(Oregon
R) embryos
Activity
Specific Activity
units (X 10m3)
units/mg
5460
212
3760
932
1820
960
720
242
62
0.2
%
100
69
3,800
10,160
21,520
62,000
272,000
GALCAT
0 0
Yield
33
18
13
4.4
1.1
AP
0.4
0.6
0.8
1 0
R#
gel electrophoresis
of D. melanogaster DNA
polymerase.
Fraction
VII (35 pg) was electrophoresed
in
two lanes of a 3.5% polyacrylamide
slab gel under nondenaturing
conditions.
One lane was stained
with Coomassie
blue (A) and the
other was sliced into 2-mm segments.
After extraction
of each slice,
an aliquot was assayed for DNA polymerase
activity
(B) as described
under “Methods.”
The marker
proteins
shown were run in adjacent
lanes. Gal, P-galactosidase;
CAT, catalase;
AP, alkaline
phosphatase.
FIG.
1. Polyacrylamide
suitable for use in a Beckman SW 41 rotor. Centrifugation
was at 40,000 x-pm at 4°C for 33 h, after which l&drop fractions
were collected from each tube. The four peak activity fractions
in each tube were pooled (Fraction VI).
Step 7. Blue Dextran-Sepharose Chromatography-Fraction VI (3 ml) was dialyzed overnight against 1 liter of 10 mu
potassium phosphate
(pH 7.6), 1 mM 2-mercaptoethanol,
1
mM PMSF, and 20% glycerol using a collodion membrane bag
with a molecular weight cut-off of 75,000. The solution was
centrifuged
at 12,000 x g for 5 min to remove a precipitate
formed during dialysis and the supernatant
fluid was loaded
onto a column (0.8 X 2.4 cm) of blue dextran-Sepharose
equilibrated
with the dialysis buffer. The column was washed
first with 4 ml of 10 mM, and then with 5 ml of 30 mu
potassium phosphate buffer; enzyme activity was eluted with
the 50 mM potassium
phosphate
buffer (all containing
2mercaptoethanol,
PMSF, and glycerol as above). The peak
activity fractions were pooled and stored at -8O’C (Fraction
VII). This fraction was stable for at least 3 months.
Physical Properties
Homogeneity--When the purified enzyme
(Fraction
VII)
was analyzed by electrophoresis
in a 3.5% nondenaturing
polyacrylamide
slab gel, a single protein band was observed
after staining with Coomassie blue (Fig. L4). An adjacent lane
containing
an identical sample was cut into 2-mm slices and
the DNA polymerase
activity that was eluted had an RM
equal to that of the protein band (Fig. 1, A and B). This
equivalence
was maintained
after electrophoresis
in 5 and
7.5% polyacrylamide
gels (results not shown). A small amount
(about 5%) of activity and protein remained at the origin of
Drosophila
DNA Polymerase
FIG. 2 (left). SDS-polyacrylamide
gel electrophoresis
of the
protein
band
obtained
following
polyacrylamide
gel electrophoresis
under
nondenaturing
condition.
A segment
of gel containing
the DNA polymerase
(Fig. 1) was denatured
and transferred
to a 7% SDS-polyacrylamide
slab gel for electrophoresis
as described
under “Methods.”
The marker
proteins
shown were run in an adjacent
lane. The four major
polypeptides
are numbered
Z to IV. MYO,
myosin; PHOS,
glycogen
phosphorylase
a; GAL, /3-galactosidase;
AP,
alkaline
phosphatase.
FIG. 3 (right). SDS-polyacrylamide
gel electrophoresis
of D.
melarwgaster
DNA
polymerase.
Fraction
VII (15 pg) was denatured and electrophoresed
on a 7% SDS-polyacrylamide
slab gel. The
four major polypeptides
and marker
proteins
are identified
as in the
legend to Fig. 2.
Molecular
Weight-The
molecular
weight of the DNA
polymerase activity was calculated by combining the sedimentation coefficient
with the Stokes radius as determined
by
Sepharose 6B gel filtration (28). The sedimentation
coefficient
of 8.7 S (Fig. 4.4) and Stokes radius of 78 A (Fig. 5), both
determined
in the presence of 0.2 M (NH&S04,
yielded a
molecular weight of 280,000. The frictional ratio was 1.81 (28),
a value which suggests that the enzyme is either asymmetric
in shape or highly solvated. The molecular weights of the four
major polypeptides
observed after SDS-polyacrylamide
gel
electrophoresis
of Fraction VII directly, or after transfer of
the protein band from the gel run under nondenaturing
conditions were 43,000, 46,000, 58,000, and 148,000 (Fig. 6).
The molecular
weight of the DNA polymerase
was also
calculated from its RM on polyacrylamide
gels with varying
degrees of cross-linking,
compared with a group of standard
proteins (29, 30). Plots of log RM uersus per cent acrylamide
yield a series of straight lines, the slopes of which are directly
proportional
to their molecular weight. The slopes were determined
for four marker proteins together with the DNA
0.63---L---r
B
“A
s-
LDH
CAT
STOKES’
o4?
ZN
8 ‘0L 3
6x
0 a
I
it
1
1
2‘-
5
0
4
8
12
16
20
24 0
FRACTION
FIG. 4. Glycerol
gradient
gaster
DNA
polymerase.
to 30% gradients
with (A) or
under “Methods.”
The two
same tubes. CAT, cat&se;
8,
FIG. 5. Determination
of the Stokes
radius
of the D. melanogaster
DNA
polymerase.
The Sepharose
6B column
was calibrated and run under the conditions
described
under “Methods.”
The
resulting
data were used to generate
the straight
line by least squares
analysis.
APO,
apoferritin;
POL, E. coli DNA polymerase
I; THY,
thyroglobulin;
LDH, L-lactic
dehydrogenase;
CAT, catalase;
GAL, pgalactosidase.
,I
E
a
0
0
RADIUS,
4
6
12
16
20
24
NUMBER
sedimentation
of the D. melanoFraction
VII was sedimented
through
10
without
(B) 0.2 M (NH&S04
as described
marker
proteins
shown were run in the
LDH,
L-lactic
dehydrogenase.
the gel, suggesting
that aggregation
or irreversible
binding to
the gel surface had occurred. When a segment from a third
identical sample lane was denatured, transferred
to and electrophoresed
on a 7% polyacrylamide
gel in the presence of
SDS, the single native protein band was resolved into four
major
polypeptides
after Coomassie blue staining (Fig. 2).
Electrophoresis
of the enzyme directly on a 7% SDS-polyacrylamide
gel yielded the same four polypeptides
after staining (Fig. 3). Densitometric
scanning of the stained gel revealed
that they accounted for about 90% of the protein applied.
Sedimentation Coefficient-Glycerol
gradient sedimentation of Fraction
VII, in the presence of 0.2 M (NH&Sod,
yielded a single peak of activity with a sedimentation
coefflcient of 8.7 S (Fig. 4A). Values of 8.6 to 9.1 S were obtained in
six determinations
on various preparations
at the stage of
Fraction VI or VII. The sedimentation
coefficient remained
unchanged when determined
in 0.5 M (NH&S04.
However, in
the absence of this salt, the sedimentation
coefficient
increased to 10.2 S (Fig. 4B), with a range of 10.1 to 11.0 S in six
determinations.
200.
150
P)
‘0
100.
%
n
s
Z
4
50.
I;
0.2
0.4
0.6
0.8
1.0
%l
FIG. 6. Determination
of the molecular
weights
of the four
major
polypeptides
of D. melanogaster
DNA
polymerase
obtained
by SDS-polyacrylamide
slab gel electrophoresis.
The
data were obtained
from the gel shown
in Fig. 2. The arrows numbered Z to IV identify
the positions
of the four major polypeptides.
MYO,
myosin;
GAL, /3-galactosidase;
PHOS,
glycogen
phosphorylase
a; AP, alkaline
phosphatase.
Drosophila
9890
345
0
1
DNA Polymerase
Ploo I r
z
a
c5
z
250
Y
s \---8
%
1
0.25
2.5
APHIDICOLIN
6
2
M,,
daltons
X 1O-5
7. Determination
of the molecular
weight
of the D. melDNA polymerase
by polyacrylamide
gel electrophoresis. The DNA
polymerase
was electrophoresed
on 3.5 (see Fig. I),
5, and 7.5% polyacrylamide
gels as described
under “Methods.”
The
Rv values of the DNA
polymerase
and marker
proteins
were determined and the calibration
curve constructed
as described
in the text.
The data were used to generate
the straight
line by least squares
analysis.
AP, alkaline phosphatase;
CAT, catalase; GAL, P-galactosidase; POL, E. coli DNA polymerase
I.
FIG.
arwgaster
1I
5.
(uahl)
FIG. 9. Inhibition
of DNA
polymerase
activity
by Aphidicolin.
Fraction
VII was assayed
in the presence
of the indicated
concentrations
of Aphidicolin.
Control
incubations
with amounts
of
dimethyl
sulfoxide
equivalent
to those added with the Aphidicolin
(0.015 to 0.3%) showed
no significant
inhibition
of DNA polymerase
activity.
The results
are expressed
as per cent of activity
in the
absence
of inhibitor.
II
TABLE
Template-primer
specificity of DNA polymerase from D.
melanogaster
Reaction
mixtures
contained
the standard
components
as described
under “Methods”
and 2.5 units of enzyme.
When homopolymer
pairs
were used as template-primers,
the mixtures
contained
100 pM [“HIdTTP
and dATP,
with 100 pM (dA),w,
or 100 pM poly(rA),
both with
10 PM or 100 pM (dT),,;
poly[d(A-T)]
was present
at 100 pM. (Concentrations
of synthetic
polymers
are per mol of nucleotide.)
dNMP
Template-primer
incorporated
pmol/30
Activated
calf thymus
Denatured,
activated
Poly[d(A-T)]
(dAhw
(dT),sn
PolyW)
. MT)12
ColEl
DNA
4
8
[MgC12]
12
,mM;
16
[MnC12].mY
20
X102
8. Dependence
of DNA polymerase
activity
on divalent
metal
cation
concentration.
Fraction
VII was assayed
in the presence of the indicated
concentrations
of MgClz
(M)
and MnCh
t-j.
polymerase
protein
band
after
electrophoresis
under nondenaturing conditions
in 3.5, 5.0, and 7.5% polyacrylamide
gels.
A calibration
graph of slope uersus molecular
weight was
constructed,
from which a molecular
weight of 550,000 was
determined
for the DNA polymerase
(Fig. 7).
of Polymerase
Activity
Reaction
Requirements-Maximum
activity required
all
four deoxyribonucleoside
triphosphates,
DNA, Mg”+, and
(NH&SO+
The optimum pH in 50 mM Tris-HCl
buffers was
8.5, the reaction rates at pH 7.0 and 9.0 being 63 and 93%,
respectively,
of the rate at the optimum.
Addition
of
(NH&SO+
at 40 mM or KC1 at 60 mM stimulated
polymerase
activity
approximately
3-fold;
60 mM NaCl produced
a 2-fold
stimulation.
A divalent
metal
cation
was
absolutely
for activity; the optima for MgC12 and MnC12 being
mM, respectively,
the former stimulating
7-fold
ciently than the latter (Fig. 8). The apparent &
was 17.5 PM. Although
2-mercaptoethanol
was not
for
activity
and
at the
standard
assay
concentration
DNA
min
470
180
15
<2
t2
t2
” Omission
of (NH&S04
and reduction
of the MgClz concentration
from
10 mM to 5 mM resulted
in approximately
one-half
the rate
observed
with activated
calf thymus
DNA.
FIG.
Characterization
DNA
calf thymus
required
12 and 0.1
more effifor dTTP
necessary
range of 0.2 to 2 pg/ml (31), was also a potent inhibitor
of the
D. melanogaster
enzyme (Fig. 9).
Template-Primer
Requirements-The
enzyme had an absolute requirement
for a template-primer.
Of the template
primers tested, activated
calf thymus DNA was the most
active. After heat denaturation
of the DNA, activity dropped
approximately
3-fold; supercoiled
ColEl
DNA was inert.
Poly[d(A-T)]
showed only slight activity and (dA)-ioo. (dT)12
was inert as a template-primer.
However, when (NH4)2S04
was omitted
from the standard
reaction
mixture
and the
MgC12 concentration
was reduced from 10 mM to 5 mM, (dA) 71X,.
(dT)12 gave approximately
one-half the activity observed with
activated DNA (Table II).
Absence of Other Activities-The
purified enzyme showed
no detectable
exonuclease .activity with native or heat-denatured P22 [“H]DNA
as substrates in the presence or absence
of the four unlabeled deoxyribonucleoside
triphosphates
(t5
pmol of “H-labeled
nucleotide
produced
by an amount of
enzyme that would catalyze the incorporation
of 1500 pmol of
[“H]dTTP
into DNA). Similarly,
no endonuclease
activity
could be detected when assayed by the conversion of supercoiled ColEl DNA to its relaxed circular or linear forms by
agarose gel electrophoresis.
There was no measurable
DNAdependent or independent
ATPase or RNA polymerase activities.
inhibited
Fraction VII slightly (25%) the activity was strongly inhibited
by NEM (>90% inhibition
at 0.1 mM NEM). Aphidicolin,
a
specific inhibitor
of mammalian
(Y-DNA polymerases
in the
DISCUSSION
We have purified
of D. melanogaster
the DNA polymerase
from early embryos
approximately
1500-fold to near-homoge-
Drosophila
DNA
neity. In previous work, proteolytic
cleavage of the enzyme
during purification,
constituted
a serious problem, resulting in
a decrease in sedimentation
coefficient without loss of catalytic activity (21, 22). In their extensive studies, Brake1 and
Blumenthal
identified
three forms of the enzyme sedimenting
at 5.5 to 7.3, 7.3 to 8.3, and 8.3 to 9.0 S (22). Each of these
forms was characterized
using fractions selected after incomplete resolution
by DEAE-cellulose
chromatogaphy
or glycerol gradient sedimentation.
During purification,
conversion
of the faster to slower sedimenting
forms was also observed,
a process that could be accelerated by treatment with trypsin,
and which was partially,
but not completely,
inhibited
by the
protease inhibitor
PMSF at 0.1 mM (21). Their work demonstrated conclusively
that the form of the enzyme purified by
Karkas et al. was an active proteolytic
degradation
product
with a sedimentation
coefficient
of 5.5 S (19). We have confirmed these findings and observed that even in the presence
of 0.1 mM PMSF, extensive purification
invariably
resulted in
the formation
of multiple peaks of activity with sedimentation
coefficients
of about 9.0, 7.4, and 5.5 S. By increasing
the
PMSF concentration
to 1 mM, including
10 mM sodium bisulfite in all buffers up to the preparative
glycerol gradient step
and working
rapidly,
we have been able to maintain
the
activity in an 8.6 to 9.1 S form that yields a single protein
band upon polyacrylamide
gel electrophoresis
under nondenaturing conditions. The mechanism
of inhibition
of protease
by sodium bisulfite is not known, but it has been found to be
effective in preventing
proteolysis
of histones during their
isolation (32).
The observation
that the relative mobility
of the single
stainable protein band observed following polyacrylamide
gel
electrophoresis
of the native enzyme is identical with that of
the polymerase
activity from the sliced gel (Fig. 1) at three
different
acrylamide
concentrations
indicates strongly that
this band contains the DNA polymerase. SDS-polyacrylamide
gel electrophoresis
of this band by transfer from the polyacrylamide gel revealed four major polypeptides
(Fig. 2), which
were also the major bands observed when the purified enzyme
was electrophoresed
directly in an SDS-polyacrylamide
gel,
constituting
about 90% of the protein applied to the gel (Fig.
3). The molecular
weight of the DNA polymerase
activity
established
by sedimentation
and gel filtration
experiments
was 280,000. The coincidence
of this figure with the sum of
the molecular weights of the four polypeptides
(295,000) might
suggest that the intact enzyme is a complex composed of a
single copy of each polypeptide.
Further work is required to
determine
whether this is indeed the case or whether the four
polypeptides
might result, for example, from proteolytic
cleavage of a single 280,000 molecular weight polypeptide
(33).
Analysis
of the homogeneous
DNA polymerase
(Y from
cultured human cells by Korn and his colleagues has shown
it to be substantially
smaller that the D. melanogaster
enzyme
with a molecular weight of 140,000 and subunits of 76,000 and
66,000 daltons (8).
Inasmuch as the apparent stoichiometry
of the four major
polypeptides
does not change with ionic strength the small
increase in sedimentation
coefficient
observed at low ionic
strength (Fig. 4, A and B) probably results from a conformational change in the enzyme rather than from the loss of an
associated polypeptide
at high ionic strength. The molecular
weight of 550,000 determined
on the basis of electrophoretic
mobility of the native enzyme on polyacrylamide
gel indicates
yet another order of complexity
in the structure of the enzyme.
Again, it may not be fortuitous that this value is approximately
twice that obtained
on the basis of sedimentation
and gel
filtration.
9891
Polymerase
A comparison
of the properties determined
so far for the D.
DNA polymerase
with those of the a-, p-, and
y(mito)-polymerases
of mammalian
cells, suggests that it most
closely resembles the a-polymerase
(reviewed in Refs. 4 to 6).
Thus, the enzyme is inhibited
by Aphidicolin,
by low concentrations
of NEM, and high ionic strengths, has a high molecular weight, an asymmetric
conformation,
a similar apparent
K, for dTTP, no detectable nuclease activity, a high turnover
number (38 molecules of dNTP/280,000-dalton
molecule/s),
and is comprised of dissimilar subunits. We have searched for
a low molecular
weight, NEM-insensitive
P-like enzyme in
both cytoplasm and nuclei of early Drosophila
embryos and
in cultured cells; thus far, without success, in confirmation
of
the report by Chang (34). It is possible, of course, that other
polymerases
are present at other developmental
stages than
examined here, as has been observed in Xenopus laevis (35).
melanogaster
Achnoculedgments-It
is a pleasure
to acknowledge
the enthusiastic participation
of Dr. G. Villani of this department
in some of these
experiments.
We are particularly
grateful
to Professor
David Hogness
for providing
us with free access to the large quantities
of Drosophila
melanogaster
embryos
required
for these studies.
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