The Quaternary Structure of DNA Polymerase epsilon  from Saccharomyces cerevisiae*

Olga ChilkovaDagger , Bengt-Harald Jonsson§, and Erik JohanssonDagger

From the Dagger  Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden and § Department of Physics and Measurement Technology/Molecular Biotechnology, Linköpings University, SE-581 83 Linköping, Sweden

Received for publication, November 20, 2002, and in revised form, January 18, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA polymerase epsilon  (Pol epsilon ) from Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication. Biochemical characterizations of Pol epsilon  have been cumbersome due to protease sensitivity and the limited amounts of Pol epsilon  in cells. We have developed a protocol for overexpression and purification of Pol epsilon  from S. cerevisiae. The native four-subunit complex was purified to homogeneity by conventional chromatography. Pol epsilon  was characterized biochemically by sedimentation velocity experiments and gel filtration experiments. The stoichiometry of the four subunits was estimated to be 1:1:1:1 from colloidal Coomassie-stained gels. Based on the sedimentation coefficient (11.9 S) and the Stokes radius (74.5 Å), a molecular mass for Pol epsilon  of 371 kDa was calculated, in good agreement with the calculated molecular mass of 379 kDa for a heterotetramer. Furthermore, analytical equilibrium ultracentrifugation experiments support the proposed heterotetrameric structure of Pol epsilon . Thus, both DNA polymerase delta  and Pol epsilon  are purified as monomeric complexes, in agreement with accumulating evidence that Pol delta  and Pol epsilon  are located on opposite strands of the eukaryotic replication fork.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pioneering studies in the SV40 DNA replication system by several research groups resulted in the reconstitution of the SV40 replication fork with mammalian proteins in vitro (1). This system showed that DNA polymerase alpha  is required for priming the DNA since it is associated with primase activity. Only DNA polymerase delta  (Pol delta )1 was required to replicate both leading and lagging strand, leaving another essential eukaryotic DNA polymerase, DNA polymerase epsilon  (Pol epsilon ), without a specific function. However, there are several lines of evidence for the presence of Pol epsilon  at the eukaryotic replication fork. Evidence for a physical interaction came from cross-linking experiments, which showed that Pol epsilon  is located at or near the replication fork (2). Genetic experiments in Saccharomyces cerevisiae have shown that Pol epsilon  participates at the replication fork. Cells grown in the presence of 6-N-hydroxylaminopurine showed a strand-specific mutation rate on the chromosome, depending on whether they carried an exonuclease-deficient Pol epsilon  or Pol delta  (3). It has also been shown that loss of the exonuclease site in either Pol delta  or Pol epsilon  alters the mutation spectra of the sup-4 gene when replicated on a plasmid (4). The altered mutational spectrum was again strand-specific. In addition, the overall mutation rate is increased about 10-fold in yeast strains that carry a pol2 mutant that is lacking proofreading capability, suggesting that Pol epsilon  contributes to the bulk synthesis of newly replicated DNA (5). Evidence for a direct role during the progression of the fork has emerged from in vitro replication assays in Xenopus extracts in a cell-free system (6).

A very surprising result was obtained when the catalytic domain of Pol epsilon  was deleted in yeast (7). The catalytic domain was not essential. The essential property of the enzyme was instead located at the C terminus of the largest subunit (7, 8). Cells that were expressing only the C terminus were viable, although they had growth problems. However, later it was shown that cells were not viable when carrying a point mutation in the conserved catalytic site of Pol epsilon  (9). Although Pol epsilon  is normally present at the fork, in the absence of the catalytic domain, another DNA polymerase, presumably Pol delta , can substitute its function.

Pol epsilon  consists of four subunits, Pol2p (256 kDa), Dpb2p (79 kDa), Dpb3p (23 kDa), and Dpb4p (22 kDa) (10). The largest subunit, Pol2p, contains the catalytic site and an essential putative zinc-finger domain at the C terminus (9). The other three subunits have an unknown function. There has been very limited biochemical characterization of the four-subunit enzyme since it was first purified in 1990 (10). This is in part due to the limited amount of enzyme in the cells, the tedious purification protocol, and the sensitivity of the enzyme to proteolytic cleavage (10, 11).

Several models of the structure of the eukaryotic replication fork have been proposed (1, 12-14). A common theme has been the coupled synthesis of the leading and lagging strands in which the replicative DNA polymerase should form a dimer as has been observed in Escherichia coli (15, 16). Earlier studies suggested the existence of a dimeric Pol delta  with two catalytic assemblies, and a model where a dimeric Pol delta  would replicate both leading and lagging strand was proposed (13, 17). However, it has now been demonstrated that Pol delta  actually is a monomer in vitro and that the previous estimates of the molecular weight of Pol delta  were misleading due to the elongated shape of the enzyme (18). The initial analysis of the four-subunit Pol epsilon  included an estimate of the molecular mass and the subunit stoichiometry (10). The molecular mass was estimated to be 295 kDa from the hydrodynamic properties of the enzyme, and the molar ratio of the four subunits was estimated at Pol2:Dpb2:Dpb3:Dpb4 = 1:1:3:4. This molar ratio predicted a molecular mass of 492 kDa, which was substantially different from that determined experimentally. The authors concluded that the enzyme was purified as a monomer with an elongated shape. Pol epsilon  has also been proposed to form a dimer based on yeast two-hybrid assays and gel filtration experiments using Pol epsilon  overproduced in a baculovirus system (9, 19). In light of these contradictory results we wished to clarify whether Pol epsilon  exists as a monomer or a dimer when overproduced in yeast. Therefore, we developed an overexpression system that allowed us to purify native Pol epsilon  from yeast, i.e. without tags. Our studies of the assembly state of this enzyme show it to be a monomeric assembly of one each of the four subunits.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains-- A protease-deficient yeast strain was used for the overexpression of Pol epsilon . PY116 (ura 3-52, trp 1Delta , his 3-11, 15, pep 4-4, prb 1-1122 (prc1-407) (CANsens), leu 2-3, 112, nuc 1: LEU 2) was transformed with plasmids pJL1 and pJL6 (see below). This strain for overexpression of Pol epsilon  was called YEJ1. PJL1 contained the POL2 gene and pJL6 contained the DPB2, DPB3, and DPB4 genes under the control of the GAL1-10 promoter. The plasmids carried a 2-µM origin and either TRP1 or URA3 as selective markers.

Plasmids-- All plasmids were based on the pRS424GAL and pRS426GAL from the pRS424-426GAL series of plasmids (a kind gift from P. Burgers). The POL2 gene was amplified from the plasmid YCplac33 POL2 (20) with the following oligonucleotides: POL2-1, 5'-TTCTAGAGCGGCCGCATGATGTTTGGCAAGAAAAAAAACAACGG-3'; POL2-2, 5'-AACACGAGCTCTCATATGGTCAAATCAGCAATACAACTCAATAA-3'. The resulting DNA fragment was digested with NotI and SacI, purified, and ligated into prs424GAL opened in NotI and SacI. The 500 nucleotides most approximate to NotI and SacI were sequenced to make sure that no mutations were inserted by KlenTaq LA during the amplification of the insert. After this, the overexpression plasmid was digested with PshAI and NgoMIV to remove most of the POL2 gene. The YCplac33 POL2 plasmid was also digested with PshAI and NgoMIV, and the 6151-nucleotide fragment containing the POL2 gene was subcloned into the opened prs424GAL-POL2 plasmid.

The resulting plasmid was named pJL1. DPB2, DPB3, and DPB4 were amplified from W303 genomic DNA with the following oligonucleotides: DPB2-1, 5'-TTCTAGGATCCATGTGTGAAATGTTTGGCTCTGGGA-3'; DPB2-2, 5'-TTCTAGAGCGGCCGCAAACGGTAGGCCAAGTAAACTGCCC-3'; DPB3-1, 5'-TTCTAGAGCGGCCGCTCAACCGTGTTGCAAAAAAAATGTCC-3'; DPB3-2, 5'-AACACGAGCTCGTAATTGTGGCACAGGCAAGCTG-3'; DPB4-1, 5'-TTCTAGAGAATTCATGCCACCAAAAGGTTGGAGAAAAG-3'; DPB4-2, 5'-AACACGTCGACATATTCAGTTGCTCTAATCGGAGC-3'. The amplified DPB2 gene was digested with BamHI and NotI, purified, and ligated into the BamHI-NotI sites of prs424GAL. The resulting plasmid was called pJL2. The amplified DPB3 gene was digested with NotI and SacI, purified, and ligated into the NotI-SacI sites of prs426GAL. This plasmid was called pJL3. The amplified DPB4 gene was digested with EcoRI and SalI, purified, and ligated into the EcoRI-SalI sites of prs426GAL. This plasmid was called pJL4. Next, DPB2, DPB3, and DPB4 were sequenced to confirm that no mutations had been inserted by the polymerase during the amplification. This was followed by the digestion of pJL3 and pJL4 with BamHI and SacI. The fragment from pJL3 that contained DPB3 was cloned into the BamHI-SacI sites of the pJL4 plasmid. This plasmid was digested with SacI and blunt-ended with Klenow. pJL2 was digested with SapI and HincII, over-hangs were filled in by Klenow, and DPB2 was ligated into the opened blunt-ended plasmid containing DPB3 and DPB4. The resulting plasmid, prs426GAL with DPB2, DPB3, and DPB4, was called pJL6.

Cell Growth-- For Pol epsilon  overexpression the YEJ1 strain was grown as previously described in (21). Briefly, the YEJ1 strain was grown in 100 ml of glycerol-lactate media overnight. After 24 h, the culture was aliquoted into six 6-liter flasks, each containing 1.2 liters of glycerol-lactate media, and left vigorously shaking at 30 °C. After another 24 h 1.2 liters of YPGL media was added to every flask. YPGL media consists of 1% yeast extract, 2% bactopeptone, 3% glycerol, 2% lactic acid, and 0.1% glucose, pH 5.5-6. After 3-4 h the protein expression was induced by adding solid galactose to a final concentration of 2%. After another 6 h the cells were harvested by centrifugation for 5 min at 4 °C. The cells were resuspended in H2O and frozen in liquid nitrogen.

Enzyme Purification-- All purification steps were carried out at 4 °C. The following buffers were used. Buffer A: 150 mM Tris-acetate, pH 7.8, 50 mM sodium acetate, 2 mM EDTA, 1 mM EGTA, 10 mM NaHSO3, 1 mM dithiothreitol, 5 µM pepstatin A, 5 µM leupeptin, 0.3 mM p-phenylmethylsulfonyl fluoride, and 5 mM benzamidine. Buffer B: 25 mM Hepes-NaOH, pH 7.6, 10% glycerol, 1 mM EDTA, 0.5 mM EGTA, 0.005% Nonidet P-40, 1 mM dithiothreitol, 5 µM pepstatin A, 5 µM leupeptin, and 5 mM NaHSO3. The concentration of sodium acetate is indicated as suffix, for example, buffer B100 = buffer B with 100 mM sodium acetate. A beadbeater was used to open 60-70 g of cells in Buffer A. Beating continued for 5 min with intervals of 1 min of cooling every 30 s. The volume of the lysate from the beadbeater was measured, and saturated ammonium sulfate was added to a final concentration of 175 mM, followed by the addition of 40 µl of 10% Polymin P per ml of extract. The lysate was incubated on ice with occasional stirring for 15 min, followed by centrifugation at 18,000 rpm in a Beckman JA25.5 rotor for 1 h. The volume of the cleared lysate was measured, and 0.28 g of solid ammonium sulfate was added per ml of extract. The ammonium sulfate was dissolved for 45 min on a magnetic stirrer, followed by centrifugation at 18,000 rpm in a Beckman JA25.5 rotor for 1 h. The ammonium sulfate precipitate was resuspended in buffer B50 and frozen. The next day, the frozen extract was thawed and dialyzed against 1000 ml of buffer B100 for 3 h, and thereafter centrifuged for 30 min. The cleared lysate was loaded onto a 20-ml phosphocellulose column equilibrated in B200. The column was washed with B200 and eluted with B750. Fractions with protein were loaded onto a MonoQ column eluted with a 20-ml linear gradient from B800 to B1200. After dialysis against 500 ml of buffer B100, peak fractions from MonoQ were loaded onto a MonoS column equilibrated in B100 and eluted with a 20-ml linear gradient from B100 to B500. Finally, the MonoS peak fraction was passed over a Superose 6 gel filtration column as described below. The protein concentration in various fractions throughout the purification was determined by the method of Bradford (22) with bovine serum albumin as a standard.

Gel Filtration Analysis-- A Superose 6 gel filtration column was equilibrated at 4 °C in Buffer C. Buffer C: 25 mM Hepes, pH 7.6, 10% glycerol, 1 mM EDTA, 0.005% Nonidet P-40, 400 mM sodium acetate, 5 mM dithiothreitol, 5 mM NaHSO3, 2 µM leupeptin, and 2 µM pepstatin A. The column was calibrated by injection of proteins with a known Stokes radius. The Stokes radii of the standard proteins were plotted against their elution volumes. The standard proteins used were: carbonic anhydrase (Ve = 18.07 ml), bovine serum albumin (Ve = 16.68 ml), catalase (Ve = 15.92 ml), ferritin (Ve = 14.82 ml), E. coli beta -galactosidase (Ve = 14.45 ml), and thyroglobulin (Ve = 12.65 ml). 0.2-20 µg of Pol epsilon  from the MonoS peak fraction was passed over the equilibrated Superose 6 column. Fractions were analyzed by SDS-PAGE, and the polymerase activity was measured. The specific activity of purified Pol epsilon  was determined to be 25-40,000 units/mg of protein, as defined by Hamatake and co-workers (10).

Glycerol Gradient Centrifugation-- 0.2-20 µg of Pol epsilon  from the MonoS peak fraction together with 0.5 µg of bovine liver catalase (Sigma) as an internal control, in a total volume of 100 µl, were layered on top of a 4-ml 15-30% linear gradient of glycerol in buffer C. The samples were spun for 16 h at 40,000 rpm in a Beckman SW60 rotor at 1 °C. Fractions were collected from the bottom of the tube, and aliquots were analyzed by SDS-PAGE, DNA polymerase assays, or Western blot analysis with Dpb4p antibodies. The rabbit Dpb4p polyclonal antibodies were raised against Dpb4p that was overexpressed and purified from E. coli. Aliquots of each fraction were also removed for spectrophotometric assay of catalase activity (23). Whole cell extracts from wild-type yeast were prepared as previously described, and cleared lysate was loaded onto the 15-30% glycerol gradient in buffer C400. The density of glycerol gradient fractions was measured refractometrically. Standard proteins with known sedimentation coefficients were used to calibrate the gradients: carbonic anhydrase (2.8 S), bovine serum albumin (4.3 S), aldolase (8.9 S), catalase (11.3 S), and ferritin (17.6 S).

Polymerase Assays-- For DNA polymerase assays an oligo(dT)16-primed (dA)300 template was used (24). A typical reaction (50 µl) contained 20 mM Tris-HCl, pH 7.5, 4% glycerol, 0.1 mg/ml bovine serum albumin, 5 mM dithiothreitol, 8 mM MgAc2, 80 µM each dATP, dCTP, and dGTP, 20 µM [3H]dTTP (400-600 cpm/pmol), 0.01 unit of poly(dA)·oligo(dT), and 1 mM spermidine and enzyme. Reactions were assembled on ice and incubated for 30 s to 5 min at 30 °C. The reactions were stopped by the addition of 150 µl of stop solution (50 mM sodium pyrophosphate, 25 mM EDTA, and 50 µg/ml salmon sperm DNA). The insoluble material was precipitated by adding 1.5 ml of 10% trichloroacetic acid, followed by a 30-min incubation on ice. The insoluble material was filtered on GF/C filters, washed 4 × 2 ml with 1 M HCl in 0.05 M sodium pyrophosphate, rinsed with ethanol, dried, and counted by liquid scintillation. One unit of enzyme activity incorporates 1 nmol of total nucleotide/hour (10).

Sedimentation Equilibrium Experiments-- The analytical ultracentrifugation experiments were carried out at 10 °C in a Beckman XL-A/XL-I analytical ultracentrifuge, equipped with an An50Ti rotor using six sector centerpieces. Sedimentation equilibrium experiments on Pol epsilon  were analyzed in Buffer D (as Buffer C but with 5 mM 2-mercaptoethanol instead of dithiothreitol) at three different initial protein concentrations, 2.1, 0.28, and 0.14 µM. Absorbance at 280 nm was measured as a function of radial position for three rotor speeds, ranging from 4000 to 7500 rpm. Equilibrium at each rotor speed was reached after 20 h. At that time, five scans were averaged for each sample at each rotor speed. The sedimentation properties of Pol epsilon  were analyzed by using the self-association model in the Beckman software package.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We chose to overexpress Pol epsilon  in S. cerevisiae for several reasons. Earlier attempts to overexpress Pol epsilon  in a baculovirus system experienced several problems (19, 25). Not only was the largest subunit of 256 kDa expressed at low levels, but in addition Pol2p was cleaved by a protease and the proteolytic fragment copurified with the holoenzyme. Finally the purified protein was tagged, and this could affect the properties of the enzyme. We chose to overexpress Pol epsilon  in S. cerevisiae without any tags added, similar to earlier overexpression studies of Pol delta  from S. cerevisiae (18).

The POL2 gene was subcloned into one plasmid and DPB2, DPB3, and DPB4 were subcloned into the second plasmid. All genes were cloned under the control of the galactose-inducible GAL1-10 promoter (21). This protocol proved successful and allowed us to purify 400 µg of Pol epsilon  from as little as 70 g of cells. The complex was purified by successive phosphocellulose, MonoQ, and MonoS chromatography, and the enzyme was finally purified to homogeneity by gel filtration column (Fig. 1) or by glycerol gradient centrifugation.


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Fig. 1.   Overexpressed Pol epsilon  was purified to homogeneity. 1 µg of Pol epsilon  from the Superose 6 column was loaded on a 4-20% linear gradient SDS-PAGE. The proteins were visualized by colloidal Coomassie staining.

Our overexpressed Pol epsilon  was purified as a four-subunit complex with all subunits co-migrating through a glycerol gradient and co-eluting from a Superose 6 column (Figs. 2B and 3B). The peak fractions from the gel filtration column and the glycerol gradient were analyzed by 10% SDS-PAGE, the stained gels were scanned, and the intensity of the protein bands was quantified. The molar ratio of the four subunits was estimated to be 1.00:1.12:1.07:1.06 (Pol2p:Dpb2p:Dpb3p:Dpb4p) (Fig. 4). In contrast, we found that the apparent molar ratio of the four subunits was ~1:1:3:4 when the SDS-PAGE was silver-stained (data not shown). Earlier estimates of the subunit stoichiometry have been based solely on silver staining, and it appears that the amounts of Dpb3p and Dbp4p may have been overestimated due to the silver staining (10, 19).


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Fig. 2.   Glycerol gradient centrifugation. A, gradient calibration was carried out with ferritin (data point 1, 17.6 S), catalase (data point 2, 11.3 S), aldolase (data point 3, 8.9 S), bovine serum albumin (data point 4, 4.3 S), and carbonic anhydrase (data point 5, 2.8 S). The sedimentation positions of the complexes are as indicated. The estimated sedimentation coefficient of Pol epsilon  was 11.88 ± 0.84 S. The sedimentation coefficient of Pol epsilon  was measured over a concentration range from 5 nM to 0.5 µM (as monomer), with no observed shift in S value. B, fractions containing Pol epsilon  were analyzed on a 10% SDS-PAGE and visualized with colloidal Coomassie staining. Catalase was added as an internal standard before loading the sample, and the catalase activity was measured in a colorimetric assay. The peak fraction containing catalase activity has been indicated with catalase. The contrast of the shown gel was enhanced to visualize Dpb3 and Dpb4.


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Fig. 3.   Gel filtration. A, the calibration of the Superose 6 column was carried out with thyroglobulin (data point 1, 85 Å), beta -galactosidase (data point 2, 69 Å), ferritin (data point 3, 61 Å), catalase (data point 4, 52.2 Å) bovine serum albumin (data point 4, 35.5 Å), and carbonic anhydrase (data point 5, 23.9 Å). The elution volumes of the complexes are as indicated. Pol epsilon  eluted at 13.8 ml, and the Stokes radius was estimated to be 74.5 ± 2.4 Å. B, fractions containing Pol epsilon  were analyzed on a 10% SDS-PAGE and visualized with colloidal Coomassie staining. The contrast of the shown gel was enhanced to visualize Dpb3 and Dpb4.


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Fig. 4.   Subunit stoichiometry of Pol epsilon . Peak fractions from the Superose 6 column were separated on a 10% SDS-PAGE and stained with colloidal Coomassie. The gel was digitized with a CanonFB1210U scanner, and the lanes were scanned and quantified using NIH Image software. The molar ratio of the subunits was Pol2p:Dpb2p:Dpb3p:Dpb4p = 1:1.12:1.07:1.06.

The sedimentation coefficient for Pol epsilon  was determined to be 11.9 S (Fig. 2). Using an antibody to Dpb4p we measured the sedimentation coefficient of Pol epsilon  when a crude extract from a wild-type strain was separated over the same glycerol gradient. The sedimentation of Pol epsilon  in a crude extract was similar to that of the purified Pol epsilon  (data not shown).

Pol epsilon  was analyzed on a Superose 6 gel filtration column (Fig. 3). The enzyme was injected over the column in the concentration range between 5 nM and 0.5 µM (as monomer), and the elution volume was 13.8 ml over the entire concentration range. The Stokes radius was determined to be 74.5 Å from the standard curve. The measured Stokes radius and the sedimentation coefficient allowed us to estimate the molecular mass, according to Siegel and Monty (26). Our calculated molecular mass of 371 kDa suggests that Pol epsilon  is purified as a monomer of a Pol2:Dpb2:Dpb3:Dpb4 complex with the molar ratio of 1:1:1:1 in the concentration range studied (Table I). The frictional coefficient f/f0 of Pol epsilon  is 1.56, suggesting a moderately elongated shape of the enzyme.


                              
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Table I
Molecular Mass of DNA polymerase varepsilon  
Mass in kDa and Stokes radii in Å. Theoretical masses were calculated from the polypeptide sequences; mass and f/f0 from s and Å were obtained from Ref. 26; mass from equilibrium sedimentation centrifugation was calculated with the Beckman software package. Indicated errors in the calculated masses reflect measurement errors only. See "Results" for details.

Sedimentation Equilibrium Experiment-- To obtain a more precise molecular mass independent of the shape of the enzyme, we carried out sedimentation equilibrium ultracentrifugation. Three different starting concentrations of Pol epsilon  ranging from 0.14 to 2.1 µM were analyzed at four different speeds until equilibrium was reached. The molecular mass of Pol epsilon  was determined to be 366 kDa under these conditions. A comparison with the predicted distribution of a monomeric versus a dimeric assembly clearly showed that Pol epsilon  was present as a monomer in the solution under all conditions investigated (Fig. 5).


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Fig. 5.   Sedimentation equilibrium experiment. Pol epsilon  with a starting concentration of 2.1 µM was centrifuged at three consecutive speeds, beginning with 4000 rpm. The theoretical curves for the monomeric and dimeric species are calculated based upon the polypeptide sequence.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have presented a method to overexpress and purify Pol epsilon  to homogeneity from S. cerevisiae. The yield and purity of the enzyme will allow us to perform a careful characterization of the enzyme. We have initiated this study by determining the molecular mass and subunit stoichiometry of the enzyme. These are essential parameters for further studies of the enzyme. From these studies and recent studies of Pol delta  we can conclude that, at least under the conditions studied, both enzymes are monomeric multisubunit assemblies with each subunit occurring once.

An attractive model of the replication fork describes how there should be coupling of leading and lagging strand synthesis. This is a model that has emerged from studies in E. coli (15, 16). It is very likely that a similar mechanism is present at the eukaryotic replication fork. Initial experiments with Pol delta  suggested that Pol delta  was a dimeric enzyme (17, 27). A model was proposed in which Pol delta  formed a dimeric unit with two catalytic domains. In this model Pol delta  would replicate both leading and lagging strand in a coordinated manner (13). This model has recently been questioned, and it has been shown that Pol delta  is a monomer with one catalytic site (18, 28). Meanwhile, Pol epsilon  was also suggested to be dimeric (19). The evidence for a dimeric enzyme originated from yeast two-hybrid assays and gel filtration experiments (9, 19). Yeast two-hybrid assays, particularly with yeast genes, can be misleading because false positives can result from a bridging interaction through a third yeast protein, as for instance was observed with the third subunit of Pol delta . Pol32 appeared to interact with itself in a two-hybrid analysis, but this interaction was actually mediated by proliferating cell nuclear antigen (18, 29). A second line of evidence for a dimeric Pol epsilon  was based upon gel filtration experiments (19). The calculated molecular mass of the protein was based upon the assumption that Pol epsilon  was globular. We have measured both the sedimentation coefficient and the Stokes radius of Pol epsilon , and from those data calculated the frictional coefficient to be 1.56 (Table I), indicating that the complex is slightly elongated. As a consequence, the molecular mass will be overestimated when determined by gel filtration only (19). The calculated molecular mass of 371 kDa from both gel filtration and density centrifugation measurements agrees well with the theoretical molecular mass of a four-subunit complex with the subunit stoichiometry of 1:1:1:1 (Table I). Finally, the sedimentation equilibrium experiments confirmed the results of our other studies where Pol epsilon  was found to exist as a monomer in solution (Fig. 5 and Table I). Thus, our results clearly support a model of Pol epsilon  as a monomer of a (Pol2:Dpb2:Dpb3:Dpb4) heterotetrameric complex.

The role of Pol epsilon  and Pol delta  at the eukaryotic replication fork is still not known with certainty. Genetic experiments in S. cerevisiae have shown that there is an asymmetry present at the replication fork (30). The current hypothesis that the coupled synthesis of the leading and lagging strand involves both Pol epsilon  and Pol delta  agrees with our observations that both Pol delta  and Pol epsilon  are monomeric assemblies. Pol delta  has been suggested to replicate the lagging strand based on a genetic interaction with the flap endonuclease (RAD27) (31). Pol epsilon  has been suggested to replicate the leading strand, based on experiments in cell-free Xenopus extracts (6). However, the fact that both Pol delta  and Pol epsilon  are purified as monomers in solution does not exclude the possibility that they dimerize in the presence of a template. The yeast two-hybrid result indicating that Dpb2p may interact with itself may suggest that Pol epsilon  could be present as a dimer in vivo (19). More experiments are required to determine which strands Pol epsilon  and Pol delta  do replicate.

We are now in a position to study the various activities of Pol epsilon  using a well defined source of the enzyme. These studies may help us understand the role of Pol epsilon  during DNA replication and DNA repair.

    ACKNOWLEDGEMENTS

We thank P. Burgers for comments on the manuscript and for providing the prs421-424GAL plasmids and pY116 strain.

    FOOTNOTES

* This work was supported by the Swedish Cancer Society, Swedish Research Council, Sven och Ebba-Christina Hagbergs Stiftelse, the Magnus Bergwalls stiftelse, and Kungliga vetenskapsakademiens stipendiefond.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.: 46-90-7866638; Fax: 46-90-7869795; E-mail: erik.johansson@medchem.umu.se.

Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M211818200

    ABBREVIATIONS

The abbreviations used are: Pol epsilon , DNA polymerase epsilon ; Pol delta , DNA polymerase delta ; s, sedimentation coefficient.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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