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J Biol Chem, Vol. 274, Issue 38, 26647-26653, September 17, 1999
From the Department of Biochemistry and Molecular Biology, New York
Medical College, Valhalla, New York 10595
The formation of a complex between DNA polymerase
Proliferating cell nuclear antigen
(PCNA)1 was originally
discovered as an antigen in autoimmune sera from patients with systemic lupus erythematosus and was reported to be found only in actively proliferating cells (1). It was later shown to be a factor that
enhanced the processivity of DNA polymerase The pol Two recent studies of yeast pol These findings are in conflict with our previous studies of the
p125-PCNA interaction, and they suggest that the binding of PCNA in
yeast and human depend on different subunit-PCNA interactions. Alternatively, it is also possible that the interaction of PCNA with
pol Ectopic Expression of p125 and PCNA in COS-7 Cells--
COS-7
cells were cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) containing 10% fetal bovine serum. These cells
were transfected with c-Myc-His-PCNA and pCMV-p125 using the calcium
phosphate method. For each 9-cm Petri dish, the transfected DNA
consisted of 5 µg of each expression vector and 10 µg of Bluescript
SK DNA. The cells were washed with phosphate-buffered saline and
scraped in 1 ml of radioimmune precipitation buffer (10 mM
Tris-HCl, pH 7.5, 120 mM NaCl, 1% Nonidet P-40, 1%
deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl
fluoride). After 15 min on ice, the lysed cells were spun at 5000 rpm
for 15 min at 4 °C. Immunoprecipitations were performed using
anti-Myc antibody (Roche Molecular Biochemicals) in 1 ml of extract.
The immunoprecipitates were separated on a 5-15% gradient SDS gel and
immunoblotted with anti-His antibody (Invitrogen) or pol Gel Filtration of Sf9 Lysates Expressing Recombinant p125
and PCNA--
Sf9 cells were grown in 75-cm2 flasks
to 75% confluence. The cells were dislodged from 25 flasks by
pipetting 5 ml of fresh medium into the flasks. Cell pellets were
collected by centrifugation at 3000 × g for 10 min.
Recombinant baculoviruses containing the human p125 coding sequence,
its deletion mutants, or PCNA were as described previously (13).
Sf9 cells were infected with either the p125 or the deleted p125
and PCNA at a multiplicity of infection of 5 for each recombinant
baculovirus. The cells were incubated in the viral inoculum for 2 h with gentle rotation every 15 min and then centrifuged at 3000 × g for another 10 min. The inoculum was removed, and cell
pellets were suspended in 150 ml of fresh medium divided into 2 flasks
and incubated for another 60 h.
The cell lysates from a total of 2 × 108 cells were
collected and processed as described previously (13). One mg of protein in a total volume of 0.5 ml was chromatographed on a Sephacryl S-300
column (1.5 × 100 cm). Fractions of 1.5 ml were collected. The
elution of pol Cross-linking of p125 and PCNA Expressed in Sf9
Cells--
The peak fraction of the PCNA-p125 complex from the S-300
chromatography was then subjected to cross-linking with Sulfo-EGS at
the indicated concentrations for 15 min at room temperature as
described previously (5). The cross-linked species were identified by
Western blotting. Western blot was performed using 78F5 and 38B5 pol
Overlay Blotting with Biotinylated PCNA--
Recombinant PCNA
was expressed in E. coli and purified to near homogeneity as
described previously (5). PCNA was labeled with biotin by reaction with
biotinamidocaproate N-hydroxysuccinamide ester (Amersham
Pharmacia Biotech). The reaction mixtures contained 0.5 mg of PCNA, 0.5 mg/ml biotinamidocaproate N-hydroxysuccinamide ester in a
total volume of 0.5 ml in 20 mM bicarbonate buffer, pH 8.6. After reaction for 60 min at room temperature, the PCNA was purified on
a G25 Sephadex column (5-ml bed volume) equilibrated in phosphate
buffered saline containing 1% bovine serum albumin. Samples containing
1-5 µg of protein were subjected to 10% SDS-PAGE and transferred to
nitrocellulose membranes. Prestained protein standards (New England
Biolabs) were used as molecular weight markers. The nitrocellulose
membranes were blocked with 5% nonfat dry milk in TBST for 45 min at
room temperature followed by three washes of TBST for 10 min each. The
blots were then incubated with biotinylated PCNA (1 µg/µl) diluted
in TBST (1:900) at 4 °C overnight. The blots were washed five times
with TBST for 15 min followed by incubation with
streptavidin-horseradish peroxidase conjugate diluted in TBST (1:5000)
for 1 h at room temperature with constant rocking. The blots were
then washed five times with TBST for 20 min each and developed using by
chemiluminescence (ECL detection system, Amersham Pharmacia Biotech).
Dot Blot Analysis of Binding of PCNA to Synthetic
Peptides--
Peptides to p21 and the N2 region of pol Pairwise Yeast Two Hybrid Interactions--
Plasmids expressing
the GAL4 fusions with the coding sequences for p125, p50 and PCNA were
constructed in the pAS2-1 and pACT2 vectors
(CLONTECH Laboratories Inc.). Sequential
transformations of Y190 were performed by the lithium acetate method
with dimethyl sulfoxide at a final concentration of 10%. pAS2-1-p125
and pAS2-p50 were transformed into Y190 and plated onto
Trp Co-immunoprecipitation of p125 and PCNA from Crude Calf Thymus and
HeLa Extracts and after Their Ectopic Expression in Cultured
Cells--
Previous studies have demonstrated that human PCNA could be
co-immunoprecipitated with the p125 catalytic subunit of pol
Next, evidence for an interaction between p125 and PCNA when they are
ectopically expressed in mammalian cultured cells was sought. Human
PCNA was expressed in Cos 7 cells with dual tags (a hexahistidine tag
and a c-Myc tag) together with p125 expressed in the pCMV vector (see
under "Experimental Procedures"). Samples of the cell extracts were
immunoprecipitated using a c-Myc antibody and were Western blotted with
pol Formation of a Physical Complex between p125 and PCNA--
The
experimental results in which p125 co-expressed with PCNA in either
baculovirus (10) or in mammalian cells (Fig. 1) indicated that p125 is
able to interact with PCNA and imply that this interaction is
independent of the presence of p50 or other subunits of pol
In order to establish that the co-elution of p125 and PCNA is not due
to fortuitous associations with unrelated proteins, cross-linking
experiments using bifunctional cross-linking agents were performed to
establish a direct p125-PCNA protein-protein interaction in the 220,000 molecular weight complex. Sulfo-EGS was used, as we have previously
shown that EGS readily cross-links PCNA (5). When the peak fractions of
the complex of p125 and PCNA obtained on gel filtration were
cross-linked with sulfo-EGS, and the formation of cross-linked species
monitored by Western blotting with antibodies against p125 (Fig.
3A, left panel) or PCNA (Fig.
3A, right panel), a rapid disappearance of the PCNA monomer
and p125 was observed with the concomitant formation of a band of 225 kDa that reacted with both p125 and PCNA antibodies. This is consistent
with the presence of p125 in complex with a PCNA trimer. It is
noteworthy that no accumulation of species that might represent p125
cross-linked to a PCNA monomer or a PCNA dimer was observed.
Control cross-linking experiments with Sf9 cell lysates
expressing PCNA or p125 alone were performed. In the case of
p125, no cross-linking with sulfo-EGS was observed under the
conditions used (not shown). In the case of PCNA, sulfo-EGS gave
exactly the same pattern of cross-linking that we had previously found for PCNA cross-linked with EGS (5), in that the major species of PCNA
that were observed after cross-linking were the monomer, the dimer, and
a smaller amount of the trimer.
In a parallel experiment, a deletion mutant of p125 ( Far Western Blotting with PCNA--
In order to obtain additional
evidence that p125 can interact with PCNA, an overlay technique for
blotting of PCNA-binding proteins was used. PCNA was labeled with
biotin as described under "Experimental Procedures." Pol
We have also examined the Yeast Two Hybrid Assay--
Human p125, p50 and PCNA coding
sequences were inserted into the pAS2-1 and pACT2 vectors and tested
for pairwise interactions using the yeast two hybrid system. For these
experiments, the yeast co-transformants were grown and the lysates
tested for The N2 Peptide Binds to PCNA--
Previous work had identified a
region of the N terminus of pol Biochemical evidence for a direct interaction of the p125
catalytic subunit with PCNA was obtained. The demonstration of a physical complex between recombinant p125 and PCNA by gel filtration and chemical cross-linking with sulfo-EGS provides rigorous evidence for this interaction. In addition, the use of biotinylated PCNA in
overlay experiments also shows that this interaction is not dependent
on the presence of other pol An important result obtained in these studies is the first biochemical
demonstration of a complex between free recombinant p125 and PCNA by
gel filtration. All the gel filtration studies were performed in the
presence of 150 mM NaCl, a standard biochemical practice to
avoid nonspecific associations. Thus the association between p125 and
PCNA takes place at ionic strengths that are near physiological. The
cross-linking of the complex with sulfo-EGS showed that there was a
rapid cross-linking to a high molecular weight species of a size that
was consistent with a p125-PCNA trimer. These results must be taken in
the context that under the conditions used we have reproducibly found
that PCNA itself is only slowly cross-linked to the trimer and that the
bulk of the reaction products are the monomer and dimer species (5). Previous observations from this laboratory have shown that PCNA in
solution is an equilibrium mixture of the dimer and trimer species (5).
The findings that the only complex present was a PCNA trimer-p125
complex, with an absence of dimeric or monomeric PCNA complexes with
p125, suggests that p125 either selectively binds to the PCNA trimeric
form or stabilizes the PCNA trimer. This preference for trimeric PCNA
is consistent with the physiologically expected interaction of pol Recently, a number of additional proteins that bind to PCNA have been
identified (4, 9, 11, 25). These findings have major implications for
an understanding the roles of PCNA. These PCNA-binding proteins fall
into three major groups: DNA replication proteins (pol An obvious question that arises is whether the N2 region of the p125
subunit of pol In Table I, the regions C-terminal to the PCNA binding motif are also
aligned. In the case of p21, this region is involved in the second
major protein-protein contact of the peptide with PCNA and forms an
anti-parallel In summary, a detailed approach was undertaken to investigate the issue
of whether the p125 polypeptide directly interacts with PCNA. Our
findings provide strong biochemical confirmation that there is a direct
interaction between the p125 subunit and PCNA, although they do not
eliminate the possibility that the pol *
This work was supported by National Institutes of Health
Grant GM 31973 and in part by the United States Army Medical Research and Materiel Command under DAMD-17-96-1-6166.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 914-594-4070;
Fax: 914-594-4058.
2
J.-Y. Mo and M. Y. W. T. Lee,
submitted for publication.
The abbreviations used are:
PCNA, proliferating
cell nuclear antigen;
pol
Direct Interaction of Proliferating Cell Nuclear Antigen with
the p125 Catalytic Subunit of Mammalian DNA Polymerase 
*
,
ABSTRACT
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(pol ) and its sliding clamp, proliferating cell nuclear antigen
(PCNA), is responsible for the maintenance of processive DNA synthesis at the leading strand of the replication fork. In this study, the
ability of the p125 catalytic subunit of DNA polymerase to engage
in protein-protein interactions with PCNA was established by
biochemical and genetic methods. p125 and PCNA were shown to co-immunoprecipitate from either calf thymus or HeLa extracts, or when
they were ectopically co-expressed in Cos 7 cells. Because pol is a
multimeric protein, this interaction could be indirect. Thus, rigorous
evidence was sought for a direct interaction of the p125 catalytic
subunit and PCNA. To do this, the ability of recombinant p125 to
interact with PCNA was established by biochemical means. p125
co-expressed with PCNA in Sf9 cells was shown to form a physical
complex that can be detected on gel filtration and that can be
cross-linked with the bifunctional cross-linking agent Sulfo-EGS
(ethylene glycol bis (sulfosuccinimidylsuccinate)). An
interaction between p125 and PCNA could also be demonstrated in the
yeast two hybrid system. Overlay experiments using biotinylated PCNA
showed that the free p125 subunit interacts with PCNA. The PCNA overlay
blotting method was also used to demonstrate the binding of synthetic
peptides corresponding to the N2 region of pol and provides
evidence for a site on pol that is involved in the protein-protein
interactions between PCNA and pol . This region contains a sequence
that is a potential member of the PCNA binding motif found in other
PCNA-binding proteins. These studies provide an unequivocal
demonstration that the p125 subunit of pol interacts with PCNA.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(pol ) and to have
key roles in both DNA replication and repair (2, 3). There have been
striking recent advances in our understanding of the structure and
functions of PCNA (4). Purification and expression of human recombinant
PCNA and its physiochemical characterization established that it was a
trimeric protein (5). The crystal structures of both yeast and human
PCNA have been determined (6, 7). Like the T4 gene 45 protein and the
subunit of Escherichia coli DNA polymerase III
holoenzyme, PCNA functions as a sliding DNA clamp that forms a closed
ring around duplex DNA (8). The binding of pol to PCNA provides an
elegant micromechanical solution to the biological need to maintain an
extraordinarily high level of processivity during the synthesis of
chromosomal DNA (8-10). Recently, it has also been found that PCNA has
a number of protein partners with which it interacts (4, 9, 11). Pol
has been shown to be involved not only in DNA replication but also in DNA repair and can be regulated by cell cycle proteins (2, 12, 13).
Thus, an important area of interest is the protein-protein interaction
sites of PCNA, because it may be the nexus for multiple protein-protein
interactions involved in replication, repair, recombination, and cell
cycle regulation.
core enzyme consists of two subunits, p125 and p50 (14).
Previous work from this laboratory has implicated the p125 subunit in
an interaction with PCNA (10). A synthetic peptide conforming to the N2
region (residues 129-149) was found to inhibit PCNA stimulation of pol
isolated from calf thymus (10). p125 and PCNA co-expressed in
Sf9 cells could be co-immunoprecipitated with an antibody to
PCNA, showing that the catalytic subunit of DNA polymerase interacted with PCNA (10). However, the recombinant p125 catalytic
subunit can only be stimulated by PCNA at most 2-3-fold, and the
presence of the p50 subunit is required to restore a significant level
of PCNA stimulation of the p125 subunit (15-17).
reported contrary results. No
evidence for a direct interaction between the Schizosaccharomyces pombe pol p125 subunit and PCNA could be found, either by
co-immunoprecipitation experiments after their co-expression in insect
cells or by a yeast two hybrid assay (18). It was concluded that no
direct interaction occurs between S. pombe p125 and PCNA. In
Saccharomyces cerevisiae, similar results were obtained
using a PCNA overlay assay. In the latter studies, the interaction of
S. cerevisiae p125 and p58, as well as the recently
identified third subunit (p55), was studied by a PCNA overlay method.
It was shown that only the third subunit (p55) of pol interacted
with PCNA (19), indicating that the interaction of pol with PCNA
involved the third subunit, whereas no evidence could be obtained for
an interaction of PCNA with either the small second subunit p58 or the
catalytic subunit.
involves multiple interactions with pol subunits. For this
reason, we have undertaken a rigorous examination of the
protein-protein interactions of the p125 subunit of pol and PCNA
using different biochemical methods. Our studies leave little doubt of
the ability of the p125 subunit to interact with PCNA.
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
monoclonal
antibody. The blot was then detected by chemiluminescence (ECL,
Amersham Pharmacia Biotech).
activity was monitored by its activity on poly(dA)/oligo(dT) as described previously (10)
monoclonal antibodies (13). Prestained protein standards (Sigma)
were used as molecular weight markers and also to provide for visual
confirmation of efficient transfer. Nitrocellulose blots were blocked
in 5% (w/v) nonfat dry milk in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% (v/v) Tween 20 (TBST) for 1 h at room temperature. The blot was then incubated with monoclonal antibodies to
pol for 12 h. at 25 °C. After three 10-min washes in TBST, the blot was incubated with anti-mouse IgG-horseradish peroxide conjugate diluted in TBST (1:10,000) for 1 h at room temperature with constant rocking. The blot was then washed five times with TBST
for 20 min each and developed by chemiluminescence (ECL detection system, Amersham Pharmacia Biotech).
p125 were
synthesized by Bio-Synthesis Co. The peptides were dissolved in water at a concentration of 5 µg/µl. Five µl of each peptide were dot blotted onto nitrocellulose membranes. Bovine serum albumin, PCNA, p125
and purified calf thymus pol (25 µg) were dot blotted onto the
same membranes. The nitrocellulose membranes were then blocked and
blotted with biotinylated PCNA as described above.
plates. Y190 stains transformed with pAS2-1-p125 or
pAS2-1-p50 were then transformed with pACT2-50 or pACT2-PCNA. After
overnight recovery in TrpLeu medium, the
transformants were plated on TrpLeu
His/3-AT plates to select for histidine prototrophy. For
the liquid assay, GAL4 and T antigen/p53 were transformed into Y190 as
positive controls. pAS2-1-p50 and pAS2-1-PCNA in Y190 were used as
negative controls. One ml of overnight yeast culture in liquid
TrpLeuHis/SD selection medium
was prepared. Four ml of YPD medium (yeast culture medium containing 20 g/liter Difco peptone, 10 g/liter yeast extract, and 2% glucose) was
added to the overnight culture and incubated for 5 h at room
temperature with shaking (230-250 rpm). The
A600 of the culture was recorded. One ml of
cells was centrifuged at 1,000 × g for 30 s and
washed once with 1 ml of Z buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4·H2O, 10 mM
KCl, 1 mM MgCl2·7H2O) followed by
another centrifugation. The cell pellet was resuspended in 100 µl of
Z buffer. The cells were disrupted by freeze-thaw (liquid nitrogen for
1 min, thawed at 30 °C for 1 min). Z buffer (0.7 ml) and
-mercaptoethanol at a final concentration of 40 mM was
added. O-Nitrophenyl -D-galactopyranoside
(160 µl, 2.2 mM in Z buffer) was added, and the reaction
was incubated at 30 °C for 3-90 min. (Positive strong interactions
were incubated for 3 min and negative interactions for 90 min.) The
reactions were terminated by addition of 0.4 ml of 1 M
Na2CO3. Cell debris was removed by
centrifugation at 10,000 × g for 2 min, and the A420 was recorded. The -galactosidase
activity was calculated. Arbitrary units of activity were calculated
as: -galactosidase units = 1000 × A420/(t × V × A600), where t = min of
incubation; V = 0.1 ml.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
from
Sf9 insect cell lysates, under conditions where both proteins were overexpressed as recombinant proteins (10). It can be argued that
the interactions observed were a consequence of the supranormal concentrations of both proteins under these conditions of
overexpression and may not reflect the behavior of the two proteins in
a normal cellular context. Studies were therefore performed to
establish whether PCNA and p125 could be co-immunoprecipitated from
mammalian tissue or cell culture extracts. The results show that p125
and PCNA can be readily co-immunoprecipitated from crude calf thymus extracts that had been partially purified on phenyl agarose, as well as
from HeLa cell lysates (Fig. 1,
A and B). Because of the multisubunit nature of
pol , these experiments do not show a direct interaction between
p125 and PCNA, but they do confirm that an interaction between pol and PCNA is readily demonstrated in cell extracts.
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Fig. 1.
Co-immunoprecipitation of p125 and PCNA with
monoclonal antibodies from calf thymus extracts or HeLa extracts.
A, lane 1, a calf thymus extract was passed
through phenyl agarose, and 50 µl of the extract was
immunoprecipitated (IP) with 20 µg of 74B1 PCNA monoclonal
antibody and blotted with monoclonal antibody 78F5 against pol .
Lane 2, a calf thymus extract was passed through phenyl
agarose, and 50 µl of the extract was immunoprecipitated with 78F5
pol antibody and immunoblotted with 20 µg of 74B1 monoclonal
antibody against PCNA. Lane 3, control containing purified
PCNA, immunoblotted with the antibody against PCNA. Western blotting
(WB) was performed using biotinylated sheep anti-mouse
immunoglobulin as second antibody followed by incubation with
streptavidin-biotinylated horseradish peroxidase complex. Color
development was performed by incubation with 4-chloro-1-naphthol and
hydrogen peroxide and terminated with sodium azide. B, HeLa
cells were lysed in 1% Nonidet P-40 lysis buffer and protease
inhibitors. The lysate was precleared with protein G agarose bead
before incubation with antibodies (Ab). HeLa lysate (2 mg of
protein) was incubated with 30 µg of monoclonal antibody for each
immunoprecipitation. Immunobeads were suspended in 150 µl of lysis
buffer and 150 µl of 2× SDS loading buffer. Lanes 1 and
4, 50 µg of untreated crude HeLa lysate. Lanes
2 and 6, 50 µl of immunoprecipitate from monoclonal
38B5 against pol . Lanes 3 and 5, 50 µl of
immunoprecipitate from monoclonal antibody 74B1 against PCNA. Samples
were subjected to 10% SDS-PAGE and transferred to nitrocellulose
membranes. The membrane was divided into two halves. Lanes
1-3 were immunoblotted with monoclonal antibody 78F5 against pol
. Lanes 4-6 were immunoblotted with monoclonal antibody
74B1 against PCNA. Western blotting was performed using horseradish
peroxidase anti-mouse immunoglobulin as a second antibody followed by
chemiluminescence detection.
antibody. The results show that p125 is
co-immunoprecipitated with c-Myc-His-tagged PCNA (not shown).
Control experiments in which the immunoprecipitates were blotted
with anti-His antibody confirmed that PCNA was present in the
immunoprecipitate. These experiments demonstrate that pol p125 can
be shown to co-immunoprecipitate with PCNA, either when endogenous pol
is present or when p125 is ectopically co-expressed with PCNA.
. In
these experiments, co-immunoprecipitation does not provide rigorous
evidence that the p125 subunit directly interacts with PCNA, as it is
possible that the p125 that is detected by co-immunoprecipitation is by
virtue of an interaction of PCNA with a pol heterodimer,
i.e., the interactions are mediated by a third
polypeptide(s). In order to provide a more rigorous test, Sf9
cells were co-infected with baculovirus vectors for p125 and for PCNA.
The Sf9 cell lysates were then subjected to gel filtration on a
Sephacryl S-300 column (Fig. 2). Assays
for pol activity showed that there were two peaks of activity, one with a relative molecular weight of 125,000 and the second with a
relative molecular weight of 220,000 (Fig. 2, upper and
middle panels). The fractions were analyzed for the presence
of p125 and PCNA by Western blotting using specific antibodies. The
results show that the 125,000 peak contained only p125, whereas PCNA
co-migrated with p125 in the 220,00 molecular weight fractions.
Furthermore, no free PCNA was detected in the range where either the
dimer or trimer form would be expected to migrate. (In previous studies of the behavior of recombinant PCNA expressed in E. coli, we
had shown that PCNA in solution is a mixture of dimers and trimers (5)). These results provide the first direct demonstration that free
p125 forms a physical complex with PCNA, and moreover, the apparent
molecular weight is consistent with a complex of p125 with a trimeric
form of PCNA, because previous work showed that the PCNA trimer
migrates with an apparent molecular weight of approximately 100,000 on
gel filtration (5).
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Fig. 2.
Gel filtration of Sf9 lysates
expressing recombinant p125 and PCNA. p125 and PCNA were
co-expressed in Sf9 cells, and the cell lysates (0.5 ml, 1 mg of
protein) were chromatographed on Sephacryl S-300 (see under
"Experimental Procedures"). The elution of pol activity was
monitored by activity assay (center panel) and for p125 and
PCNA by Western blotting using a mixture of monoclonal antibodies to
p125 and PCNA (bottom panel). The column was calibrated
using ferritin (molecular weight 440,000), catalase (232,000), aldolase
(158,000), and bovine serum albumin (67,000), shown as 1-4
in the upper panel. The approximate molecular weights of the
two peaks of pol activity were 220,000 and 125,000, as shown by the
arrows in the upper panel.
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Fig. 3.
Cross-linking analysis of the p125/PCNA
complex. Samples of the peak fraction number 66, (40 µl, 20 ng
of protein) from the S-300 chromatography (Fig. 3) were incubated with
sulfo-EGS at the indicated concentrations (mM) for 15 min
at room temperature and then subjected to SDS-PAGE followed by Western
blotting using a monoclonal antibody against p125 (A, left
panel) and a monoclonal antibody against PCNA (A, right
panel). Arrows a and b show p125
immunoreactive bands of 225 and 125 kDa, respectively; arrow
c corresponds to the cross-linked PCNA dimer, and arrow
d corresponds to the PCNA monomer. B shows the
determination of the relative molecular masses of the bands
a-d (open squares); prestained protein standards (New
England Biolabs. Inc.) were used as markers (solid circles).
C, a similar cross-linking experiment was performed using a
deletion mutant of p125 ( 
2-249) in which the N-terminal 249 residues were deleted (13). The upper panel shows the
Western blot of the 97 kDa immunoreactive p125 (2-249) (
), which
was not depleted during the course of the experiment. In addition, no
significant formation of higher molecular weight species was observed
(not shown). The lower panel shows the immunoblot with
antibody against PCNA, where 1-3 show the positions of the
monomer, dimer, and trimer forms of PCNA, respectively, without
evidence for formation of cross-links with p125 (2-249).
2-249) in
which the N-terminal 248 residues were removed (13, 20) was
co-expressed with PCNA and subjected to cross-linking. In this case,
only the monomer, dimer, and trimer species of PCNA were observed, as
for PCNA alone, as shown in Fig. 3C. Blotting with pol antibodies also showed that there were no cross-links formed with the
N-terminal deletion mutant of pol (Fig. 3C, upper
panel). These results are consistent with our previous
observations that the N-terminal region of p125 is required for its
interaction with PCNA.
was
subjected to SDS-PAGE and then transferred to nitrocellulose membranes.
The membranes were then blotted with biotinylated PCNA (see under
"Experimental Procedures") and visualized using a chemiluminescence
method. A number of experiments were performed; they showed that only
the p125 band provided a strong reaction with biotinylated PCNA. When
immunoaffinity purified calf thymus pol enzyme was blotted with
biotinylated PCNA, a strong reaction was seen with the p125 catalytic
subunit (Fig. 4A, left panel).
The p50 subunit of pol did not interact with PCNA in the overlay
experiments (Fig. 4A, left panel). The inability of the p50
subunit to interact with PCNA was confirmed using purified recombinant
p50 subunit (Fig. 4A, left panel). This experiment was
repeated using recombinant pol heterodimer produced by
overexpression of p125 and p50 in Sf9 cells. The same results
were obtained, namely that p125 but not p50 reacted with biotinylated
PCNA (Fig. 4B). Because the overlay depends on an
interaction with a polypeptide band separated by SDS-PAGE, this
positive interaction demonstrates that the p125 subunit interacts with
PCNA in a manner that is independent of the presence of the other
subunits of pol . In Fig. 4A, it is noted that the
overlay of immunoaffinity purified pol with PCNA reveals a doublet
of 70 kDa. In other studies, partial protein sequence was obtained of
this band, and a BLAST search identified this polypeptide as KIAA0039
(GenBankTM). This was found to be a mammalian counterpart
of S. pombe
Cdc27.2
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Fig. 4.
PCNA overlay analysis of pol . PCNA was labeled with biotin by reaction
with biotinamidocaproate N-hydroxysuccinamide ester (see
under "Experimental Procedures"). Samples were run on SDS-PAGE gels
(10% acrylamide), transferred to nitrocellulose, and then blotted
using biotinylated PCNA/streptavidin-horseradish peroxidase conjugate.
The blots were visualized using a chemiluminescence method (ECL
detection system, Amersham Pharmacia Biotech). A, the
left panel shows an experiment using purified calf thymus
pol obtained by immunoaffinity affinity chromatography (21) that
had been further purified on heparin-agarose. This enzyme () was
analyzed by PCNA overlay together with recombinant p50 (p50)
overexpressed in E. coli (16) and purified to
near-homogeneity (2.5 µg of protein were used). The positions of the
p125 and p50 subunits of pol are shown by the
arrowheads. Also shown is the position of a p70 polypeptide
that interacts with PCNA. The center panel shows the
Coomassie Blue-stained SDS-PAGE gel of the same preparations, and the
right panel is a Western blot using a mixture of monoclonal
antibodies to p125 and p50 to show the identity of the polypeptide
bands. B, p125 and p50 were overexpressed in Sf9
cells and purified to near homogeneity (P. Zhang and M. Y. W. T. Lee, unpublished data). The left panel shows the
elution of pol activity from the final Mono-Q chromatography step,
assayed in the absence (
) and presence (
) of PCNA. The
center panel shows a Western blot of the peak fractions 65, 70, and 75 with a mixture of antibodies to the p125 and p50 subunits.
The right panel shows the overlay blot with biotinylated
PCNA. The arrowheads show the migration positions of p125
and p50 subunits.
2-249 N-terminal deletion mutant of p125,
in which the N2 region is absent (13), by PCNA overlay. The results
(Fig. 5A) show that this
deletion mutant is not recognized by overlay with biotinylated PCNA. In
parallel, experiments were performed using deletion mutants
186-321, 336-715 (core region deleted), 675-1107, and
778-1047 (C-terminal regions deleted) expressed in Sf9 cells
(Fig. 5B). All of these deletion mutants, covering
essentially the entire p125 sequence from residue 186 (Fig.
5C), interacted with PCNA. These results, taken with the inability of the 2-249 deletion mutant to bind to biotinylated PCNA, restricts the binding region on p125 to within the first 186 residues.
View larger version (50K):
[in a new window]
Fig. 5.
PCNA overlay of recombinant p125 and its
N-terminal deletion mutants. A, p125 and its N-terminal
deletion mutant ( 2-249) in which residues 2-249 are deleted were
expressed in Sf9 cells and analyzed by PCNA overlay. The three
lanes are the three peak fractions of the recombinant
proteins during high pressure liquid chromatography gel filtration that
were subjected to SDS-PAGE and overlay analysis with biotinylated PCNA.
The left panels show the Western blots of the fractions for
p125 and the 97 kDa 2-249 deletion mutant. The right
panels show the overlay with biotinylated PCNA. B,
Sf9 cells (1 × 108) were infected with
recombinant baculovirus vectors for 186-321, 336-715,
675-1107, or 778-1047. Cell lysates (50 µl) from each of the
cultures were then subjected to SDS-PAGE and analyzed by overlay with
PCNA as for A. Lanes 1-4, represent 186-321,
336-715, 675-1107, and 778-1047, respectively.
C, map of the deletion mutants 186-321, 336-715,
675-1107, and 778-1047.
-galactosidase activity. The results were compared on the
basis of relative specific activities (arbitrary units of
-galactosidase activity per unit of cell culture density). The
results are shown in Fig. 6. This analysis confirms the biochemical data that p125 and PCNA interact and
that p50 and PCNA do not interact.
View larger version (21K):
[in a new window]
Fig. 6.
Liquid assay for yeast two hybrid
interactions between p125 and PCNA. The p125, p50, and PCNA
sequences were tested for pairwise interactions in the yeast two hybrid
system as described under "Experimental Procedures." The data are
shown as arbitrary units of -galactosidase activity per unit of cell
culture density. T-antigen and p53 constructs were used as controls for
a known pair of interacting proteins. The following pairs were tested:
p125-p50, p125-PCNA, and p50-PCNA.
, the N2 region
(GVTDEGFSVCCHIHGFAPYFY, residues 129-149) as being involved in the
interaction of pol with PCNA. This was based on the ability of a
synthetic peptide with this sequence to inhibit the PCNA stimulation of
pol (10). However, these experiments were performed with purified
pol , and in the context of evidence that p125 does not interact
with PCNA in other systems, it could be argued that these findings were
due to an interference of the peptide with an interaction between p125
and an intermediary protein that leads to loss of PCNA response.
Experiments were performed to test for a direct interaction of PCNA
with the N2 peptide as well as with several variants with selected
alanine replacements by dot blot analysis using biotinylated PCNA (Fig. 7). The peptides were bound to
nitrocellulose and then blotted with biotinylated PCNA. The results
(Fig. 7) show that immobilized N2 peptides, but not the mutant N2
peptide in which the three terminal YFY residues were replaced with
alanine (GVTDEGFSVCCHIHGFAPAAA), are blotted by biotinylated
PCNA. p21 peptide and p125 were also blotted in this dot blot assay
(Fig. 7). The loss of interaction of the mutant in which the three
terminal aromatic residues were changed to alanine is highly
significant because it provides supportive evidence that the N2 region
contains a variant of the PCNA binding motif (see under
"Discussion").
View larger version (112K):
[in a new window]
Fig. 7.
Dot blot analysis of binding of PCNA using
biotinylated PCNA. Synthetic peptides/proteins were dot blotted
onto nitrocellulose membranes and tested for binding to PCNA using
biotinylated PCNA as described under "Experimental Procedures." The
proteins tested were bovine serum albumin (BSA), recombinant
p125 subunit expressed in Sf9 cells (p125), and
immunoaffinity purified calf thymus pol (pol ). The
synthetic peptides that were tested are listed below the blot.
CT is a peptide to the C terminus of p125 (residues
1091-1107) that we have used for the preparation of monoclonal
antibody 38B5. p21 is the p21-derived peptide (139-160)
that contains the consensus PCNA binding motif
(underlined).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits, and the use of deletion
mutants of p125 restricts the location of the interaction site to the
N-terminal 186 residues of p125. Further evidence for a region on p125
that is involved in the interaction with PCNA was obtained by the use
of synthetic peptides to the N2 region (residues 129-149). These
findings confirm and extend previous studies from this laboratory that
show that p125 directly interacts with pol and that the N2 region
in p125 can be implicated in the protein-protein interaction with PCNA.
The need for a more detailed investigation of whether there is a direct
interaction between p125 and PCNA was raised by studies of pol -PCNA
interactions in yeast, in which only negative evidence for an
interaction of the p125 and PCNA has been obtained. Tratner et
al. (18) reported that recombinant S. pombe p125 did
not interact with hemagglutinin epitope-tagged PCNA when they were
co-expressed in insect cells and tested for co-immunoprecipitation of
35S-labeled proteins or when tested for pairwise
interactions by the yeast two hybrid system. In S. cerevisiae, a third subunit of pol encoded by the POL32 gene
has been identified (22, 23). A 32P-tagged PCNA containing
a fused PKA site at the N terminus was used in overlay experiments
against p125 (POL3), p58 (POL31), and p55 (POL32) subunits. The results
showed that only the p55 subunit bound to the tagged PCNA and no
evidence of interaction of either p58 (the p50 homologue) or p125 with
PCNA was found (19). The different results that were obtained in the
yeast system could be due to differences in the experimental conditions used, as noted by Eissenberg et al. (19). The present
studies confirm that, unlike the yeast proteins, human PCNA and p125
co-immunoprecipitate with either p125 or PCNA antibodies when they are
co-expressed in Sf9 cells, COS 7 cells, and are also
co-immunoprecipitated from calf thymus or HeLa extracts. As already
noted, co-immunoprecipitation methods do not eliminate the possibility
that positive results are due to the intervention of an intermediary polypeptide(s).
with PCNA, the function of which is associated with a trimeric state.
It is also consistent with current models of the assembly of the
replication complex, in which replication factor C first loads PCNA
onto DNA, following which pol is recruited (8). Recent findings
(24) have shown that replication factor C disengages from PCNA upon
sliding clamp formation and that the loaded PCNA clamp was able to
recruit and bind polymerase and stimulate DNA replication.
, replication
factor C and pol 
, flap endonuclease 1, and DNA ligase), DNA repair
proteins (xeroderma pigmentosum G and methyl(5-cytosine)methyl
transferase), and cell cycle regulatory proteins (p21 and p57). These
proteins contain a short PCNA binding motif in which there is a
conserved glutamine and two conserved aromatic residues. The motif in
question lies at the N terminus of the p21 peptide that forms an
-helical region in which the two aromatic residues interact with the
large hydrophobic pocket of PCNA, whereas the C-terminal region forms
an anti-parallel sheet with the interdomain connector loop of PCNA
(7).
harbors a sequence that corresponds to the PCNA
binding motif that has been identified in these PCNA-binding proteins.
The alignment of the N2 sequence of pol from human and yeast with
the PCNA binding motifs of a number of proteins is shown in Table
I, which shows the eight-residue motif
bounded by the conserved glutamine and the aromatic residues. The main features of the motif are the conserved glutamine at position 1, the
presence of an aliphatic residue (leucine or isoleucine) at position 4, and a pair of aromatic residues at positions 7 and 8. Comparison of the
N2 sequence shows that it possesses the aromatic residues and the
aliphatic residue at position 4, but does not have the conserved
glutamine. Thus, this region of the N2 sequence does not carry a
complete consensus with the known PCNA binding motif. However, this
glutamine residue is not conserved in p57, which also binds PCNA (Table
I). Evidence obtained in this study using peptides to the N2 region of
pol provides strong evidence that it contains a variant of the PCNA
binding motif (Table I). The loss of binding when the aromatic residues
are mutated in the N2 peptide does provide significant evidence that this region of the N2 sequence may be a member of the PCNA binding motif family of sequences.
Alignment of the N2 region of pol with the PCNA binding motif
-sheet with the interdomain connector loop of PCNA
(7). In previous studies, it had been shown that the interdomain
connector loop of PCNA is important for the interaction of pol , and
it was proposed that the N2 region of pol may interact with PCNA in
a manner that may be similar to the interaction of p21 with PCNA (26,
27). There is a group of PCNA-binding proteins in which there is a
preponderance of basic residues in this region (Table I), as previously
noted by Warbrick et al. (28). However, it is also seen that
there is a second group of sequences, in which there is a high
percentage of proline residues. This includes pol from human and
yeasts, human uracil DNA glycosylase, methyl 5'-cytosine methyl
transferase, the human Tigger sequences (28), and S. pombe
RAD2. The positions of the prolines is well conserved in these eight
examples (Table I). The presence of this "proline-rich" motif
provides additional support for the view that the N2 sequence belongs
to the family of PCNA binding sequences. Furthermore, it should not be
forgotten that from the example of p21 that the
QXXI/LXXFF motif reflects an interaction only
with the large hydrophobic pocket of PCNA. Although examples also exist
that indicate that this interaction alone (Drosophila POGO
and S. pombe Cdc27) is sufficient for PCNA binding, the
presence of the two features of the C-terminal region in the form of
the proline rich motif and the basic motif suggests that these may represent sequences that are suited to the formation of a -sheet with the interdomain connector loop of PCNA.
holoenzyme has multiple
sites of interaction with PCNA through one or more of its subunits. The
latter possibility is one that could be facilitated by the trimeric
nature of PCNA, because this provides for extended interactions of
individual PCNA subunits with different subunits of pol .
FOOTNOTES
Current address: Dept. Cell Biology and Anatomy, University of
Miami School of Medicine, Miami, FL 33101.
ABBREVIATIONS
, polymerase ;
EGS, ethylene
glycol-bis(sulfosuccinimidyl- succinate);
PAGE, polyacrylamide gel electrophoresis.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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