Characterization of the p125 Subunit of Human DNA Polymerase delta  and Its Deletion Mutants
INTERACTION WITH CYCLIN-DEPENDENT KINASE-CYCLINS*

Sheng-Ming WuDagger , Peng ZhangDagger §, Xiao Rong ZengDagger , Shan-Jian ZhangDagger , Jinyao MoDagger §, Bao Qing LiDagger §, and Marietta Y. W. T. LeeDagger §

From the Dagger  Department of Biochemistry and Molecular Biology, University of Miami, Miami, Florida 33101 and the § Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The catalytic subunit of human DNA polymerase (pol) delta  was overexpressed in an active, soluble form by the use of a baculovirus system in insect cells. The recombinant enzyme was separated from endogenous DNA polymerases by phosphocellulose, Mono Q-Sepharose, and single-stranded DNA-cellulose chromatography. Recombinant DNA pol delta  was also purified by immunoaffinity chromatography. The enzymatic properties of the purified catalytic subunit were characterized. The enzyme was active and possessed both DNA polymerase and associated 3' to 5' exonuclease activities. NH2-terminal deletion mutants retained polymerase activity, whereas the core and COOH-terminal deletion mutants were devoid of any measurable activities. Coinfection of Sf9 cells with recombinant baculovirus vectors for pol delta  and cyclin-dependent kinase (cdk)-cyclins followed by metabolic labeling with 32Pi showed that the recombinant catalytic subunit of pol delta  could be hyperphosphorylated by G1 phase-specific cdk-cyclins. When cdk2 was coexpressed with pol delta  in Sf9 cells, pol delta  was found to coimmunoprecipitate with antibodies against cdk2. Experiments with deletion mutants of pol delta  showed that the NH2-terminal region was essential for this interaction. Coimmunoprecipitation and Western blot experiments in Molt 4 cells confirmed the interaction in vivo. Preliminary experiments showed that phosphorylation of the catalytic subunit of pol delta  by cdk2-cyclins had little or no effect on the specific activity of the enzyme.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

DNA polymerase (pol)1 delta  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 alpha  and pol beta . The 125-kDa subunit of human pol delta  (p125) has been identified as the catalytic subunit (4). Pol delta  is a member of a family of DNA polymerases which includes DNA polymerase alpha , pol epsilon , 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 delta  (7). In addition, there are several regions in the NH2 and COOH termini which are conserved among human pol delta , yeast pol delta , and yeast and human pol epsilon  (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 delta , 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 delta , consistent with its role as the leading strand polymerase (8). Although there have been some mutagenesis studies of the yeast pol delta  (9), little has been done with human or mammalian pol delta , largely because of the lack of a suitable expression system. To facilitate structure-function studies of pol delta , it is desirable to have an expression system for the production of the recombinant protein. The expression of the human pol delta  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 delta . In addition, we have obtained novel evidence that pol delta  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 delta  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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 delta  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 delta containing the corrected full-length pol delta  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 beta -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 delta  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 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 delta  (2, 14).

HPLC-- The combined fractions from the phosphocellulose column which contained recombinant pol delta  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 delta  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 delta  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 delta  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 delta  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 delta  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 delta  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 delta , Cyclins, and Cdks and 32Pi Labeling-- Sf9 cells (107) were grown to exponential stage. Pol delta , 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 delta  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 delta  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 delta  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 delta .

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression of Pol delta  p125-- The expression of human pol delta  in Sf9 cells infected with recombinant baculovirus was analyzed by immunoblotting with a pol delta  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 delta  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 delta  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 delta . 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 delta  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 delta  by SDS-PAGE (20 µl/lane) followed by immunoblotting. Lanes are marked according to time of harvest.

The recombinant pol delta  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 delta  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 delta  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).

Purification of Recombinant Pol delta -- 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 delta  from Sf9 is the presence of endogenous DNA polymerases (15), which could compromise studies of the enzymatic properties of human recombinant pol delta . 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 delta , 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 delta  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 delta  (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).

The peak fractions that immunoblotted with pol delta  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 delta  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 delta . 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 delta  (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 delta  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.

Immunoaffinity Purification of Recombinant Pol delta -- We have shown previously that calf thymus pol delta  can be isolated by immunoaffinity chromatography using monoclonal antibody 78F5 coupled to AvidChrom hydrazide (14). Crude Sf9 cell extracts were chromatographed on a pol delta  immunoaffinity column ("Experimental Procedures"). The column was washed with buffer containing 50 mM NaCl, and pol delta  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 delta  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 delta . Panel A, an extract from cells infected with recombinant baculovirus was chromatographed on a pol delta  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 delta  (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 delta .

                              
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Table I
Purification of recombinant DNA pol delta  p125
Assays was performed using poly(dA) · oligo(dT) template.

Characterization of Recombinant p125-- The enzymatic properties of the recombinant pol delta  catalytic subunit were compared with those of native calf thymus pol delta , 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 delta  catalytic subunit were similar to those of native pol delta and the Sf9 polymerases in that they were inhibited by aphidicolin (Fig. 8A) and resistant to 2-(p-n-butylanilino)-9-(2-deoxy-beta -D-ribofuranosyl)adenine 5'-triphosphate (not shown). A well known characteristic of calf thymus pol delta  is its sensitivity to inhibition by N-ethylmaleimide; recombinant pol delta  was inhibited in a manner similar to calf thymus pol delta , 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 delta  (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 delta  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 delta : comparison with native calf thymus pol delta  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 delta  (closed circles), native calf thymus pol delta  (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 delta . 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 delta .

Recombinant pol delta  was stimulated by Mn2+ in a manner similar to that already known for calf thymus pol delta . 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 delta  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 delta  belongs to the alpha -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 alpha , the NH2-terminal regions of pol delta  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 delta  (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 (Delta 186-321) and p97 (Delta 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 delta  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.

Evidence for the Phosphorylation of Pol delta  by Cyclin-dependent Protein Kinases-- Sf9 cells were coinfected with recombinant viruses harboring pol delta  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 delta  monoclonal antibodies followed by SDS-PAGE and autoradiography as described previously (13). The results (Fig. 10) showed that pol delta  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 delta  was coexpressed with these cdk-cyclins was about 10-fold greater than when pol delta  was expressed on its own. The relative phosphorylation of pol delta  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 delta  was expressed alone. The relative intensity of cdk4-cyclin D1 coinfected with pol delta  was lower than that of pol delta  alone. Our results indicate that pol delta  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 delta  in Sf9 insect cells. The indicated cdk-cyclins and pol delta  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 delta  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 delta  p125 polypeptide were determined by densitometry.

Activity of Phosphorylated and Unphosphorylated Forms of Pol delta -- The effects of coexpression of p125 with cdk2-cyclin E, cdk2-cyclin A, and cdk4-cyclin D3 on the activity of pol delta  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 delta  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 delta  activities of the peak fractions. The presence of the cdk-cyclins in the eluates was confirmed by immunoblot (not shown).

Coimmunoprecipitation of Cdk2 and Pol delta -- It was found that pol delta  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 delta  (not shown). The interaction of pol delta  with cdk2 was investigated further by examination of the coimmunoprecipitation of deletion mutants of pol delta  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 delta , 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 delta  to bind to cdk2. Sf9 cells (about 107) were coinfected with pol delta  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 delta  monoclonal antibodies.

Coimmunoprecipitation of Pol delta  with Members of the Cdk-Cyclins-- The coimmunoprecipitation of pol delta  with cdk2 could also be observed in cultured Molt 4 cell extracts when cell extracts were immunoprecipitated with pol delta  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 delta  antibody also showed that cdk2 was coimmunoprecipitated with pol delta  (Fig. 12, last lane). When cyclin E was used as the precipitating antibody, the coimmunoprecipitation of pol delta  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 delta  interacts with cdk2 and a cyclin in vivo and point to the existence of macromolecular complexes between pol delta  and the cdk-cyclins.


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Fig. 12.   Coimmunoprecipitation of pol delta  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).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The studies reported here show that the catalytic subunit of DNA pol delta  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 delta  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 delta  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 delta preparations containing only the catalytic subunit have suggested that it is not PCNA-responsive (18, 19), whereas our previous studies of recombinant pol delta  expressed in vaccinia virus have indicated a weak response (2-3-fold stimulation). The baculovirus-expressed pol delta  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 delta  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 delta  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 delta  is phosphorylated in vivo during the cell cycle and is maximal near the G1/S transition (25). The primary structure of pol delta  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 delta  is the central enzyme in eukaryotic DNA replication and is tethered to DNA by a direct interaction with the PCNA clamp, which converts pol delta from a distributive into a highly processive enzyme for DNA synthesis (31, 32). Thus, the finding that pol delta  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 alpha -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 alpha -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 delta  showed that the tertiary structure of pol delta  is not required for this interaction and that the binding region is located in the NH2-terminal 249 residues of pol delta . The NH2-terminus of yeast and mammalian pol delta  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 delta  (5). The binding site of pol delta for PCNA has been mapped to the N2 region (13). The data presented also provide the first evidence for complexes that involve pol delta  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 delta . The present findings show that the interaction of pol delta  with cdk2 and cdk4 needs to be investigated further, in addition to the issue of the cellular role of phosphorylation of pol delta  by the cdk-cyclins.

There are many levels at which phosphorylation could affect pol delta  function other than the simple modulation of enzyme activity in a simple assay. This is apparent because physiologically pol delta  is part of a holoenzyme and part of an extended multiprotein complex. Current findings that p21, a potent inhibitor of G1 cdks, and pol delta  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 delta  is a substrate for the cyclin-dependent protein kinases. This was shown by the coexpression of baculovirus vectors for pol delta  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 delta , possibly triggering its phosphorylation at different sites or times of the cell cycle. Coimmunoprecipitation of pol delta  deletion mutants with cdk2 also established the site of interaction (Fig. 11). Although the regulation of pol delta  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 delta  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 delta  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 delta  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 delta  and for that matter other replication proteins. The putative kinase consensus sequences in pol delta  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 delta .

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM31973 and in part by the United States Army Medical Research and Materiel Command under DAMD-17-96-1-6166.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 914-594-4070; Fax: 914-594-4058.

1 The abbreviations used are: pol, polymerase; p125, 125-kDa subunit of human pol delta ; cdk, cyclin-dependent kinase; AcMNPV, A. californica multiple nuclear polyhedrosis virus; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PCNA, proliferating cell nuclear antigen.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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