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INTRODUCTION |
DNA polymerase (pol)1
is the central enzyme in eukaryotic DNA replication (1) and also
serves an important role in DNA repair (2). Isolation of the calf
thymus (3) and human (4) enzymes has shown that it consists of at least
two core subunits of 125 and 50 kDa. The hallmarks of this polymerase
are that it has an intrinsic 3' to 5' exonuclease activity,
distinguishing it from pol
and pol
. The 125-kDa subunit of
human pol
(p125) has been identified as the catalytic subunit (4).
Pol
is a member of a family of DNA polymerases which includes DNA
polymerase
, pol
, the herpesvirus DNA polymerases, and
bacteriophage T4 polymerase (5, 6). Examination of the regions of
conserved sequence has led to the identification of domains that are
potentially required for DNA interaction, deoxynucleotide interaction,
as well as the 3' to 5' exonuclease activity of pol
(7). In addition, there are several regions in the NH2 and COOH
termini which are conserved among human pol
, yeast pol
, and
yeast and human pol
(5, 7).
Studies of the replication of SV40 DNA in vitro have led to
the identification of a number of accessory proteins, which, together with pol
, are required for the formation of a replication complex at the replication fork. These include PCNA, which functions as a
sliding clamp and enhances the processivity of pol
, consistent with
its role as the leading strand polymerase (8). Although there have been
some mutagenesis studies of the yeast pol
(9), little has been done
with human or mammalian pol
, largely because of the lack of a
suitable expression system. To facilitate structure-function studies of
pol
, it is desirable to have an expression system for the
production of the recombinant protein. The expression of the human pol
catalytic subunit has been achieved in mammalian cells using a
vaccinia virus vector (10). In this study we report the expression of
p125 in Sf9 cells using a baculovirus vector as well as methods
for separating the recombinant protein from endogenous DNA polymerases
in baculovirus-infected Sf9 cells. Deletion mutants of p125 were
also characterized to investigate the domain structure of pol
. In
addition, we have obtained novel evidence that pol
p125 is
phosphorylated by the cyclin-dependent kinase (cdk)-cyclin
complexes and also can be coimmunoprecipitated with cdk2 when they are
coexpressed in Sf9 insect cells. The interaction of pol
with
the cyclins and cdks was also confirmed by coimmunoprecipitation and
Western blot experiments in Molt 4 cells. Preliminary experiments showed that phosphorylation has moderate or little effect on the activity of the catalytic subunit.
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EXPERIMENTAL PROCEDURES |
Materials--
Sf9 cells were purchased from Invitrogen
and were maintained at 27 °C in TNM-FH insect medium supplemented
with 10% fetal calf serum and 50 µg/ml gentamycin. Cells were
propagated both as adherent monolayers and as nonadherent suspension
cultures. These cells were used as the hosts for the propagation of
wild type Autographa californica multiple nuclear
polyhedrosis virus (AcMNPV) and recombinant baculoviruses. Cyclin and
cdk recombinant baculoviruses were gifts of Dr. Charles Sherr (St.
Jude's Hospital, Memphis, TN). BaculoGoldTM-linearized
baculovirus DNA was purchased from Pharmingen. The baculovirus transfer
vector P2bac was purchased from Invitrogen. Plasmid pALTER-1 was
purchased from Promega.
Construction and Screening of Recombinant Baculoviruses--
The
coding sequence of pol
which was used in these studies was derived
from the cDNA originally isolated by Yang et al. (7).
This coding sequence was inserted into the pALTER vector and corrected
by site-directed mutagenesis so that His-119, Asn-173, and Gly-776 were
mutated to Arg-119, Ser-173, and Arg-776 to conform to the genomic
sequence (10, 11). The plasmid pALTER-pol
containing the corrected
full-length pol
coding sequence (3.5 kilobases) was excised from
the pALTER plasmid by BamHI/HindIII digestion,
gel purified, and inserted into
BamHI/HindIII-digested baculovirus transfer
vector p2bac. The recombinant p2bac plasmids were cotransfected into
Sf9 cells with wild type baculovirus DNA according to Ausubel
et al. (12). Wild type BaculoGoldTM-linearized
AcMNPV DNA (1 µg), recombinant plasmid DNA (3 µg), cationic
liposome solution (25 µl), and 1 ml of Grace's insect medium
containing no supplements were mixed by vortexing for 10-15 s and
incubated at room temperature for 15 min. The transfection mixture was
then layered onto Sf9 cells growing on 60-mm plates. After 4 days at 27 °C, the medium was aspirated and analyzed for virus
production by plaque assay. The recombinant baculoviruses were
identified as occlusion-negative plaques with a dissecting microscope.
Because the BaculoGoldTM-linearized virus DNA contains a
lethal deletion and a lacZ gene, the small portion of
nonrecombinant virus plaques stained blue on 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside plates, whereas all recombinants
produced colorless plaques on these plates. After three rounds of
plaque purification, pure recombinant baculoviruses were obtained.
Occlusion-negative viral stocks were prepared from the final
supernatants, titered, and stored at 4 °C. Deletion mutants of pol
were constructed as described in Ref. 13.
Infection of Sf9 Cells with Recombinant Baculovirus and
Preparation of Cell Extracts--
Recombinant viral stocks (0.5 ml)
were added to a multiplicity of infection between 5 and 10 for the
infection of log phase Sf9 cells for 1 h. The inoculum was
then removed from the plates, and 8 ml of fresh complete TNM-FH insect
medium was added. The infected Sf9 cells were allowed to grow
for 2 days at 27 °C and were harvested 48 h postinfection.
Cells were harvested from 80 100-mm plates and collected by
centrifugation. The cell pellets were washed twice with ice-cold
phosphate-buffered saline, pH 7.4. Subsequent manipulations were
carried out at 4 °C. The cells from 80 plates (about 8 × 108 cells) were suspended in 5-cell pellet volumes (50 ml)
of lysis buffer (40 mM Tris-HCl, pH 7.8, 0.25 M
sucrose, 0.1 M NaCl, 0.1% Nonidet P-40, 0.1 mM
EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 10 mM
benzamidine-HCl). Cells were disrupted by passage through a French
press at 1,000 p.s.i. The lysate was centrifuged at 27,000 × g for 30 min. The supernatant was removed and saved as the
soluble extract, and the pellet was suspended in 20 ml of lysis buffer
plus 0.5 M NaCl and sonicated three times for 20 s
each at 50 watts on ice. The extract was again centrifuged at
27,000 × g for 30 min, and the supernatant was
designated as the high salt-solubilized fraction. Protein concentrations of the first and second extracts were 12 and 9 mg/ml,
respectively. The pellet was then dissolved in 1 ml of 8 M
urea. The two fractions (low and high salt extracts) were then combined
and dialyzed against TGEED buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.1 mM EGTA, I
mM dithiothreitol).
Phosphocellulose Chromatography--
The dialyzed lysates were
loaded onto a phosphocellulose column (5 × 7 cm) equilibrated in
TGEED buffer. The column was eluted with a linear gradient of 50-1
M NaCl in TGEED buffer in a total volume of 2 liters.
Fractions of 10 ml each were collected and assayed for DNA polymerase
activity. Western blots were also performed using 38B5, a monoclonal
antibody against the COOH-terminal region of pol
(2, 14).
HPLC--
The combined fractions from the phosphocellulose
column which contained recombinant pol
p125 were dialyzed against
TGEED buffer, pH 7.8, passed through an 0.45-µm syringe filter, and injected onto a Mono Q HR 5/5 column. The enzyme was eluted with a
linear gradient of 0-1 M NaCl for 20 min at 1 ml/min.
Single-stranded DNA-cellulose Chromatography--
Fractions from
the Mono Q column were dialyzed against HEPES buffer (20 mM
HEPES, 5 mM MgCl2, 10% glycerol, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, pH
7.5) and were then loaded onto a single-stranded DNA-cellulose column
(0.5 × 6 cm) equilibrated with HEPES buffer. The column was
washed with the same buffer, and a gradient of 50-500 mM
NaCl in a total volume of 40 ml was applied. Fractions of 1 ml were collected and analyzed by SDS-PAGE, Western blotting, and assays for
pol
activity.
Immunoaffinity Chromatography--
Monoclonal antibody 78F5 was
coupled onto AvidChrom hydrazide (Sigma) as described by Jiang et
al. (14). The column (1 × 10 cm) was equilibrated with TGEE
buffer (50 mM Tris-HCl, 0.1 mM EGTA, 0.5 mM EDTA, 10% glycerol, pH 7.8). The column was washed with
the same buffer containing 50 mM NaCl, and pol
was
eluted with 0.2 M NaCl in TGEE buffer. Fractions of 1 ml
were collected and analyzed as described above.
DNA Polymerase Assays--
Sparsely primed poly(dA)·oligo(dT)
was used as the template as described by Lee et al. (3). The
standard reaction for the poly(dA)·oligo(dT) assay contained 0.25 optical density units/ml poly(dA)·oligo(dT) (20:1), 200 µg/ml
bovine serum albumin, 5% glycerol, 10 mM
MgCl2, 25 mM HEPES, pH 6.0, 100 cpm/pmol
[3H]TTP, and 0.2-0.4 unit of pol
in the presence or
absence of 0.2 µg of PCNA in a total volume of 100 µl. Reaction
mixtures were incubated for 60 min at 37 °C and were terminated by
spotting onto DE81 papers that were then washed four times with 0.3 M ammonium formate, pH 7.8, once with 95% ethanol, and
counted as described previously (4).
Assay for 3' to 5' Exonuclease Activity--
The assay was
performed by measuring the release of [3H]dTMP from
[3H]dT50 as described previously (3). The
assay contained 2 µM [3H]dT50
(200-300 cpm/pmol), 25 mM HEPES buffer, pH 7.4, 5 µg of bovine serum albumin, 5 mM MgCl2, and 0.2-0.4
unit of pol
in a total volume of 60 µl. Reaction mixtures were
incubated for 30 min at 37 °C and were terminated by spotting 20 µl onto DE81 filter papers. Filters were washed four times with 0.3 M ammonium formate, pH 7.8, and once with 95% ethanol and
counted as described previously (3).
Western Blot Analysis--
The recombinant proteins expressed in
Sf9 cells infected with recombinant baculoviruses were analyzed
by Western blotting with pol
monoclonal antibody 38B5 (2, 14).
Extracts of Sf9 cells prepared as described above were subjected
to SDS-PAGE in 5-15% gradient gels that were then transferred to
nitrocellulose membranes. Prestained protein standards (Sigma) were
used as molecular weight markers and also to provide visual
confirmation of efficient transfer. The nitrocellulose blots were
blocked with 5% nonfat dry milk in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, for 1 h at room temperature. The blots
were then incubated with monoclonal antibody against pol
for
12 h at 25 °C. After three 10-min washes in 50 mM
Tris-HCl, pH 7.5, 0.15 M NaCl, the blots were incubated with biotinylated sheep anti-mouse immunoglobulin for 1 h at
27 °C followed by incubation with streptavidin-biotinylated
horseradish peroxidase complex. Color development was performed by
incubation with 4-chloro-1-naphthol and hydrogen peroxide and
terminated with sodium azide.
Coinfection of Sf9 Cells with Pol
, Cyclins, and Cdks
and 32Pi Labeling--
Sf9 cells
(107) were grown to exponential stage. Pol
, cyclin, and
cdk recombinant baculoviruses (0.5 ml) were added as indicated. The
cells were infected at room temperature for 1 h. The recombinant baculoviruses were removed, replaced with growth medium, and the cells
were grown for an additional 2 days at 27 °C before labeling with
32Pi. Infected Sf9 cells were
transferred into a 15-ml tube for 32Pi
labeling. After centrifugation and removal of growth medium, the cells
were resuspended in 2 ml of fresh phosphate-free medium containing 200 µCi of 32Pi (specific activity 3,000 Ci/mmol)
and incubated at 37 °C for 2 h. The cells were centrifuged at
3,000 × g for 5 min. The supernatant was removed, and
the cells were washed twice with phosphate-buffered saline. The cells
were sonicated for 30 s in 40 mM Tris-HCl, pH 7.8, 0.25 M sucrose, 0.5 M NaCl, 0.1% Nonidet P-40,
0.1 mM EGTA, 1 mM EDTA, 1 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and
10 mM benzamidine-HCl. The crude cell extracts were
transferred to microtubes and centrifuged at 15,000 × g for 30 min. About 20 mg of total protein was used for
immunoprecipitation in the presence of 20 µg of 78F5 pol
monoclonal antibody (2, 14) and 40 µl of protein A-Sepharose slurry
at 4 °C overnight. The Sepharose beads were washed twice with
sonication buffer and boiled for 5 min in 50 µl of SDS sample buffer.
The proteins released from the beads were then subjected to SDS-PAGE
and autoradiography.
Immunoprecipitation and immunoblotting of Molt 4 Cells with Pol
and Members of the Cyclin and Cdk--
4 × 107
exponentially growing Molt 4 cells were prepared and lysed with 300 µl of Nonidet P-40 buffer (50 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride, 150 mM NaCl,
and 1% Nonidet P-40). The lysates were precleaned with protein A beads
(50 µl of a 10% suspension) by rotating at 4 °C for 30 min. The
supernantants were removed by centrifugation and transferred to a fresh
tube. The antibody used for immunoprecipitation was then added in the presence of 50 µl of fresh protein A beads and incubated at 4 °C for 1 h. Anti-pol
monoclonal antibody (20 µg), PCNA
monoclonal antibody (20 µg), anti-cyclin E and A antibodies (100 µl
of hybridoma cell supernatant), and anti-cdk2 polyclonal antibody (2 µl) were used for the experiments. The extracts were then centrifuged
and washed with Nonidet P-40 buffer three times. After SDS-PAGE, the separated proteins were transferred to a nitrocellulose membrane and
Western blotted with antibodies to cdk2, cdk5, or pol
.
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RESULTS |
Expression of Pol
p125--
The expression of human pol
in
Sf9 cells infected with recombinant baculovirus was analyzed by
immunoblotting with a pol
monoclonal antibody (38B5; see
"Experimental Procedures"). The infected cells were disrupted by
passage through a French press in 0.1 M KCl and centrifuged
to provide the first extract. The pellet was reextracted by sonication
in 0.5 M KCl (second extract). The pellet was then
dissolved in l ml of 8 M urea. Immunoreactive protein was
found to be present in the two salt extracts but not in the urea
extract when equal amounts of protein were loaded from each fraction
(Fig. 1). These experiments showed that
pol
was expressed as a soluble protein that can be extracted
completely by 0.5 M KCl. Immunoblots of the corresponding
extracts of Sf9 cells infected with wild type AcMNPV using the
same antibody showed the absence of immunoreactive polypeptide (not
shown). The time course of pol
expression was examined by
immunoblot analysis of cells taken at intervals after infection with
recombinant virus (Fig. 2). For these
experiments the 0.1 and 0.5 M KCl extracts were combined.
Very little p125 immunoreactivity was observed at 12 h
postinfection, and the peak of expression was found to be between 36 and 48 h (Fig. 2).

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Fig. 1.
Immunoblot of extracts of Sf9 cells
infected with recombinant baculovirus. Extracts of Sf9
cells infected with recombinant baculoviruses were prepared as
described under "Experimental Procedures." The cells were disrupted
and extracted in 50 ml of lysis buffer containing 0.1 M
NaCl and then with 20 ml of lysis buffer containing 0.5 M
NaCl. The pellet was then dissolved in 1 ml of 8 M urea.
These three extracts (60 or 30 µg of protein/lane) were
then analyzed by SDS-PAGE (5-15% acrylamide). Western blotting was
performed using monoclonal antibody 38B5 against human pol .
Lanes 1, 5, and 9 are high molecular
weight standards as marked; lanes 2-4 are 60 µg of the
0.1 M NaCl, 0.5 M NaCl, and 8 M
urea extracts, respectively. Lanes 6-8, same as lanes
2-4 but with 30 µg of protein/lane; lane 10, low
molecular weight protein standards as marked.
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Fig. 2.
Time course of pol expression in
Sf9 cells. Sf9 cells infected with recombinant virus
were harvested at 12, 24, 36, 48, and 60 h after infection. The
cells were lysed and extracted as described under "Experimental
Procedures." The 0.1 M and 0.5 M NaCl
extracts were combined and analyzed for the expression of pol by
SDS-PAGE (20 µl/lane) followed by immunoblotting.
Lanes are marked according to time of harvest.
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The recombinant pol
was immunoblotted using a series of
peptide-specific antibodies (Fig. 3) as
described by Hao et al. (5). The different peptide-specific
antibodies (N1, N2, N3, N4, N5, C1, and C2) recognized the recombinant
p125 expressed in the baculovirus system. This experiment provided
additional confirmation of the identity of the overexpressed protein.
Note that the immunoblots (Fig. 3) for p125 appear as a doublet. As we
will show, p125 could be purified to a single polypeptide of 125 kDa,
although it was often observed as a doublet. A similar behavior was
encountered in the isolation of the calf thymus enzyme. At present the
most likely explanations are that this may reflect posttranslational
modification of the enzyme by phosphorylation or partial
proteolysis.

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Fig. 3.
Immunoblot of crude recombinant pol extract using peptide-specific antibodies. Sf9 cells were
infected with recombinant baculovirus, and the cell extracts were
immunoblotted using polyclonal antibodies against specific peptides
derived from the NH2- and COOH-terminal regions of the pol
sequence (13). These were as follows: N1 (84-101), N2 (129-149),
N3 (244-262), N4 (276-295), N5 (312-331), C1 (1047-1068), and C2
(1069-1090). The figure shows a composite of individual blots, each of
which shows two lanes, the left lane being the
prestained protein standards and the right lane, the
Sf9 cell extracts (20 µl, 50 µg of protein).
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Purification of Recombinant Pol
--
Cells from 80 100-mm
plates of Sf9 cells infected with recombinant baculovirus were
harvested as described under "Experimental Procedures." A potential
complication for the isolation of the recombinant human pol
from
Sf9 is the presence of endogenous DNA polymerases (15),
which could compromise studies of the enzymatic properties of human
recombinant pol
. We have circumvented this by passing the crude
extract through a phosphocellulose column ("Experimental
Procedures"). When the crude extract was chromatographed on a
phosphocellulose column, two peaks of activity were detected using
poly(dA)·oligo(dT) as a template. One peak eluted at about 0.4 M NaCl and the second at 0.6-0.7 M NaCl (Fig.
4, center panel). To determine
which of the peaks was the overexpressed pol
, immunoblots were
performed using monoclonal antibody 38B5. Only the first peak of
activity (fractions 80-120) was immunoblotted; the second peak
(fractions 120-160) did not contain immunoreactive protein (Fig. 4,
top panel). The second peak also corresponded to the peak of
polymerase activity eluted at about 0.7 M KCl when extracts of Sf9 cells infected with wild type AcMNPV baculovirus were
chromatographed (Fig. 4, bottom panel). DNA polymerase
isolated from the calf thymus was reported to elute between 235 and 320 mM KCl (3). The second peak was presumed to be endogenous
DNA polymerase in baculovirus-infected Sf9 cells, which has been
reported to elute from phosphocellulose at high salt concentrations
(15).

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Fig. 4.
Phosphocellulose chromatography of Sf9
cell extracts infected with recombinant baculovirus. A cell
extract from Sf9 cells infected with recombinant baculovirus was
chromatographed on phosphocellulose as described under "Experimental
Procedures." The fractions were assayed for DNA polymerase activity
using poly(dA)·oligo(dT) as template (center panel). The
fractions containing the two peaks of activity (80-170) were
immunoblotted using an antibody against pol (38B5) as shown in the
top panel. BC refers to the extract before
chromatography. A cell extract from Sf9 cells infected with the
control baculovirus was also chromatographed on phosphocellulose, and
the fractions were assayed for DNA polymerase activity as shown in the
bottom panel. Immunoblots of the peak fractions failed to
show any immunoreactive protein (not shown).
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The peak fractions that immunoblotted with pol
antibody were
pooled, dialyzed, and chromatographed on a Mono Q HPLC column. The
column was eluted with a salt gradient as described under "Experimental Procedures" (Fig. 5).
Assay of the fractions revealed a peak of DNA polymerase activity which
eluted at about 350 mM NaCl. Calf thymus DNA pol
elutes
at 260 mM KCl under the same conditions (3, 4). The
preparation contained a 125-kDa polypeptide that was immunoblotted by
antibody 38B5 (Fig. 5, inset). The recombinant p125 was
purified to near homogeneity by passage through a single-stranded DNA-cellulose column ("Experimental Procedures"). DNA polymerase activity and exonuclease activities were assayed and found to coelute
(Fig. 6). The enzyme was found to be
nearly homogeneous as shown by Coomassie Blue staining of SDS-PAGE of
the peak fraction (Fig. 6, inset).

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Fig. 5.
Mono Q chromatography of recombinant pol
. The peak fractions from the phosphocellulose chromatography
step were combined and subjected to HPLC on a Mono Q 5/5 column (see
"Experimental Procedures"). The enzyme was eluted with a linear
gradient of 0-1 M NaCl in 20 min at 1 ml/min. The
fractions were assayed for DNA polymerase activity (closed
circles). The elution of protein is shown by the absorbance at 280 nm (squares). The inset shows the SDS-PAGE of
fractions 12 and 14, which were stained for protein (left
panel) and immunoblotted using a monoclonal antibody against pol
(right panel).
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Fig. 6.
Single-stranded DNA-cellulose
chromatography. The fractions from the peak of the Mono Q column
which immunoblotted with the pol antibody were combined, dialyzed
against buffer, and loaded onto a single-stranded DNA-cellulose column
as described under "Experimental Procedures." Fractions of 1 ml
were collected and assayed for DNA polymerase activity
(circles) and for exonuclease activity (inverted
triangles). The inset shows the SDS-PAGE of fraction
16, which was stained for protein.
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Immunoaffinity Purification of Recombinant Pol
--
We have
shown previously that calf thymus pol
can be isolated by
immunoaffinity chromatography using monoclonal antibody 78F5 coupled to
AvidChrom hydrazide (14). Crude Sf9 cell extracts were
chromatographed on a pol
immunoaffinity column ("Experimental Procedures"). The column was washed with buffer containing 50 mM NaCl, and pol
was eluted by 0.2 M NaCl
as shown by analysis for DNA polymerase and exonuclease activities
(Fig. 7A) and Western blotting
(Fig. 7A, inset). The enzyme obtained was still
impure (Fig. 7A, inset) as determined by SDS-PAGE
gels stained for protein. Sf9 cells infected with wild type
virus were also passed through this immunoaffinity column, and no
detectable DNA polymerase activity was recovered (Fig. 7A).
This demonstrated that DNA polymerase activities from the Sf9
cells infected with wild type virus did not bind to the column. Note
that the overexpressed p125 catalytic subunit could be eluted from the
immunoaffinity column by simply using 0.2 M KCl, whereas
calf thymus DNA pol
holoenzyme is eluted at 0.4 M NaCl
and 30% ethylene glycol (14). The peak fractions were combined and
rechromatographed on the same column. This allowed for the isolation of
the recombinant p125 in a nearly homogeneous form (Fig. 7B).
Starting with 800 mg of total protein in the crude extract, about 0.11 mg of nearly homogeneous protein was recovered, presenting a
purification of 153-fold and a final specific activity of 1,200 units/mg of protein using poly(dA)·oligo(dT) as a template (Table
I).

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Fig. 7.
Immunoaffinity chromatography of recombinant
pol . Panel A, an extract from cells infected with
recombinant baculovirus was chromatographed on a pol immunoaffinity
column as described under "Experimental Procedures." The column was
eluted with 50 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.1 mM EGTA, and 200 mM
NaCl. Fractions of 1 ml were collected. The fractions were assayed for
DNA polymerase activity (solid circles) and for 3' to 5'
exonuclease activity (solid squares). The inset
shows the SDS-PAGE of fractions 6 and 8 stained for protein with
Coomassie Blue. The same fractions were immunoblotted using an antibody
against pol (lanes 6' and 8'). An extract
from cells infected with control baculovirus was also chromatographed
on the same column and assayed for DNA polymerase activity (solid
triangles). Panel B, the active fractions from the
first immunoaffinity chromatography (panel A) were pooled,
dialyzed against the equilibration buffer, and rechromatographed on the
same column. DNA polymerase and exonuclease activities were assayed as
in panel A. The inset shows the SDS-PAGE of the
peak fractions stained for protein and also immunoblotted using an
antibody against pol .
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Characterization of Recombinant p125--
The enzymatic properties
of the recombinant pol
catalytic subunit were compared with those
of native calf thymus pol
, which had been isolated by
immunoaffinity chromatography (14), and with the endogenous DNA
polymerase activity from Sf9 cells infected with wild type
AcMNPV (Fig. 8). The latter was the
partially purified preparation obtained after phosphocellulose
chromatography (see Fig. 4, bottom panel). The activities of
the recombinant pol
catalytic subunit were similar to those of
native pol
and the Sf9 polymerases in that they were
inhibited by aphidicolin (Fig. 8A) and resistant to
2-(p-n-butylanilino)-9-(2-deoxy-
-D-ribofuranosyl)adenine 5'-triphosphate (not shown). A well known characteristic of calf thymus
pol
is its sensitivity to inhibition by
N-ethylmaleimide; recombinant pol
was inhibited in a
manner similar to calf thymus pol
, whereas the Sf9
polymerase was significantly more resistant to
N-ethylmaleimide (Fig. 8B). The inhibition by low
levels of salt is another characteristic of calf thymus pol
(Fig.
8C). Recombinant p125 differed from the calf thymus enzyme
in that it was less sensitive to inhibition. The Sf9 DNA
polymerase activity was not inhibited but slightly stimulated at 100 mM KCl and was only inhibited at much higher salt
concentrations (Fig. 8C). The heat inactivation of the three
polymerases was also examined. The enzyme was heated to 45 °C and
assayed for polymerase activity at the indicated times. DNA polymerase
from calf thymus and the p125 subunit displayed a similar behavior
when heat-treated and were much less sensitive to heat than the
Sf9 polymerase (Fig. 8D).

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Fig. 8.
Characterization of recombinant pol :
comparison with native calf thymus pol and endogenous DNA
polymerases in baculovirus-infected Sf9 cells. Effects of
different compounds and conditions were assayed using
poly(dA)·oligo(dT) as a template. Assay conditions were as described
under "Experimental Procedures" for the DNA polymerase activities
of recombinant pol (closed circles), native calf thymus
pol (closed squares), and endogenous DNA polymerase from
wild type baculovirus overexpressed in Sf9 cells (open
triangles). PCNA was added in the assays for calf thymus pol .
The endogenous DNA polymerase from wild type baculovirus overexpressed
in Sf9 cells was the material obtained after phosphocellulose
chromatography as in Fig. 4, bottom panel. Panel
A, effect of aphidicolin; panel B, effect of
N-ethylmaleimide; panel C, effect of KCl;
panel D, effect of heat treatment at 45 °C for varying
amounts of time; panels E and F, effects of
Mn2+ and Mg2+, respectively, on the DNA
polymerase activity of recombinant pol .
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Recombinant pol
was stimulated by Mn2+ in a manner
similar to that already known for calf thymus pol
. Optimal
activation was observed between 0.3 and 0.5 mM
Mn2+, whereas optimal activity of the Sf9 polymerase
was obtained at about 3 mM Mn2+ (Fig.
8E). Maximal activation of both calf thymus and recombinant pol
by Mg2+ was reached at about 5 mM,
whereas the Sf9 polymerase activity was stimulated maximally at
20 mM Mg2+ (Fig. 8F). These
experiments showed that the properties of the recombinant p125 subunit
were quite consistent with those of the calf thymus native enzyme.
Deletion Mutagenesis of p125--
Extensive compilation and
alignment of DNA polymerase sequences from a broad phylogenetic
spectrum, i.e. from both prokaryotes and eukaryotes, have
shown that these fall into two major protein families (16, 17). DNA pol
belongs to the
-like or B family of DNA polymerases (16). A
distinguishing feature of this family is the presence of a conserved
core region containing six distinct conserved regions, I-VI, which are
thought to contain the catalytic domain for polymerase activity. Unlike
pol
, the NH2-terminal regions of pol
possess
several regions (N1-N5) that are conserved in the Epstein-Barr virus
and herpesvirus DNA polymerases (5).
Deletion mutants of the full-length human pol
(1,107 residues) were
constructed. These were p97, in which the N1 and N2 regions of the
NH2 terminus (2-249) were deleted; p109, in which N3, N4,
and part of the N5 region including the ExoI domain
(186-321) were deleted; p82, in which regions IV, A, B, II, VI, and
III (336-715) were deleted; and p94, in which regions C, V, CT-1, CT-2, CT-3, and ZnF1 (778-1,047) were deleted (7). These were purified
to near homogeneity by phosphocellulose, Mono Q, and single-stranded
cellulose chromatography as described above. SDS-PAGE of the mutants
(Fig. 9) showed that these had the
expected molecular weights. Assays for enzyme activity showed that only
p109 (
186-321) and p97 (
2-249) retained DNA polymerase
activity. The p82 and p94 mutants had negligible activities (Table
II). This is expected as most of the core
region involved in deoxynucleotide interaction was deleted in p82,
whereas most of the COOH-terminal domain responsible for DNA
interaction was deleted in p94 (Fig. 9).

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Fig. 9.
Expression of deletion mutants of pol p125. Deletion mutants were constructed as described in Ref. 13.
These mutants were purified to homogeneity by phosphocellulose, Mono Q,
and single-stranded DNA-cellulose chromatography. The protein staining
of the purified mutants after SDS-PAGE are shown. The map of the
deletions is shown on the right.
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Table II
Relative specific activities of recombinant p125 and its deletion
mutants
Enzymes were purified to near homogeneity as described under
"Experimental Procedures" and assayed for DNA polymerase activity
using poly(dA) · oligo(dT) as template.
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Evidence for the Phosphorylation of Pol
by
Cyclin-dependent Protein Kinases--
Sf9 cells
were coinfected with recombinant viruses harboring pol
and
different pairs of recombinant baculoviruses harboring cdk-cyclins. The
cdk-cyclin pairs were cdk2-cyclin A, cdk2-cyclin E, cdk4-cyclin D1,
cdk4-cyclin D2, cdk4-cyclin D3, cdc2-cyclin A, and cdc2-cyclin B1.
After 48 h of infection, the cells were labeled with
32Pi for 2 h at 37 °C in low phosphate
medium, sonicated, and analyzed by immunoprecipitation using a mixture
of pol
monoclonal antibodies followed by SDS-PAGE and
autoradiography as described previously (13). The results (Fig.
10) showed that pol
was
hyperphosphorylated when it was coexpressed with the G1
phase-specific cdk-cyclins, cdk4-cyclin D3 or cdk2-cyclin E. The
relative intensity of phosphorylation when pol
was coexpressed with
these cdk-cyclins was about 10-fold greater than when pol
was
expressed on its own. The relative phosphorylation of pol
after
coinfection with the S or G2/M-specific cdc2-cyclins
(cdc2-cyclin A or cdc2-cyclin B1) was about 20% of that of the
G1/S-specific cdk-cyclins. Cdk2-cyclin A and cdk4-cyclin D2
gave phosphorylation intensities that were similar to the control values obtained when pol
was expressed alone. The relative
intensity of cdk4-cyclin D1 coinfected with pol
was lower than that
of pol
alone. Our results indicate that pol
is phosphorylated by cdk4-cyclin D3 and cdk2-cyclin E and is a likely substrate of these
G1/S-specific cdk-cyclins.

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Fig. 10.
In vivo phosphorylation of recombinant
pol in Sf9 insect cells. The indicated cdk-cyclins and
pol were coexpressed in Sf9 cells by coinfection as
described under "Experimental Procedures." The cells were labeled
metabolically with 32Pi, and the cell lysates
were immunoprecipitated with 20 µg of pol monoclonal antibody and
40 µl of protein A-Sepharose slurry. The immunoprecipitates were
subjected to SDS-PAGE and then autoradiographed (upper
panel). Relative intensities of the pol p125 polypeptide were
determined by densitometry.
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Activity of Phosphorylated and Unphosphorylated Forms of Pol
--
The effects of coexpression of p125 with cdk2-cyclin E,
cdk2-cyclin A, and cdk4-cyclin D3 on the activity of pol
were
assessed by examination of the activities in the lysates after gel
filtration on an HPLC column (Table III).
There were no striking effects on the specific activities of the pol
catalytic subunit assayed using poly(dA)·oligo(dT) as a template
(Table III). Immunoblots for the cdk-cyclins in the fractions confirmed
that these were also present in the fractions.
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Table III
Specific activities of p125 coinfected with different combinations of
cdk-cyclins
Lysates obtained from equal amounts of coinfected cells were
precipitated with 50% ammonium sulfate. The precipitates were
dissolved in TGEED buffer containing 150 mM KCl, and equal
volumes (0.5 ml) of each were loaded onto a Superose 6 HPLC gel
filtration column (see "Experimental Procedures"). The results show
the protein concentration and pol activities of the peak fractions.
The presence of the cdk-cyclins in the eluates was confirmed by
immunoblot (not shown).
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Coimmunoprecipitation of Cdk2 and Pol
--
It was found that
pol
could be coimmunoprecipitated with cdk2 from Sf9 cell
extracts when they were coexpressed in experiments in which the
extracts were immunoprecipitated with antibody against cdk2 and
immunoblotted with antibody against pol
(not shown). The
interaction of pol
with cdk2 was investigated further by examination of the coimmunoprecipitation of deletion mutants of pol
with cdk2. The results (Fig. 11) showed
that all of the deletion mutants tested were coimmunoprecipitated with
the exception of the mutant in which the NH2 terminus
(residues 2-249) were deleted. These results demonstrate that there is
likely a direct interaction between cdk2 and pol
, although the
possibility that this interaction is mediated by a third protein cannot
be discounted.

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Fig. 11.
Analysis of the ability of the deletion
mutants of pol to bind to cdk2. Sf9 cells (about
107) were coinfected with pol deletion mutants and cdk2
recombinant baculoviruses as indicated. The levels of expression of
these mutants were similar as determined by immunoblotting of the
Sf9 cell lysates. About 10 mg of total protein from each cell
lysate was used for immunoprecipitation with cdk2 polyclonal antibody
and SDS-PAGE. The separated proteins were transferred to a
nitrocellulose membrane and immunoblotted with a mixture of
NH2- and COOH-terminal pol monoclonal antibodies.
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Coimmunoprecipitation of Pol
with Members of the
Cdk-Cyclins--
The coimmunoprecipitation of pol
with cdk2 could
also be observed in cultured Molt 4 cell extracts when cell extracts
were immunoprecipitated with pol
antibody and Western blotted with antibody to cdk2 (Fig. 12, first
lane). The reciprocal experiment using cdk2 as the precipitating
antibody followed by immunoblotting with pol
antibody also showed
that cdk2 was coimmunoprecipitated with pol
(Fig. 12, last
lane). When cyclin E was used as the precipitating antibody, the
coimmunoprecipitation of pol
was observed. The
coimmunoprecipitation of cdk2 and cdk5 by PCNA antibody was also
observed under the same experimental conditions (Fig. 12). These
experiments show that pol
interacts with cdk2 and a cyclin in
vivo and point to the existence of macromolecular complexes
between pol
and the cdk-cyclins.

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Fig. 12.
Coimmunoprecipitation of pol with
members of the cdk-cyclin system. Molt 4 cells were lysed by
sonication. About 10 mg of total protein was immunoprecipitated with
the first antibody (Im. Ab) plus protein A-Sepharose and
then Western blotted with a second antibody (WB Ab). The
common band in the last three lanes is an
artifact (IgG heavy chain).
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DISCUSSION |
The studies reported here show that the catalytic subunit of DNA
pol
can be expressed in Sf9 cells in an active form and can
be isolated by a conventional purification protocol or by an
immunoaffinity chromatography procedure. Isolation of the recombinant protein was aided by the use of antibodies against pol
which did
not cross-react with the endogenous DNA polymerase in
baculovirus-infected Sf9 cells. We took advantage of an
immunoaffinity chromatography procedure to purify the recombinant pol
in a facile manner and to ascertain that it was separated from any
endogenous DNA polymerases. The properties of the overexpressed p125
catalytic subunit were compared with those of the native enzyme. Assays
of the enzyme activity using poly(dA)·oligo(dT) as a template showed
that the specific activities of the preparations were only about 1,200 units/mg (Table I) compared with about 25,000 units/mg protein for the
calf thymus holoenzyme (14). This difference is likely the result of
the lack of, or of a greatly attenuated response to PCNA by the free
catalytic subunit. Other studies of pol
preparations containing
only the catalytic subunit have suggested that it is not
PCNA-responsive (18, 19), whereas our previous studies of recombinant
pol
expressed in vaccinia virus have indicated a weak response
(2-3-fold stimulation). The baculovirus-expressed pol
shows little
or no response to PCNA, whereas the response is restored by the
presence of the p50 subunit (20-22). In other aspects, the enzymatic
behavior of the recombinant p125 is very similar to that of the
holoenzyme.
Studies of deletion mutants show that deletions (amino acids 2-249 or
186-321) in the NH2 terminus retain polymerase activity. Deletions in the core region (amino acids 336-715) and the deletion of
regions C and V in the core as well as most of the COOH-terminal region
including the zinc finger motifs (778-1047) had no assayable activity
(Table II). This is consistent with numerous other studies that
indicate that the core region of this family of polymerases is involved
in the binding of the incoming dNTP substrate (23, 24) and contains the
catalytic center for DNA polymerase activity. The retention of
enzymatic activity by the NH2-terminal deletion mutants is
consistent with the existence of a domain structure in which the
NH2-terminal region does not function in catalysis. That
this is likely is also consistent with the structure of T4 polymerase,
which contains most of the conserved core but only part of the
NH2-terminal region that includes a motif required for the
exonuclease activity (5).
The present studies provide the first evidence that the catalytic
subunit of pol
is itself a substrate for
cyclin-dependent protein kinases and that this is specific
for the G1 cdk-cyclins because other cdk-cyclin
combinations were less effective in phosphorylating pol
when they
were coexpressed in Sf9 insect cells. Although the in
vivo kinase activity of cdk-cyclin overexpressed in Sf9 insect cells may not reflect actual cellular events in the mammalian cell cycle, the involvement of G1 phase cdk-cyclins is
consistent with our previous observations that pol
is
phosphorylated in vivo during the cell cycle and is maximal
near the G1/S transition (25). The primary structure of pol
shows a number of potential phosphorylation sites for the cdks,
including six sites possessing the (S/T)P motif: serines 207 and 788 and threonines 83, 150, 238, and 640 (25). It is well known that in
mammalian cells the key regulators of the transition from
G1 to S phase of the cell cycle include the G1
cyclins-three D type cyclins (D1, D2, D3) and cyclin E (26). Cyclin E
expression is periodic, peaks at the G1/S transition, and
regulates S phase commitment together with its catalytic subunit cdk2.
Unlike cyclin E, expression of D type cyclins is cell lineage-specific
and highly mitogen-dependent, rising on growth factor
stimulation and declining rapidly on growth factor withdrawal (27, 28).
The current model for G1 cdk-cyclin functions is that
cyclin D binds directly to the tumor suppressor gene product pRb,
targeting cdk4 to its substrate, and resulting in phosphorylation of
pRb during middle to late G1 phase. This reverses the
growth-suppressive effects of pRb by releasing transcriptional factor
E2F from its inhibitory constraint; the untethered E2F factor is then
able to activate a series of genes required for DNA replication (26).
The G1 cdk-cyclins are also thought to phosphorylate other
key substrates resident at the DNA replication origin to trigger the
actual onset of DNA replication once cells pass the restriction point
(29, 30). Pol
is the central enzyme in eukaryotic DNA replication
and is tethered to DNA by a direct interaction with the PCNA clamp,
which converts pol
from a distributive into a highly processive
enzyme for DNA synthesis (31, 32). Thus, the finding that pol
is a
substrate for the G1 cdk-cyclins is of significance as it
provides a potential linkage for the cell cycle control of DNA
synthesis. However, our studies do not reveal any major effects of
phosphorylation on the activity of the p125 catalytic subunit, and only
small increases (<2-fold) were observed after co-expression with
cdk-cyclins (Table III). Pol
-primase has also been shown to be
phosphorylated, and phosphorylation does not or only moderately changes
its enzymatic properties (33-35). However, the ability of pol
-primase to initiate SV40 DNA replication in vitro was
found to be inhibited markedly after phosphorylation by cyclin
A-dependent kinases (36).
Examination of the interaction of cdk2 with the deletion mutants of pol
showed that the tertiary structure of pol
is not required for
this interaction and that the binding region is located in the
NH2-terminal 249 residues of pol
. The
NH2-terminus of yeast and mammalian pol
harbors several
highly conserved regions (N1-N5) that are also present in herpes and
Epstein-Barr viral polymerases (5). These conserved regions are likely
protein-protein interaction sites for pol
(5). The binding site of
pol
for PCNA has been mapped to the N2 region (13). The data
presented also provide the first evidence for complexes that involve
pol
and the cdk-cyclins. The targeting of the cdks to a substrate has some precedence since the G1 cdk-cyclins are known to
form complexes with pRb. The obvious question is whether this has any functional physiological significance in relation to the
phosphorylation or regulation of pol
. The present findings show
that the interaction of pol
with cdk2 and cdk4 needs to be
investigated further, in addition to the issue of the cellular role of
phosphorylation of pol
by the cdk-cyclins.
There are many levels at which phosphorylation could affect pol
function other than the simple modulation of enzyme activity in a
simple assay. This is apparent because physiologically pol
is part
of a holoenzyme and part of an extended multiprotein complex. Current
findings that p21, a potent inhibitor of G1 cdks, and pol
compete for the same sites in the interdomain connector loop of
PCNA (37, 38) add even more complexity to these questions. Xiong
et al. (39, 40) observed that PCNA is in a quaternary complex that includes cyclin D, cyclin-dependent kinases
(cdk2, cdk4, cdk5), and p21. No phosphorylation of PCNA and p21 was
detected, suggesting that neither of them is the primary substrate of
phosphorylation. Thus, there are many possible permutations and
speculations possible as to how regulatory systems could emerge from
this melange of potential complexes. We have obtained preliminary
evidence that pol
is a substrate for the
cyclin-dependent protein kinases. This was shown by the
coexpression of baculovirus vectors for pol
with several different
cdk-cyclin combinations in Sf9 cells (Fig. 10) and
coimmunoprecipitation Western blot studies in Molt 4 cells (Fig. 12).
These results suggest that more than one cyclin might regulate pol
,
possibly triggering its phosphorylation at different sites or times of
the cell cycle. Coimmunoprecipitation of pol
deletion mutants with
cdk2 also established the site of interaction (Fig. 11). Although the
regulation of pol
by protein phosphorylation has yet to be
demonstrated firmly, this possibility provides a potential mechanism
that might provide for the temporal regulation of DNA synthesis in
concert with the cell cycle.
Although the present evidence indicates that the phosphorylation status
of the catalytic subunit of DNA polymerase
may have no significant
effect on its activity, the question of whether phosphorylation has any
physiological relevance in affecting or regulating the biological
function of polymerase
still needs to be answered. A role of
phosphorylation or binding of the kinase in affecting the properties of
the polymerase in vivo in modulating the function of pol
in DNA replication or repair cannot be excluded. In this regard, note
that significant difference was observed when replication protein A is
phosphorylated in SV40 DNA replication (41-43) and nucleotide excision
repair systems (42). Further studies are needed to answer the question
of the regulatory consequences of phosphorylation of pol
and for
that matter other replication proteins. The putative kinase consensus
sequences in pol
also show that it could be a substrate for
DNA-dependent protein kinase. The latter kinase
phosphorylates serine or threonine residues that are followed or
preceded by glutamine residues (S/T)-Q or Q-(S/T). It remains to be
determined whether other kinases, e.g. DNA-dependent protein kinase, are also involved in the
phosphorylation of the catalytic subunit of pol
.