The Quaternary Structure of DNA Polymerase
from Saccharomyces cerevisiae*
Olga
Chilkova
,
Bengt-Harald
Jonsson§, and
Erik
Johansson
¶
From the
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 |
DNA polymerase
(Pol
) from
Saccharomyces cerevisiae consists of four subunits (Pol2,
Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication.
Biochemical characterizations of Pol
have been cumbersome due to
protease sensitivity and the limited amounts of Pol
in cells. We
have developed a protocol for overexpression and purification of Pol
from S. cerevisiae. The native four-subunit complex was
purified to homogeneity by conventional chromatography. Pol
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
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
. Thus, both DNA polymerase
and Pol
are purified as
monomeric complexes, in agreement with accumulating evidence that Pol
and Pol
are located on opposite strands of the eukaryotic
replication fork.
 |
INTRODUCTION |
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
is required for priming the DNA since it
is associated with primase activity. Only DNA polymerase
(Pol
)1 was required to
replicate both leading and lagging strand, leaving another essential
eukaryotic DNA polymerase, DNA polymerase
(Pol
), without a
specific function. However, there are several lines of evidence for the
presence of Pol
at the eukaryotic replication fork. Evidence for a
physical interaction came from cross-linking experiments, which showed
that Pol
is located at or near the replication fork (2). Genetic
experiments in Saccharomyces cerevisiae have shown that Pol
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
or Pol
(3). It has also been shown
that loss of the exonuclease site in either Pol
or Pol
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
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
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
(9). Although Pol
is normally present at the fork, in the absence of the catalytic
domain, another DNA polymerase, presumably Pol
, can substitute its function.
Pol
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
with two catalytic assemblies, and a
model where a dimeric Pol
would replicate both leading and lagging
strand was proposed (13, 17). However, it has now been demonstrated
that Pol
actually is a monomer in vitro and that the
previous estimates of the molecular weight of Pol
were misleading
due to the elongated shape of the enzyme (18). The initial analysis of
the four-subunit Pol
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
has also been
proposed to form a dimer based on yeast two-hybrid assays and gel
filtration experiments using Pol
overproduced in a baculovirus
system (9, 19). In light of these contradictory results we wished to
clarify whether Pol
exists as a monomer or a dimer when
overproduced in yeast. Therefore, we developed an overexpression system
that allowed us to purify native Pol
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 |
Strains--
A protease-deficient yeast strain was used for the
overexpression of Pol
. PY116 (ura 3-52, trp 1
, 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
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
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
-galactosidase (Ve = 14.45 ml), and
thyroglobulin (Ve = 12.65 ml). 0.2-20 µg of
Pol
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
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
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
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
were analyzed by using the
self-association model in the Beckman software package.
 |
RESULTS |
We chose to overexpress Pol
in S. cerevisiae for
several reasons. Earlier attempts to overexpress Pol
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
in S. cerevisiae without any tags added, similar to
earlier overexpression studies of Pol
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
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 was purified to homogeneity. 1 µg of Pol 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
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 was 11.88 ± 0.84 S. The sedimentation coefficient of Pol
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 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), -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 eluted at 13.8 ml, and the Stokes radius was
estimated to be 74.5 ± 2.4 Å. B, fractions containing
Pol 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
. 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
was determined to be 11.9 S
(Fig. 2). Using an antibody to Dpb4p we measured the sedimentation coefficient of Pol
when a crude extract from a wild-type strain was
separated over the same glycerol gradient. The sedimentation of Pol
in a crude extract was similar to that of the purified Pol
(data
not shown).
Pol
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
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
is 1.56, suggesting a moderately elongated shape of the
enzyme.
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Table I
Molecular Mass of DNA polymerase
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.
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|
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
ranging from 0.14 to 2.1 µM were analyzed at four different speeds until
equilibrium was reached. The molecular mass of Pol
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
was present as a monomer in the solution
under all conditions investigated (Fig.
5).

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Fig. 5.
Sedimentation equilibrium
experiment. Pol 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.
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 |
DISCUSSION |
We have presented a method to overexpress and purify Pol
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
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
suggested that Pol
was a dimeric enzyme (17, 27). A model was proposed in which Pol
formed a dimeric unit with two catalytic domains. In this model Pol
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
is a monomer with one catalytic site (18, 28).
Meanwhile, Pol
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
. 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
was based upon gel
filtration experiments (19). The calculated molecular mass of the
protein was based upon the assumption that Pol
was globular. We
have measured both the sedimentation coefficient and the Stokes radius
of Pol
, 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
was found to exist as a
monomer in solution (Fig. 5 and Table I). Thus, our results clearly
support a model of Pol
as a monomer of a (Pol2:Dpb2:Dpb3:Dpb4)
heterotetrameric complex.
The role of Pol
and Pol
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
and
Pol
agrees with our observations that both Pol
and Pol
are
monomeric assemblies. Pol
has been suggested to replicate the
lagging strand based on a genetic interaction with the flap endonuclease (RAD27) (31). Pol
has been suggested to replicate the
leading strand, based on experiments in cell-free Xenopus extracts (6). However, the fact that both Pol
and Pol
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
could be present as a dimer in vivo (19). More
experiments are required to determine which strands Pol
and Pol
do replicate.
We are now in a position to study the various activities of Pol
using a well defined source of the enzyme. These studies may help us
understand the role of Pol
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
, DNA
polymerase
;
Pol
, DNA polymerase
;
s, sedimentation
coefficient.
 |
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