(Received for publication, October 22, 1996, and in revised form, February 4, 1997)
From the Departments of Biochemistry and Molecular Biology and Medicine, University of Miami School of Medicine, Miami, Florida 33101
DNA polymerase is a heterodimer consisting of
a catalytic subunit of 125 kDa and a small subunit of 50 kDa (p50). We
have overexpressed p50 in Escherichia coli and have
characterized the recombinant protein. p50 was readily overexpressed
using the pET vector as an insoluble protein. A procedure was developed
for its purification and renaturation. Examination of the
physicochemical properties of renatured p50 showed that it is a
monomeric protein with an apparent molecular weight of 60,000, a Stokes
radius of 34 Å, and a sedimentation coefficient of 4.1 S. Its physical
properties were indistinguishable from p50 expressed as a soluble
protein using the pTACTAC vector. Examination of the effects of
recombinant p50 on the activity of DNA polymerase
showed that p50
is able to slightly stimulate (about 2-fold) the activity of the
recombinant 125-kDa catalytic subunit using poly(dA)·oligo(dT) as a
template in the absence of proliferating cell nuclear antigen. In the
presence of proliferating cell nulear antigen, activity is stimulated
about 5-fold. Seven stable hybridoma cell lines were established that produced monoclonal antibodies against p50. One of these antibodies (13D5) inhibited the activity of calf thymus DNA polymerase
. This
antibody, when coupled to a solid support, also was found to provide a
method for the immunoafffinity purification of recombinant p50 and of
DNA polymerase
from calf thymus or HeLa extracts. Immunoprecipitation and enzyme-linked immunosorbent assays also confirmed that p50 interacts with the catalytic subunit of DNA polymerase
.
DNA polymerase (pol)1 is involved in both DNA replication (1) and DNA repair (2) in mammalian
cells. Its activity is stimulated by PCNA, a DNA sliding clamp which
provides the processivity required for its role in DNA replication (3,
4). In addition to PCNA, a number of other accessory proteins are
required for the assembly of a functional replication complex at the
leading strand of the replication fork. These include the multi-subunit replication factor C (RF-C) complex and replication protein A (RP-A), a
single-stranded DNA-binding protein (5). pol
isolated from calf
thymus (6) and human placenta (7) is a heterodimer of 125- and 50-kDa
polypeptides. The catalytic activity had been clearly demonstrated to
be associated with the 125-kDa subunit (7), and preparations of
mammalian pol
have been reported that contain only the active
125-kDa subunit (8, 9). Goulian et al. (9) isolated two
preparations of mouse DNA polymerase
, one of which consisted of a
single polypeptide of 123-125 kDa and was unresponsive to PCNA
stimulation. It was suggested that the 50-kDa polypeptide is required
for the stimulation of DNA polymerase
by PCNA (9). However, yeast
pol
catalytic subunit overexpressed in Escherichia coli
was stimulated 2.5- to 3-fold (10). Human pol
catalytic subunit
expressed in the vaccinia virus system also showed a slight increase in
activity in the presence of PCNA (11). This increase in activity is
significantly lower than the 34-fold stimulation of the native human
DNA polymerase
core reported previously in our laboratory (7). In
this study we report the expression of human p50 in E. coli
and its biochemical characterization.
The cDNA of p50 was isolated by PCR
cloning using primers based on the reported sequence of human p50 (12).
The coding sequence was inserted into both the pET21a (Novagen) and
pTACTAC vectors (13, 14). PCR amplification was used for the insertion
of the coding sequence of human p50 between the NdeI and
HindIII sites of the vectors. The primer
5-CAGGAGTGTGCATATGTTTTCTGAGC-3
was used for
the 5
end of the coding sequence with an engineered NdeI
site (underlined residues) at the initiating methionine codon (bold
residues). The antisense primer at the 3
end
(5
-CCACAAGCTTGAGTCAGGGGCC-3
) had a
HindIII site (underlined residues) after the termination codon (bold residues). The primers were phosphorylated with T4 polynucleotide kinase before use. The PCR conditions used were 95 °C
for 1.5 min, 45 °C for 2 min, and 72 °C for 3 min, for 30 cycles.
The product was a single band of about 1.4 kilobases, which was
subsequently purified on a Centricon 100 column followed by
phenol/chloroform extraction. After digestion with NdeI and HindIII, the PCR product was ligated into the pET vector
that had been previously digested with NdeI and
HindIII and purified by agarose gel electrophoresis. The
construct was then used to transform E. coli
DH5
-competent cells. The correctness of the inserted DNA was
confirmed by DNA sequencing. A single colony from E. coli
DH5
cells harboring the pET21a/p50 construct was used to inoculate a
5-ml culture in Terrific medium (1.2% tryptone, 2.4% yeast extract,
0.4% glycerol, 0.017 M KH2PO4,
0.072 M K2HPO4, 50 µg/ml
ampicillin). After overnight growth at 37 °C, the 5-ml culture was
used to inoculate a 2-liter culture in LB medium and grown at 37 °C
for 4-6 h until the absorbance reached 0.6 at 600 nm.
Isopropyl-
-thiogalactoside was then added to a concentration of 0.5 mM, and the culture was grown for an additional 6 h at 37 °C. A similar procedure was used for the insertion of the coding sequence for p50 into the pTACTAC vector (14, 15).
The cells from 1 liter of culture harboring the p50-pET21a plasmid were harvested by centrifugation at 3,000 × g, 4 °C for 30 min, and frozen overnight. The frozen cells were suspended in lysis buffer (25 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 1 mM EDTA, 0.1 mM EGTA, 10 mM benzamidine HCl, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol (DTT), 0.1% Triton X-100, 0.1 M NaCl) and disrupted using a French press. The suspension was centrifuged at 27,000 × g at 4 °C for 30 min. The pellet was resuspended in 50 ml of lysis buffer containing 0.5 M NaCl, and sonicated. The suspension was centrifuged at 27,000 × g in 4 °C for 30 min. The resulting pellet was then sonicated in 10 ml of 200 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1 mM EDTA, 1 mM DTT, and 6 M urea to solubilize the pellet and was centrifuged at 12,000 × g at 4 °C for 30 min. The resulting supernatant (5 ml, 60 mg of protein) was loaded onto a Sephacryl S300HR column (2.5 × 72 cm) equilibrated with 20 mM Tris-HCl, pH 7.8, 1 mM EDTA, 0.1 mM EGTA, 0.5 M NaCl, and 2 M urea. Fractions (4 ml) were collected, and the elution of p50 was monitored by SDS-PAGE and protein staining. Renaturation was performed by HPLC chromatography in a buffer containing 0.25 M NaCl instead of 2 M urea. A portion of the peak fraction (0.45 ml) was injected onto a Waters Protein Pak 300SW column equilibrated with 20 mM Tris-HCl, pH 7.8, 5% glycerol, 1 mM EDTA, 0.1 mM EGTA, 0.25 mM NaCl, and 1 mM DTT. Fractions of 0.5 ml were collected and analyzed for p50 by immunoblotting using a monoclonal antibody.
Generation of Monoclonal Antibodies against Recombinant p50 and Preparation of a p50 Immunoaffinity ColumnA panel of murine
hybridoma cell lines that produced antibodies against p50 was developed
by immunizing three female BALB/c mice with recombinant p50 purified to
the Sephacryl S300HR step. The mice were injected intraperitoneally
with 100 µg of p50 emulsified with complete Freund's adjuvant. Two
weeks later the mice were given a booster injection of another 100 µg
of p50 with incomplete Freund's adjuvant. The booster was repeated at
monthly intervals. One of the mice exhibited a high titer after two
booster injections and was sacrificed 3 days after the final booster
injection. The procedure for fusion of the spleen cells to produce
hybridomas was as described by Lee et al. (16). Western
blotting of hybridoma cell supernatants was used to screen for
positives. SDS-PAGE and Western blotting were performed as described
previously (17). The antigen used for screening was the native pol enzyme isolated by immunoaffinity chromatography from the calf thymus
(17). Positive hybridoma cultures were isolated by limiting dilution three times. Seven stable hybridomas were established. Antibodies were
isolated by purification on protein A-Sepharose columns.
Purified antibody 13D5 was coupled onto AvidChrom hydrazide (Sigma) as described by Jiang et al. (17). A cell lysate from 6 liters of an induced culture of E. coli harboring the p50-pTACTAC plasmid (6 g of protein in 300 ml) was precipitated by the addition of ammonium sulfate to 50% saturation. The pellet was then resuspended in TGEE buffer (40 mM Tris-HCl, pH 7.8, 10% glycerol, 1 mM EDTA, 0.1 mM EGTA). The solution (1.7 g of protein in 200 ml) was then applied to a 5-ml 13D5 immunoaffinity column. The column was washed with 50 ml of TGEE buffer containing containing 0.4 M NaCl. The p50 was eluted with 0.1 M triethylamine, pH 11.5, that was immediately neutralized with 1 M Tris-HCl, pH 6.8. Peak fractions containing p50 were pooled (1.2 mg of protein in 19 ml) and diluted with TGEE to 150 ml. The partially purified protein was again subjected to immunoaffinity chromatography. The final p50 eluate was pooled and dialyzed against three changes of TGEE buffer containing 1 mM DTT, and concentrated by centrifugation in Centricon membrane filters to a final volume of 1.5 ml containing 0.26 mg of protein.
Immunoprecipitation of PolCells from a 1-liter culture
of HeLa cells were sonicated in 10 ml of lysis buffer (40 mM Tris-HCl, pH 7.8, 0.25 M sucrose, 0.1 M NaCl, 1.0 mM EDTA, 0.1 mM EGTA,
10 mM benzamidine HCl, 1 mM
phenylmethylsulfonyl fluoride, and 0.1% Nonidet P-40). The extract was
centrifuged at 20,000 × g and at 4 °C for 30 min to remove cell debris. The supernatant was then batch-adsorbed onto 4-ml
of protein A-agarose equilibrated with TGEEN buffer (40 mM Tris-HCl, pH 7.8, 10% glycerol, 1 mM EDTA, 0.1 mM EGTA, 0.15 M NaCl, 0.1% Nonidet P-40) and
rotated for 16 h at 4 °C to remove the nonspecific binding
proteins. The protein A-agarose-treated HeLa cell extract (400 µg of
protein) was incubated with 20 µg of p50 monoclonal antibody at
0 °C for 60 min in a total volume of 200 µl of TGEEN buffer. Fifty
µl of a 25% protein A-agarose suspension was added, and the
suspension was rotated at 4 °C for 12 h. The affinity beads
were then collected by a brief centrifugation for 15 s. The beads
were washed 4 times with TGEEN buffer (1 ml each wash). Bound proteins
were released from beads by heating for 2 min at 100 °C in 50 µl
of SDS-PAGE sample buffer (60 mM Tris-HCl, pH 6.8, 10%
glycerol, 5% mercaptoethanol, and 2% SDS) and were used for Western
blot analysis using 78 F5 pol monoclonal antibody.
DNA polymerase activity was
assayed using poly(dA)·oligo(dT) in the presence or absence of PCNA
as described by Lee et al. (7). 3-5
-Exonuclease and
5
-3
-exonuclease assays were performed as described by Lee et
al. (6).
ELISA assays were performed according to Dornreiter et al. (18) and Lee et al. (19) with minor modifications. ELISA plates were coated with 300 ng/well p125 protein in 50 µl of phosphate-buffered saline buffer, pH 7.5, for 1 h at room temperature. In a parallel experiment, ELISA plates were coated with BSA as a negative control. After washing twice with phosphate-buffered saline buffer, the ELISA wells were blocked with 5% (w/v) BSA in phosphate-buffered saline buffer overnight, and the wells were washed twice with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40 (Buffer A). Increasing amounts of p50 in 50 µl of buffer A were added, and the plates were shaken gently for 1 h at 4 °C. The unbound proteins were removed by washing six times with buffer A. For detection of p50, 1 µg of monoclonal antibody (17D2) in 50 µl of buffer A containing 3% (w/v) BSA was incubated with the plates for 1 h at room temperature. The plates were washed six times with buffer A followed by a 40-min incubation with a horseradish peroxidase-conjugated anti-mouse IgG (1:1000, Amersham) in Buffer A containing 3% BSA. The reaction was developed with tetramethylbenzidine base (TMB-ELISA, Pierce) for 5-10 min and quantitated at 450 nm with a microtiter plate reader (Bio-Rad). In a reciprocal experiment, 300 ng of p50 and BSA were immobilized on ELISA plates and blocked with 5% (w/v) BSA. After washing, increasing amounts of p125 were added to the ELISA plates. Monoclonal antibody against the p125 (1 µg, 7B7) was used for the detection of p125 as described above.
The cDNA for the
p50 subunit was cloned by PCR amplification from a human liver gt11
cDNA library using primers based on the reported cDNA sequence
of the human p50 subunit (12). The cDNA clone consisted of a
1519-nucleotide sequence with an open reading frame of 469 amino acid
residues encoding the identical amino acid sequence as reported by
Zhang et al. (12). The p50 protein was readily overexpressed
in E. coli when the pET21a vector was used (see
"Experimental Procedures"). As has been commonly observed for a
number of overexpressed proteins, the recombinant p50 protein was
expressed as an insoluble form in inclusion bodies. The p50 was
extracted from the inclusion bodies using 6 M urea, and
approximately 60 mg of protein could be obtained from a 1-liter cell
culture. The major protein component of the extract was p50, as
determined by SDS-PAGE and immunoblotting (not shown). The solubilized
protein was then purified by gel filtration in the presence of 2 M urea on a Sephacryl S300HR column. During this process
the p50 protein remained in solution in 2 M urea. A portion of the peak fraction was then subjected to HPLC gel permeation chromatography in the presence of 0.25 M NaCl to remove the
urea. This process was found to allow the apparent renaturation of the p50, as it now behaved as a soluble protein as shown by the HPLC elution (Fig. 1A). The presence of the p50
protein was determined by immunoblotting using 13D5, a p50 monoclonal
antibody (see "Experimental Procedures"). It is seen that most of
the protein is in the included fraction of the column with a small
amount eluting in the void volume.
Effect of p50 on Pol
To test whether this
solubilized p50 material had any effect on the p125 catalytic subunit
of pol , samples of the fractions were added to a standard assay for
pol
activity in the presence of PCNA using purified recombinant
p125 obtained by expression in Sf9 cells. The results show that the
renatured p50 was able to stimulate pol
activity by roughly
2.5-fold and that this stimulatory effect coincided with the peak of
p50 protein (Fig. 1B). To further establish that this was a
functional effect of p50 on pol
activity, samples of the peak
fraction from the HPLC run (fraction 30, Fig. 1A) were used
to determine whether this stimulation was
concentration-dependent. The results (Fig. 1C) showed that a response could be seen in the range of 0.1-1 µg of
p50/assay. The renatured recombinant p50 did not have any effect on the
3
-5
-exonuclease activity of pol
(not shown) and did not possess
DNA binding ability as it did not bind to double- or single-stranded
DNA cellulose columns. Analysis of the renatured p50 for 3
-5
- and
5
-3
-exonuclease activities showed that these were absent.
The p50
obtained after HPLC gel permeation chromatography was characterized to
determine whether it behaved as a globular protein. The apparent
molecular mass was determined to be ~60 kDa by HPLC gel permeation
chromatography. A Stokes radius of 34 Å was obtained when the data
were plotted against the Stokes radii of the protein standards (Fig.
2A). This can be compared with the Stokes
radius of bovine serum albumin, a globular protein, which is 36 Å. The
sedimentation coefficient of p50 was found to be 4.1 S by glycerol
gradient ultracentrifugation (Fig. 2B). These data confirm
that the renatured p50 behaves as a monomeric protein and has
physicochemical properties consistent with it having refolded into a
native globular state. The isoelectric point of p50 was determined in
acrylamide gels to be 5.3 (not shown).
Expression of p50 as a Soluble Protein
Because of concerns
that the renatured p50 might not represent a native folded protein, we
expressed p50 in the pTACTAC vector, which has been shown to allow
expression of several proteins in a soluble form when induced at room
temperature rather than at 37 °C (13-15). The results showed that
this vector allowed the expression of p50 in a soluble state, although
the levels were much lower than in the pET21a vector. The soluble p50
expressed using the pTACTAC vector therefore required a much more
extensive purification than the material from the pET vector, where the inclusion bodies were largely composed of p50 itself. This problem was
solved by the use of an immunoaffinity column using a monoclonal antibody against p50, 13D5 (see below). The cell lysate from 6 liters
of culture was partially purified by 50% ammonium sulfate precipitation and passed through a monoclonal anti-p50 column as
described under "Experimental Procedures." The yield of soluble protein was 0.26 mg starting from 6 liters of cells and provided a near
homogeneous preparation of p50 (Fig. 3). The p50
expressed using the pET21a and pTACTAC vectors and that of p50 in the
calf thymus pol core were all immunoblotted by monoclonal antibody 13D5, which provided additional confirmation of the identity of the
recombinant proteins (Fig. 4).
The abilities of the p50 expressed in the pTACTAC vector as a soluble
protein and of the renatured p50 isolated after expression in the pET
vector to affect pol activity were compared (Fig. 5). The two proteins were essentially indistinguishable
in their behavior. These results show that the procedure used for
renaturation of the insoluble p50 results in a protein that is
functionally similar to the protein expressed in a soluble state. The
recombinant pol
p125 activity was stimulated about 2-fold by the
addition of p50. The combined effect of p50 and PCNA provided about a
5-fold stimulation of the free p125 activity. Because of concerns that the small effects of the p50 on the pol
activity were nonspecific, recombinant p50 was heat-denatured. This heat-treated p50 and BSA were
used as negative controls (Fig. 6). Examination of their effects as a function of added protein and time showed that the heat-denatured protein had no significant effects when compared with
untreated p50 and that the increase in activity is not due to a
stabilization of the p125 subunit by the addition of protein to the
solution (Fig. 6).
Interaction of the Pol
The physical
interaction of p50 with pol p125 was demonstrated by an ELISA
procedure (Fig. 7). When the plates were coated with p50
and the response was tested by the addition of p125 and a p125
antibody, a concentration dependence of absorbance could be detected.
Similar results were obtained from the reciprocal experiment, in which
p125 was bound to the ELISA plates and probed with p50 and a p50
antibody (Fig. 7). These results establish that recombinant p50 does
interact with p125.
Generation of Monoclonal Antibodies to p50
Monoclonal
antibodies to p50 were generated (see "Experimental Procedures").
The p50 protein obtained by extraction from inclusion bodies and
purified by gel filtration in 2 M urea was used as the
antigen, and hybridomas were screened by the ability of their cell
supernatants to immunoblot the p50 subunit of calf thymus pol isolated by immunoaffinity chromatography (17). Seven stable hybridoma
cell lines were selected and established, all of which produced
monoclonal antibodies that strongly immunoblotted p50. The monoclonal
antibodies were typed as to their immunoglobulin class by the
Ouchterlony double-diffusion technique (20). Three antibodies were of
the IgG1 class (12B6, 16A8, and 17D2), two were of the IgG2b class
(13D3 and 13D5), one was an IgG2a (13D8), and one was IgG3 (13E6). All
of the monoclonal antibodies were able to co-immunoprecipitate the p125
subunit of pol
from HeLa cell extracts. This is illustrated in Fig.
8, in which HeLa extracts were immunoprecipitated with
13D5 (p50) monoclonal antibody and then immunoblotted with a pol
monoclonal antibody (38B5). The effects of these monoclonal antibodies
on the activity of pol
core purified from calf thymus by
immunoaffinity chromatography was examined. Only one antibody, 13D5,
was found to be inhibitory (Fig. 9). This inhibitory
antibody did not inhibit the 3
-5
-exonuclease activity of pol
isolated from the calf thymus (not shown). None of the antibodies
inhibited the polymerase activity of the recombinant pol
p125
overexpressed in the Sf9 cells.
Monoclonal antibody 13D5 was found to be suitable for the preparation
of an immunoaffinity support for the isolation of p50. The purified
protein was coupled to AvidChrom hydrazide as described by Jiang
et al. (17). The column was useful for the isolation of
recombinant p50 expressed as a soluble protein in E. coli
(see above). This column also allowed the isolation of pol from
calf thymus extracts (Fig. 10).
Mammalian DNA polymerase is a heterodimer consisting of a
catalytic subunit of 125 kDa and a small subunit of 50 kDa (6, 7, 17).
Reports of pol
preparations consisting of the free pol
catalytic subunits from rabbit reticulocytes (8), mouse (9), and
Drosophila (21) supported the possibility that the presence
of p50 is required for PCNA sensitivity of the catalytic subunit. The
pol
catalytic subunits of both yeast (10) and mammalian p125 (11)
have been overexpressed and exhibit only a slight response to PCNA.
Highly purified human placental pol
preparations purified by
conventional means are stimulated at least 10-20-fold and may reach
above 30-fold depending on the assay conditions (7, 11). In the studies
reported here, we find that the p125 subunit of pol
overexpressed
in baculovirus is stimulated by PCNA less than 2-fold. Thus, our
findings are consistent with other reports on the behavior of the
isolated p125 subunit in regard to either a lack of, or a low level of, sensitivity to PCNA. However, in the presence of p50 pol
activity is stimulated about 5-fold by PCNA suggesting that the 50-kDa subunit
is required for the stimulation of DNA polymerase
by PCNA.
The effects of p50 on recombinant p125 are small, and the failure to
induce a strong PCNA sensitivity suggests that the reconstitution of a
native enzyme was not achieved. Nevertheless, there clearly is an
interaction between the two recombinant proteins based on the
functional effects of p50 on p125, and the demonstration of a
protein-protein interaction via the ELISA system. Several reasons for
the failure to observe a high level of PCNA responsiveness are
possible. First, either one of the recombinant proteins could be
improperly folded or lacking in a required post-translational modification. Second, the proper folding of both proteins may require
their co-expression or may require the action of specific chaperone
proteins. Third, the formation of the core enzyme may require the
presence of other subunits. With regard to the first issue, we have
characterized recombinant p50 to confirm that it behaves as a globular
protein based on its physicochemical properties and by comparison of
its functional effects on pol p125 with that of a soluble form of
p50 isolated by expression in the pTACTAC vector. At the present time
it is not known if p50 is subject to post-translational modification,
although we have preliminary evidence that p50 is phosphorylated
in vitro.2 With regard to the
second issue, one possible avenue would be to co-express the two
proteins in the baculovirus system. It should be noted that our
attempts at separation of the two subunits from the native enzyme were
unsuccessful and could only be achieved after denaturation of the
complex, which showed that their interaction was extremely strong.
Alternatively, the low level of PCNA responsiveness could be due to a
situation where only a small fraction of p125 is reconstituted with
p50.
It seems unlikely that p50 fulfills a simple structural role in
maintaining pol function, based on studies of yeast genes that
encode p50 homologues. A BLAST search of the p50 protein sequence
against the GenBankTM data base showed that it has
significant identity with a Saccharomyces cerevisiae gene,
HYS2, which encodes a protein of 487 residues corresponding
to a molecular mass of 55 kDa (22). Alignment of the protein sequences
shows that there is a 34% identity between human p50 and the Hys2
protein so that the latter is a likely candidate for the yeast
counterpart of the mammalian p50 subunit of pol
. Genetic analysis
of the HYS2 gene (22) showed that it is essential for DNA
replication in S. cerevisiae. Additionally, HYS2
mutants display supersensitivity to hydroxyurea, increased levels of
mitotic chromosome loss, and recombination. More recently, MacNeill
et al. (23) cloned a Schizosaccharomyces pombe
gene, cdc1+, which displays significant sequence
identity (34%) to both the p50 subunit of pol
and to
HYS2. A physical interaction of the Cdc1 protein with the
yeast pol
protein was demonstrated by in vitro
co-immunoprecipitation. Thus, genetic studies have pointed to an
important requirement for the pol
small subunit in yeast, since
cdc1 and HYS2 mutations lead to defective DNA
synthesis and/or repair as well as hypersensitivity to hydroxyurea and
methyl methane sulfonate. Cellular recognition of these defects in
replicated DNA leads to the abnormalities in cell cycle progression
22, 23).
The findings in both budding and fission yeast that genetic screens for
genes required for cell cycle progression have yielded genes encoding
the small subunit of pol are highly indicative of an important
function for this subunit. The p50 subunit could be critical in
providing for the interaction of pol
with other replication
proteins, or it may be that regulation of pol
may be mediated
through p50. This function may also involve the interaction of the
small subunit of pol
with another protein which is encoded by
cdc27+, another gene that is required for cell
cycle progression (23). It was demonstrated that
cdc1+ overexpression is able to rescue mutants
of cdc27+ and that the gene product of
cdc27 + interacts with the Cdc1 protein. In the
mammalian system, no counterpart of the Cdc27 protein has been
reported, although as noted by MacNeill et al. (23), its
association with the core enzyme might not survive the multiple
chromatography steps required for its purification. In this regard, it
may be noted that during the immunoaffinity purification of pol
from calf thymus (17), there is a persistent appearance of a
polypeptide of 40 kDa. Further investigation will be required to
determine the functional role of p50 as well as the identification and
isolation of the proteins that interact with p50. The availability of
overexpressed p50 as well as the antibodies which we have characterized
may be useful tools for these studies.