ABSTRACT
DNA polymerase
[delta]
is usually isolated as a heterodimer composed of a 125 kDa catalytic subunit
and a 50 kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA
synthesis. We have recently demonstrated that the catalytic subunit of human
DNA polymerase
[delta]
(p125) expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric calf thymus
DNA polymerase
[delta]
, is not responsive to stimulation by PCNA. To determine whether the lack of
response to PCNA of the recombinant catalytic subunit is due to the absence of
the small subunit or to differences in post-translational modification in insect cells
versus mammalian cells, we have co-expressed the two subunits of human DNA polymerase
[delta]
in insect cells. We have demonstrated that co-expression of the catalytic and small subunits of human DNA polymerase
[delta]
results in formation of a stable, fully functional heterodimer, that the
recombinant heterodimer, similar to native heterodimer, is markedly stimulated
(40- to 50-fold) by PCNA and that the increase in activity seen in the presence
of PCNA is the result of an increase in processivity. These data establish that
the 50 kDa subunit is essential for functional interaction of DNA polymerase
[delta]
with PCNA and for highly processive DNA synthesis.
DNA polymerase [delta] is an essential DNA polymerase that is required to replicate chromosomal DNA in eukaryotic cells (
1
) and may also function in repair (
2
). Although studies with the reconstituted
in vitro
SV40 DNA replication system have suggested that DNA polymerase [delta] is required for leading strand synthesis at the viral replication fork
as well as for completing Okazaki fragments initiated by the DNA polymerase [alpha]/primase complex (
3
), the role of DNA polymerase [delta] in cellular DNA replication is still unresolved (
4
)
By itself, DNA polymerase [delta] is a non-processive DNA polymerase and requires its accessory protein, proliferating cell nuclear
antigen (PCNA), for highly processive synthesis on primed single-stranded templates such as poly(dA)[middot]oligo(dT) (
5
,
6
). Results of kinetic and binding studies have shown that PCNA increases the activity and
processivity of DNA polymerase [delta] by stabilizing the interaction of the enzyme with the template/primer (
7
,
8
), possibly by forming a trimeric closed ring structure which encircles DNA and provides a sliding clamp for attachment of DNA polymerase [delta] (
9
).
When purified to homogeneity from fetal calf thymus, DNA polymerase [delta] was found to have a molecular weight of 173 000 and to be comprised of
subunits of 125 and 48 kDa, present in equimolar amounts (
7
,
10
). Both the polymerase and 3' -> 5' exonuclease activities are catalyzed by the 125 kDa subunit
(
11
,
12
); the function of the small subunit, however, is unknown. Although DNA
polymerase [delta] is usually isolated as a heterodimer from both higher and lower
eukaryotes, a single subunit form of the enzyme has been purified from
Drosophila melanogaster
(
13
) and both one- and two-subunit forms of the enzyme have been isolated from mouse cells (
14
). Interestingly, the catalytic subunit of DNA polymerase [delta] isolated from either
D.melanogaster
or mouse cells is unresponsive to PCNA, suggesting that the small subunit is
required for DNA polymerase [delta]-PCNA interaction.
We have recently expressed the recombinant catalytic subunit of human DNA
polymerase [delta] in baculovirus-infected insect cells and found that it was unresponsive to
stimulation by PCNA (
15
), similar to the catalytic subunits isolated from mouse cells and
D.melanogaster
embryos. Similar results were reported for the recombinant mouse catalytic
subunit expressed in
Escherichia coli
(
16
) and the recombinant
Schizosaccharomyces pombe
catalytic subunit expressed in insect cells (
17
). However, our results differed from those reported for the recombinant
Saccharomyces cerevisiae
catalytic subunit expressed in
E.coli
(
18
) and the recombinant human protein expressed in monkey cells (
19
). The DNA polymerase activity and processivity of the latter polypeptides were found to be stimulated by PCNA, although the extent of stimulation (2- to 5-fold) was less than that seen with the native enzymes. To determine
whether the lack of response to PCNA of the recombinant human catalytic subunit
expressed in insect cells is due to the absence of the small subunit or to
differences in post-translational modification in insect cells versus mammalian cells, we have
co-expressed the two subunits of human DNA polymerase [delta] in baculovirus-infected insect cells and compared the properties of the
recombinant heterodimer with those of the recombinant catalytic subunit and the
native heterodimer isolated from calf thymus.
Glutathione-Sepharose 4B, Source 15Q, Sephacryl S-300 and pGEX-4T-1 were obtained from Pharmacia Biotech. Protein A- Sepharose 4B Fast Flow was from Sigma.
Spodoptera frugiperda
cells (Sf9), wild-type baculovirus AcMNPV, recombinant [beta]-galactosidase virus, the transfer vector pBlueBacIII, cationic
liposomes and PCR primers for amplification of inserts in recombinant baculovirus were
obtained from Invitrogen Corp. Fetal bovine serum, gentamycin and pleuronic F-68 were from Gibco-BRL. Grace's insect medium was prepared by the Cell Culture
Facility, University of Miami. Calf thymus DNA polymerase [delta] was prepared as described by Downey and So (
20
). PCNA was prepared from fetal calf thymus as described in Tan
et al.
(
5
). Construction of recombinant baculovirus AcN-p125-14, which expresses the125 kDa subunit of human DNA polymerase [delta], and purification of the recombinant protein are described
in Zhou
et al.
(
15
). Rabbit polyclonal antisera to a peptide near the C-terminus of the catalytic subunit (R804) or to the recombinant small
subunit (R527) of human DNA polymerase [delta] were prepared as described (
8
).
The coding sequences for the small subunit of human DNA polymerase [delta] were excised from an M13mp19 clone, HDSF/HDSR-4 (
21
), by
Bam
HI digestion and the purified fragment was inserted into the
Bam
HI site of the pBlueBacIII transfer vector. The resulting construct, pBlueBac-p50, was co-transfected with linearized wild-type baculovirus AcMNPV DNA into Sf9 cells using cationic
liposome-mediated transfection according to the supplier's protocol and the
recombinant virus was plaque purified. The presence of an insert in a putative
recombinant virus was verified by PCR using primers complementary to the
polyhedrin gene. Recombinant virus AcN-p50-1 was amplified in Sf9 cells to a titer of 10
8
plaque forming units/ml.
Sf9 cells were grown to confluence in T-25 Falcon flasks at 27oC in Grace's medium supplemented with 10% fetal bovine serum, 50 [mu]g/ml gentamycin and 0.1% pleuronic F-68 and infected at a multiplicity of infection (MOI) of 10
with either wild-type virus AcMNPV, recombinant [beta]-galactosidase virus, recombinant viruses AcN-p125-14 or AcN-p50-1 or co-infected at a MOI of 5 with AcN-p125-14 and AcN-p50-1. Cells
were harvested at various times post-infection by centrifugation at 1200
g
, washed twice with serum-free Grace's medium and proteins analyzed by SDS-PAGE and immunoblotting.
Both AcN-p125-14 and AcN-p50-1 were used to co-infect 3 l of Sf9 cells (1.5 * 10
6
cells/ml) at a MOI of 5. After 60 h the cells were harvested, washed with serum-free Grace's medium, resuspended in 120 ml ice-cold hypotonic buffer (20 mM HEPES, pH 7.6, 10 mM sodium bisulfite,
1 mM dithiothreitol and 1 mM EDTA) containing a mixture of protease inhibitors (10 [mu]g/ml aprotinin, 5 [mu]g/ml leupeptin, 10 [mu]g/ml pepstatin A, 100 [mu]g/ml bacitracin, 250 [mu]g/ml soybean trypsin inhibitor, 0.4 mM phenylmethylsulfonyl fluoride and 10 mM benzamidine hydrochloride)
and subjected to Dounce homogenization. After centrifugation at 1200
g
for 15 min, the supernatant was again centrifuged at 100 000
g
for 75 min. The supernatant was adjusted to 100 mM NaCl, brought to 30%
saturation with ammonium sulfate and left on ice for 2 h. The precipitate was collected by centrifugation at 27 000
g
for 30 min, dissolved in 20 ml buffer A (20 mM HEPES, pH 7.6, 10% glycerol, 0.5
mM EDTA, 1 mM dithiothreitol and 0.4 mM phenylmethylsulfonyl fluoride)
supplemented with the mixture of protease inhibitors described above, adjusted
to a conductivity corresponding to 100 mM NaCl with buffer A and loaded on a
Source 15Q column (1.6 * 7.5 cm) equilibrated in buffer A containing 100 mM NaCl. The column was
washed with 3 bed vol of the same buffer and protein was eluted with a 130 ml
linear gradient of 100-800 mM NaCl in buffer A. Recombinant heterodimer, detected by PCNA-dependent DNA polymerase activity and by immunoblot analysis, eluted between 200 and 245 mM NaCl. The peak fractions were pooled and 1.5 ml were loaded on a
Sephacryl S-300 column (1.6 * 80 cm) equilibrated with buffer A containing 100 mM NaCl. The
column was eluted at a flow rate of 30 ml/h and 1 ml fractions were collected. The peak fractions
were pooled and concentrated on a small (1.0 * 1.3 cm) Source 15Q column.
Protein samples were separated by 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane. After incubation for 30 min with blocking buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl and 2% non-fat dry milk), the membrane was incubated overnight with
rabbit polyclonal antisera to p125 (R804) and p50 (R527) and washed three times
with blocking buffer. The membrane was then incubated with horseradish
peroxidase-conjugated goat anti-rabbit IgG for 1.5 h at room temperature, washed three times with
blocking buffer and twice with Tris-buffered saline (50 mM Tris-HCl, pH 7.4, 100 mM NaCl). Color was developed in 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution (0.4 mg/ml DAB, 0.009% H
2
O
2
and 100 mM Tris-HCl, pH 7.5).
To raise antibodies against the catalytic subunit of DNA polymerase [delta] for immunoprecipitation studies, a 1007 bp
Bam
HI-
Bgl
II fragment from pGEX-5X-1-p125 (
15
) was subcloned into the
Bam
HI site of pGEX-4T-1 to create a fusion protein between glutathione S-transferase (GST) and the N-terminal one third (amino acids 1-335) of the catalytic subunit of human DNA
polymerase [delta]. The fusion protein was expressed in
E.coli
strain DH5[alpha], purified on glutathione-Sepharose beads and used to immunize rabbits. Antiserum was
affinity purified by sequential passage through columns containing GST and GST
fusion protein respectively, cross-linked to glutathione-Sepharose beads according to Koff
et al.
(
22
). Bound antibody was eluted with 100 mM glycine, pH 2.4, and collected into a one tenth volume of 2 M Tris-HCl, pH 8.0. Affinity purified antibody to p125 (1 mg) was cross-linked to 1 ml protein A-Sepharose beads using dimethylpimelidate (
23
) to produce anti-p125 beads. Control beads were prepared by cross-linking pre-immune rabbit IgG to protein A-Sepharose.
Sf9 cells were grown to confluence in T-75 Falcon flasks, infected with wild-type virus, AcN-p125-14 or AcN-p50-1 or co-infected with AcN-p125-14 and AcN-p50-1, harvested
48 h post-infection and washed twice with serum-free Grace's medium. Cells were suspended in 1 ml lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, 250 [mu]g/ml soybean trypsin inhibitor and 0.4 mM
phenylmethylsulfonyl fluoride), incubated on ice for 30 min and cell extracts
recovered by centrifugation at 27 000
g
for 10 min. Aliquots (500 [mu]l) of cell extracts were mixed with 10 [mu]l anti-p125 beads or control beads and incubated at 4oC for 1 h with rocking. The beads were washed three times with lysis buffer, resuspended in 30 [mu]l SDS-PAGE buffer and analyzed by Western blotting.
DNA polymerase activity was assayed with poly(dA)[middot]oligo(dT) (10:1 nucleotide ratio) as template/primer in the presence and
absence of calf thymus PCNA as described (
15
).
Processivity was determined by measuring the sizes of products synthesized on a (dA)
4000
[middot](dT)
16
(80:1 nucleotide ratio) template/ primer under conditions where <1 residue of dTMP is incorporated per primer terminus, as described in Zhou
et al.
(
15
).
Recombinant baculoviruses that express the catalytic subunit (
15
) and the small subunit (this report) of human DNA polymerase [delta] were constructed and used to infect or co-infect Sf9 cells. Figure
1
shows an analysis by Western blotting (panel A) of extracts from uninfected and
baculovirus-infected Sf9 cells. Cells infected with AcN-p125-14 or co-infected with AcN-p125-14 and AcN-50-1 produced a protein of ~125 kDa that was
immunoblotted by antiserum to a peptide near the C-terminus of the catalytic subunit of mammalian DNA polymerase [delta]. Similarly, cells infected with AcN-p50-1 or co-infected with AcN-p50-1 and AcN-p125-14 produced a protein of ~50 kDa that was
immunoblotted by antiserum to recombinant human p50. Extracts from uninfected
insect cells or cells infected with wild-type virus or recombinant [beta]-galactosidase virus did not contain any proteins that cross-reacted with the antibodies to either the catalytic or small
subunit of mammalian DNA polymerase [delta].
Affinity purified antibody to the catalytic subunit of human DNA polymerase [delta] (anti-p125) was found to immunoprecipitate both subunits of DNA
polymerase [delta] from cell extracts. As shown in Figure
2
, treatment of HeLa cell extracts with anti-p125 beads, but not with control beads, resulted in co-immunoprecipitation of the 125 and 50 kDa subunits of human DNA
polymerase [delta], as detected by Western blotting. Similar treatment of infected insect cell extracts showed that the anti-p125 antibody immunoprecipitated p125 from cells infected with AcN-p125-14 or co-infected with AcN-p125-14 and AcN-p50-1, but not from cells
infected with wild-type virus or with AcN-p50-1. More importantly, p50 was immunoprecipitated with antibody
to p125 from cells co-infected with AcN-p125-14 and AcN-p50-1, but not from cell infected with AcN-p50-1 alone. These results suggest that co-expression of the recombinant
catalytic and small subunits of human DNA polymerase [delta] in insect cells results in the formation of a stable heterodimer.
Similar results were obtained with affinity purified antibody to human p50, i.e. anti-p50 antibody co-immunoprecipitated p125 from cells co-infected with AcN-p125-14 and AcN-p50-1, but not from cells infected with
AcN-p125-14 alone (data not shown).
Recombinant DNA polymerase [delta] heterodimer was partially purified from Sf9 cells co-infected with AcN-p125-14 and AcN-p50-1 using ammonium sulfate precipitation and
chromatography on Source 15Q and Sephacryl S-300, as described in Materials and Methods. Figure
3
A shows the elution profile of the recombinant heterodimer on Sephacryl S-300, as detected by PCNA-dependent DNA polymerase activity with poly(dA)[middot]oligo(dT) as template/primer. After concentration of the active
fractions on a small Source 15Q column, the polypeptides present were resolved
by SDS-PAGE and analyzed by silver staining (Fig.
3
B) and Western blotting (Fig.
3
C). The results showed that the fractions with PCNA-dependent DNA polymerase activity contained both the 125 and 50 kDa
recombinant polypeptides in approximately equimolar ratio.
The effect of increasing concentrations of PCNA on the activity of the
recombinant heterodimer, the recombinant catalytic subunit and native calf
thymus DNA polymerase [delta] is shown in Figure
4
A. As previously reported (
15
), recombinant p125 expressed in insect cells was not responsive to PCNA.
However, both the recombinant heterodimer and native DNA polymerase [delta] were stimulated by increasing concentrations of PCNA, being maximally stimulated (45- to 50-fold) at a concentration of ~800 ng/ml. That the stimulation of enzymatic activity is
due to increased processivity is shown in Figure
4
B. In the absence of PCNA all three proteins were essentially distributive, incorporating <30 nt/enzyme binding event. In the presence of PCNA, the heterodimeric forms of
DNA polymerase [delta], either recombinant or native (Fig.
3
, lanes 4 and 6), were highly processive, whereas the catalytic subunit (Fig.
3
, lane 5) was non-processive. These reactions were carried out under conditions where the
enzymes incorporated <1 residue dTMP/primer terminus and thus the sizes of the products represent the
results of single binding events. The trace of short product in Figure
4
B (lane 4) likely indicates the presence of a small amount of free p125 in the
recombinant heterodimer preparation. This may account for the slightly lower
stimulation of the recombinant heterodimer by PCNA (45-fold) as compared with the native heterodimer (50-fold) shown in Figure
4
A.
We recently reported that the recombinant catalytic subunit of human DNA
polymerase [delta] expressed in insect cells is not responsive to stimulation by PCNA and
suggested that the small subunit may be required for functional interaction of
the enzyme with PCNA (
15
). These results were consistent with reports on the lack of response to PCNA of
isolated native catalytic subunits from mouse cells and
D.melanogaster
embryos (
13
,
14
) and with reports of recombinant mouse protein expressed in
E.coli
(
16
) and recombinant
S.pombe
protein expressed in insect cells (
17
). However, they differed from two reports in which recombinant catalytic
subunits were found to be responsive to PCNA, i.e.
S.cerevisiae
protein expressed in
E.coli
(
18
) and human protein expressed in monkey cells (
19
).
In the present studies we have demonstrated that co-infection of insect cells with recombinant baculoviruses expressing the
catalytic (125 kDa) and small (50 kDa) subunits of human DNA polymerase [delta] results in the formation of a heterodimer, as evidenced by co-elution of the 125 and 50 kDa polypeptides on gel filtration, as
well as by co-immunoprecipitation of the 125 and 50 kDa polypeptides from co-infected insect cell extracts by affinity purified antibody to p125. The recombinant heterodimeric DNA polymerase [delta], similar to the native heterodimer isolated from calf
thymus, was found to be markedly stimulated by PCNA (40- to 50-fold) and the increase in activity seen in the presence of PCNA was
found to be the result of increased processivity. Since the recombinant
catalytic subunit alone was unresponsive to PCNA, this establishes that the
small subunit is essential for functional interaction of DNA polymerase [delta] with PCNA and for highly processive DNA synthesis. The reasons why, in
some studies, recombinant catalytic subunit alone was found to be stimulated by
PCNA are not obvious. In the case of human protein expressed in monkey cells (
19
), it is possible that the recombinant human protein formed a heterodimer with
monkey cell p50, accounting for the observed stimulation by PCNA. It is more
difficult to explain the effect of PCNA on the yeast catalytic subunit
expressed in
E.coli
(
18
). However, this protein was denatured and renatured and it is possible that the
refolded protein had undergone a conformational change which enabled it to bind
to and be stimulated by PCNA. At present it is not clear whether PCNA directly
interacts with p50 or whether the binding of p50 to p125 leads to an increased
interaction of the catalytic subunit with PCNA, possibly as a result of a
conformational change.
It has recently been reported that
cdc1
+
of
S.pombe
encodes a 51 kDa protein (
27
) with significant sequence similarity to the small subunit of human and bovine
DNA polymerase [delta] (
21
) as well as to the Hys2 protein of
S.cerevisiae
(
28
).
cdc1
+
was shown to interact genetically with
pol3
+
, which encodes the catalytic subunit of
S.pombe
DNA polymerase [delta], and physical interaction of the Cdc1 and Pol3 proteins was demonstrated
in vitro
, suggesting that Cdc1 is the small subunit of
S.pombe
DNA polymerase [delta].
cdc1
+
was found to be essential for cell cycle progression but not for bulk DNA
replication.
cdc1
mutants were found to have an extended S phase and to be supersensitive to
hydroxyurea and methylmethane sulfonate, consistent with a role in DNA
replication and/or repair. Interestingly,
cdc1
+
also interacted genetically with
cdc27
+
and Cdc1 interacted physically with Cdc27, a protein of unknown function that
is also required for cell cycle progression. Clearly, further studies on the
protein- protein interactions of the small subunit of DNA polymerase [delta] will be necessary to clarify its role in cellular DNA replication
and repair.
The subunit structure of DNA polymerase [delta] is very similar to that of HSV-1 DNA polymerase, i.e. both are heterodimers with subunits of
similar sizes (
7
,
10
,
24
). The catalytic subunits, but not the small subunits, of both polymerases share
significant homology (
21
,
25
). It is worth mentioning that the small subunit of HSV-1 DNA polymerase, a product of the
UL42
gene, functions as an accessory factor to increase the processivity of the
catalytic subunit, analogous to the effect of PCNA on DNA polymerase [delta] (
26
). DNA polymerase [delta], on the other hand, requires PCNA for processive DNA synthesis, but its
effect is dependent on association of the catalytic subunit with the small
subunit of the enzyme. The finding that the processivity of DNA polymerase [delta] in the presence of PCNA may be modulated by the small subunit suggests
the possibility that the small subunit may also modulate the interaction of DNA
polymerase [delta] with other replication proteins.
This work was supported by grant DK26206 from the National Institutes of Health.
The advice of Dr James Crute in designing a purification protocol is gratefully
acknowledged.
*To whom correspondence should be addressed at: Department of Medicine,
University of Miami School of Medicine, PO Box 016960 (R99), Miami, FL 33101,
USA. Tel: +1 305 243 6304; Fax: +1 305 243 4519; Email:
aso@mednet.med.miami.edu
REFERENCES
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