From the Departments of Biochemistry and Molecular Biology and Medicine, University of Miami, School of Medicine, Miami, Florida 33101
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ABSTRACT |
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Proliferating cell nuclear antigen (PCNA) is
required for processive DNA synthesis catalyzed by DNA polymerase (pol
) and polymerase
. We have shown that the epitope of a human
PCNA inhibitory monoclonal antibody (74B1), which inhibits the PCNA
stimulation of DNA synthesis catalyzed by pol
, maps to residues
121-135, which overlap the interdomain connector loop of PCNA
(residues 119-133). We have mutagenized residues 122-133 of human
PCNA. The mutant proteins were expressed in Escherichia
coli and purified to near-homogeneity. The interactions of the
mutants with antibody 74B1 were examined; mutation of Gly-127 abolished
the recognition by antibody 74B1 in a Western blot analysis, confirming
the epitope assignment of 74B1. Mutations of Val-123, Leu-126, Gly-127,
and Ile-128 affected the ability of PCNA to stimulate DNA synthesis by
pol
in several different assays. These mutations affected the
interactions between PCNA and pol
as determined by enzyme-linked immunosorbent assays. These mutants were also affected in their abilities to form a ternary complex with a DNA template-primer, as
determined by electrophoretic mobility gel shift assays. The findings
show that the interdomain connector loop region is involved in binding
of pol
. This same region is involved in the binding of p21, and our
findings support the view that the mechanism of inhibition of DNA
synthesis by p21 is due to a competition for PCNA binding to pol
.
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INTRODUCTION |
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DNA polymerase (pol
)1 is a central enzyme
involved in the replication of mammalian chromosomal DNA and is also
involved in DNA repair (1, 2). Studies of in vitro SV40
replication has established the role of pol
, as well as an
understanding of the complex number of proteins that are involved in
eukaryotic DNA replication (2). It is now considered that pol
is
involved in the lagging-strand synthesis, while pol
is involved in
leading-strand synthesis and also for completion of lagging strand
synthesis (2). A key element of the function of a replicative DNA
polymerase is the ability for extended processive synthesis of DNA.
This ability is conferred on replicative polymerases by accessory
proteins, which function as molecular sliding clamps. These clamps form toroidal rings that encircle the DNA strand and also interact with the
polymerase. This function in Escherichia coli is fulfilled by the
subunit (3), and in mammalian cells by PCNA (proliferating cell nuclear antigen), which is an essential factor for the eukaryotic DNA replication and functions as a processivity factor for pol
(4,
5). The basis for the functions of the sliding clamps of both E. coli and PCNA has been elucidated by the determination of their
crystal structures, which reveals them to be toroidal proteins that
encircle the DNA strand (6, 7). PCNA is a homotrimer. Each subunit
consists of two structurally equivalent domains giving the trimer a
six-fold symmetry. PCNA is loaded onto double-stranded DNA by the
action of RF-C (8), followed by the loading of pol
through an
interaction between PCNA and pol
to form a ternary pol
-PCNA-template-primer complex. However, little is known about the
structural elements involved in the interaction of PCNA with pol
.
PCNA is also implicated in DNA repair (9, 10) and cell cycle control
processes (11). PCNA has been shown to interact with p21 (12, 13). The
latter is a cell cycle checkpoint protein, which inhibits
cyclin-dependent protein kinases, and has also been shown
to inhibit DNA synthesis in vitro (12, 13). The mechanism
for the latter effects has been shown to be due to the ability of p21
to bind directly to human PCNA (hPCNA); p21, however, does not inhibit
the repair functions of pol (14, 15). A more recent study showed
that a peptide derived from the C terminus of p21 when added at higher
levels inhibited both DNA replication and nuclear excision repair (16).
The nature of the interaction of p21 with PCNA has been determined at
the atomic level by solution of the three-dimensional structure of a
complex of hPCNA with a 22-residue peptide derived from the C terminus
of p21 (17).
Recently, we reported that an inhibitory monoclonal hPCNA antibody
(74B1), inhibits the ability of hPCNA to stimulate DNA synthesis
catalyzed by pol in vitro using poly(dA)·oligo(dT) as a template. The epitope of this antibody has been mapped to residues
121-135 of human PCNA (18). The crystal structure shows that this
region is located in the loop that connects the two conformationally
conserved domains in the PCNA monomer (17). In this study, we report a
mutational study of this loop region to investigate the structural
basis for the interactions of hPCNA with pol
by the use of
site-directed mutagenesis in combination with electrophoretic mobility
shift, processivity gel, and ELISA assays.
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EXPERIMENTAL PROCEDURES |
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Polymerase , Replication Protein A, and Replication Factor C
Proteins--
Calf thymus pol
was purified by immunoaffinity
chromatography as described by Jiang et al. (19). Human
replication protein A (RPA) expressed in E. coli (a gift
from Dr. M. S. Wold, University of Iowa, Iowa City, IA) was
purified according to Henricksen et al. (20). RF-C was a
generous gift from Dr. M. O'Donnell, Howard Hughes Medical Institute,
Cornell University Medical College, New York, NY.
Construction of Site-directed Mutants of PCNA-- The coding sequence for human PCNA was inserted between XbaI and SacI sites in pALTER-1 plasmid (Promega). Site-directed mutagenesis was performed as described previously (21). Primers used for generation of the mutants were: D122A, GTTGTTCAACAGCTAAATCCAT; V123A, CAAGTTGTTCAGCATCTAAATC, E124Q, TTCCAAGTTGTTGAACATCTAAA; Q125E, AATTCCAAGTTCTTCAACATC, L126S, TGGAATTCCACTTTGTTCAAC; G127A, GTTCTGGAATTCCAAGTTGTTC; I128A, CTGTTCTGGAGCTCCAAGTTG; P129A, CTCCTGTTCTGCAATTCCAAG; E130Q, TGTACTCCTGTTGTGGAATTCC; Q131E, AGCTGTACTCCTCTTCTGGAATTC; E132Q, CACAGCTGTACTGCTGTTCTGG; Y133F, TACACAGCTGAACTCCTGTTC. Underlined residues are those mutated from the wild type sequence. Mutations were verified by DNA sequencing. NdeI-HindIII cassettes containing the mutated PCNA sequences were recloned into the bacterial expression vector pTACTAC (22).
Expression and Purification of the PCNA Mutants--
Constructs
containing wild type and mutant PCNA were transformed into E. coli DH5 cells for overexpression of protein. Overnight cell
cultures (5 ml) were used to inoculate 1-liter cultures (Terrific media) and grown at 37 °C until the A600
reached a value of 0.3. Isopropyl-1-thio-
-D-galactopyranoside was then added to
a concentration of 0.3 mM, and the cultures were grown for
another 16-18 h at 26-28 °C. The cells were harvested, and the
PCNA proteins were purified as described by Zhang et al.
(23).
Assays for Enzyme Activities--
DNA polymerase activity was
assayed with poly(dA)·oligo(dT) as described by Jiang et
al. (19) and with singly primed M13 template essentially as
described by Burgers 1995 (24). M13mp18 single-stranded DNA was primed
with a 40-mer oligonucleotide complementary to nts 7041-7080 of the
M13 genome. The primer was annealed to M13 single-stranded DNA as
described by Podust et al. (25). The standard 30-µl
reaction mixture contained calf thymus pol (500 ng), RPA (850 ng),
RF-C (25 ng), PCNA (250 ng), 40 mM Tris-HCl (pH 7.8), 8 mM magnesium acetate, 0.2 mg/ml bovine serum albumin, 1 mM dithiothreitol, 100 µM each dATP, dCTP,
and dGTP, 25 µM [3H] dTTP, 0.5 mM ATP, 100 ng of singly primed M13mp18 DNA. The complete
reaction mixtures were incubated at 37 °C for 30 min.
SDS-PAGE and Western Blotting-- SDS-PAGE and Western blotting were performed as described by Jiang et al. (19).
Electrophoresis Mobility Shift Assays--
Calf thymus pol was purified by immunoaffinity chromatography as described by Jiang
et al. (19), followed by high performance liquid
chromatography gel filtration on a SEC-250 column (Bio-Rad). Wild type
hPCNA and mutants (150 ng) plus calf thymus pol
(5 ng) were
incubated at room temperature for 15 min in 10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 50 mM KCl, and 5% glycerol
in a total volume of 20 µl. The 5
32P-labeled 30-21-mer
template-primer (0.8 ng) was added, and the mixture was incubated for
an additional 10 min at room temperature after which the reaction
mixtures were subjected to nondenaturing polyacrylamide gel
electrophoresis (26). Acrylamide concentrations of 6% were used, and
gels were prerun for 1 h at 80 V. Electrophoresis was performed
for 2.5 h at 100 V with a running buffer consisting of 45 mM Bis-Tris, pH 7.5, 45 mM boric acid, and 0.1 mM EDTA. After electrophoresis, gels were dried and
subjected to autoradiography.
ELISA Assays--
ELISA assays were performed according to
Dornreiter et al. (27) with minor modifications. ELISA
plates were coated with 500 ng/well hPCNA mutant proteins in 50 µl of
phosphate-buffered saline (PBS), pH 7.5 for 4 h at 4 °C. After
washing twice with PBS buffer, the wells were blocked with 3% (w/v)
bovine serum albumin in PBS buffer overnight at 4 °C and washed
three times with PBS buffer. Monoclonal antibody 74B1 specific to PCNA
(Fig. 3) or pol (Fig. 6) was added at a concentration of 500 ng/well in 50 µl PBS and incubated at room temperature for 2 h.
The unbound antibody or protein was removed by washing six times with
PBS buffer. For detection of the binding between hPCNA mutant proteins and 74B1 monoclonal antibody, horseradish peroxidase conjugated anti-mouse IgG (1:2000, Amersham) in PBS buffer was added. For detection of pol
, 0.5 µg of monoclonal antibody (78F5) in 50 µl
of PBS buffer was incubated with the plates for 1 h at room temperature. The plates were washed six times with PBS buffer, followed
by a 40-min incubation with horseradish peroxidase-conjugated anti-mouse IgG (1:2000, Amersham Corp.) in PBS buffer. The reaction was
developed with tetramethylbenzidine base (TMB-ELISA, Pierce) for 2-5
min and quantitated at 450 nm with a microtiter plate reader
(Bio-Rad).
CD Spectra-- Near and far UV CD spectra of wild type hPCNA and hPCNA mutants were determined with a JASCO J720 spectropolarimeter. Twenty spectra were collected for each sample at a speed of 100 nm/min. and subsequently averaged. A cell with a path length of 1 cm was used for near UV spectra (250-320 nm), and one with a path length of 0.1 cm was used for the far uv spectra (200-250 nm). Sample protein concentration was 0.25 mg/ml. Data were fitted re-iteratively to appropriate equations with the SigmaPlot curve fitting program.
Chemical Cross-linking-- The chemical cross-linking of wild type and mutant PCNAs was performed with ethylene glycol bis(succinimidyl succinate) (Pierce) as described by Zhang et al. (23). The reactions were performed at room temperature for 2 min, and the products were analyzed by Western blotting.
Processivity Assay--
The effect of hPCNA mutants on the
processivity of pol was analyzed by polyacrylamide gel
electrophoresis of the reaction products as described by Prelich
et al. (5). 5
32P-end-labeled
(dT16) was annealed to poly(dA). The reaction mixtures (60 µl) contained 0.25 OD units/ml 32P-labeled
poly(dA)·oligo(dT) [40:1], 40 mM Tris-HCl, pH 6.5, 5 mM MgCl2, 2 mM dithiothreitol, 10%
glycerol, 0.1 mg/ml bovine serum albumin, 80 µM dTTP, 150 ng of hPCNA, and 1 unit of pol
. After incubation at 37 °C for 30 min, reactions were terminated by the addition of 10 µl of 10 mg/ml
of salmon testis DNA in 20 mM EDTA. The DNA was
precipitated with ethanol and dissolved in deionized formamide, 10 mM EDTA, and 0.1% xylene cyanol. The samples were heated
at 100 °C for 2 min, cooled on ice, and subjected to electrophoresis
on 8% polyacrylamide, 8 M urea gels.
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RESULTS |
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Expression and Purification of PCNA Mutants--
We have shown
previously that the region between residues 121-135 of human PCNA
(hPCNA) contains the epitope for an inhibitory monoclonal antibody,
74B1, by the use of overlapping peptides that were tested for their
ability to inhibit the immunofluorescence flow cytometry assay of
cellular PCNA (18). This region is contained in the extended loop
region (residues 119-133) that extends across the surface of hPCNA and
connects the two conformationally conserved regions of PCNA that
constitute the basic structure of the hPCNA monomer (17). To assess the
importance of this loop in the interaction of hPCNA with pol , we
systematically mutated residues 122-133. The following mutants were
constructed: D122A, V123A, E124Q, Q125E, L126S, G127A, I128A, P129A,
E130Q, Q131E, E132Q, and Y133F. All of the PCNA mutants were expressed
as recombinant proteins in E. coli as described by Zhang
et al. (23) (see "Experimental Procedures"). The hPCNA
mutant proteins were expressed at levels comparable to that of the wild
type hPCNA as soluble proteins. The purification of hPCNA mutant
proteins was monitored by SDS-PAGE, and all were purified to
near-homogeneity as judged by SDS-PAGE (Fig.
1).
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Chemical Cross-linking-- The ability of the hPCNA mutants to engage in normal subunit-subunit interactions was tested by chemical cross-linking using ethylene glycol bis(succinimidyl succinate), which we have shown previously to result in the rapid formation of a cross-linked dimeric species as well as smaller amounts of higher oligomers (23). All the hPCNA mutants behaved like the wild type hPCNA (data not shown), i.e. they retained the ability for monomer-monomer protein interactions. These results show that the mutations in the loop region did not appear to affect subunit interactions of PCNA, a possibility that could lead to loss of function. The CD spectra of the hPCNA mutants in the near and far UV were examined and were not significantly different from the wild type, indicating that there were no gross secondary and tertiary structural changes in the PCNA mutants (not shown).
Effects of Mutations in the Interdomain Connector Loop on the
Immunoreactivity of PCNA--
Because we had shown previously that an
antibody which inhibited the PCNA stimulation of pol possessed an
epitope that mapped to the interdomain connector loop (18), we tested
the immunoreactivity of the mutants. This inhibitory antibody, 74B1,
was used to Western blot the hPCNA mutants. All of the mutants could be
Western blotted by the inhibitory monoclonal antibody (74B1), with the
exception of the mutant G127A (Fig. 2,
lower panel). This result supports the previous assignment
of the epitope of antibody 74B1 to residues 121-135 of PCNA by the use
of overlapping peptides (18), and also suggests that Gly-127 is
an important determinant in the antibody epitope. As a control, a
commercially available PCNA monoclonal antibody (mAb19F4) (Fig. 2,
upper panel) was also tested. This antibody, whose epitope
was mapped to residues 111-125 (18), was able to detect all of the
mutants.
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Functional Analysis of the PCNA Mutants--
The functional
behavior of the hPCNA mutants in terms of their effects on in
vitro DNA synthesis catalyzed by pol were examined with both
poly(dA)·oligo(dT) and singly primed M13 templates (Fig. 4, A and B). The
concentration dependence of the hPCNA mutants for the stimulation of
pol
activity on the poly(dA)·oligo(dT) template was examined.
Eight of the 12 hPCNA mutants showed little or no difference in their
ability to stimulate DNA synthesis catalyzed by pol
, and exhibited
similar concentration dependences as the wild type hPCNA (Fig.
4A). Mutant L126S was able to stimulate pol
in this
assay to a level similar to that of the wild type, but exhibited a very
clear difference in concentration dependence. The mutants V123A, I128A,
and G127A, in that order, showed a severe loss of ability to stimulate
pol
activity (Fig. 4A). The results obtained when the
mutants were tested with the singly primed M13 assay in which DNA
synthesis is dependent on RF-C and RPA are shown in Fig. 4B.
The results were qualitatively similar to those observed with the
poly(dA)·oligo(dT) template, in that the same mutants showed the most
significant losses of activity. These results suggest that none of
these mutants is specifically impaired in their abilities to interact
with RF-C. In addition, we measured the RF-C-catalyzed loading of PCNA
onto DNA by the ability of PCNA to stimulate of the ATPase activity of
RF-C. ATPase assays were performed with all the mutants as described by
Fukuda et al. (28), and all of the mutants exhibited a
similar ability to stimulate the ATPase activity of RF-C as wild type
hPCNA (not shown).
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Interaction of the PCNA Mutants with pol --
The interaction
of the hPCNA mutants with pol
was tested by ELISA. In this assay,
hPCNA mutants were immobilized on microtiter plates, and the binding of
pol
was detected using antibody mAb78F5. The results are shown in
Fig. 6. Mutants G127A and I128A exhibited the weakest interaction with pol
. Mutants Q131E and E132Q were also
weak in their interaction with pol
in comparison to the wild
type.
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Electrophoretic Mobility Shift Assays (EMSA)--
To gain further
insight into the interactions of the mutant PCNAs with pol , we
performed EMSA as described under "Experimental Procedures." In
these assays, the ability of PCNA to enhance the binding of pol
to
a labeled DNA template-primer by a shift of the primer in nondenaturing
gel electrophoresis is examined. The results of this analysis are shown
in Fig. 7. Formation of the complexes was
dependent on the simultaneous presence of pol
, PCNA, and the model
template-primer (Fig. 7, lanes 1-5). The formation of pol
-PCNA-template-primer complexes by gel shift assays was detected
with wild type PCNA and with 5 of the 12 mutants (D122A, E124Q, Q125E,
E130Q, and Y133F). Mutants V123A, L126S, G127A, I128A, P129A, Q131E,
and E132Q did not form pol
-PCNA-template-primer complexes in this
assay (Fig. 7). Several EMSA analyses were performed with different
concentrations of mutants, and the results were reproducible.
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DISCUSSION |
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We have systematically mutated each of the residues between 122 and 133 to further define the structural elements in PCNA that are
involved in its interaction with pol . The choice of this region was
based on our previous assignment of the epitope of the inhibitory
monoclonal antibody 74B1 to amino acid residues 121-135 of hPCNA by
the use of peptides (18). To confirm that this was a rational basis for
the functional evaluation of the mutants, we first characterized their
immunochemical properties. Our results confirm the assignment of the
epitope of antibody 74B1. One of the 12 PCNA mutants, G127A, could not
be Western blotted with the 74B1 antibody (Fig. 2). This lack of
interaction of the mutant G127A with the monoclonal antibody 74B1
provides solid evidence that the region between residues 122-133 of
hPCNA indeed harbors the epitope for 74B1. The ELISA assays showed that within this connector loop region, residues Asp-122, Glu-124, and
Gly-127 to Glu-130 are also involved in the interaction between antibody 74B1 and hPCNA. It is surprising that mutation of the Gly-127
exhibited such a pronounced effect on the interaction between hPCNA and
pol
. However, any mutation of glycine, because of the insertion of
a large R group, may be sufficient to disrupt the local structure of
the epitope that is required for interaction with the monoclonal
antibody. Thus, our findings confirm the epitope assignment of
monoclonal antibody 74B1 and extend our previous work, in terms of the
interpretation of the inhibitory effects of this antibody as being due
to its binding to a region involved in protein-protein interaction with
pol
. These findings are significant because the inhibition of PCNA
stimulation of pol
by this antibody can now be confirmed to be due
to its direct interference with the binding of PCNA to pol
.
The analysis of the effects of mutations on residues 122-133 in the
interdomain connector of PCNA on the interaction with pol was
examined in this study at several levels. These studies provide further
evidence that this region of hPCNA is directly involved in binding to
pol
. Mutation of Gly-127 and Ile-128 resulted in severe defects in
the stimulation of pol
. Two other mutants, V123A and L126S,
exhibited less severe defects in the stimulation of pol
activity.
This loss of activity was established to be due to a weakened
interaction between pol
and PCNA by an ELISA assay in the case of
Gly-127 and Ile-128. EMSAs that measured the ability of the PCNA and
pol
to form a ternary complex with a model substrate template
proved to be a much more sensitive index of the effects of mutant on
PCNA-pol
interactions, as complex formation was lost with the same
four mutants but also with P129A, L126S, and V123A. Our findings are
summarized in Table I. This shows quite
clearly that mutations of Gly-127 and Ile-128 had a pronounced impact
on the ability of human PCNA to stimulate the human pol
activity. A
possible explanation for the pronounced impact of mutation of Gly-127
is that it causes a change in the local structure of the interdomain
connector loop of hPCNA, which makes this region of hPCNA less
accessible to pol
. The interaction profile of the PCNA mutants with
monoclonal antibody 74B1 is consistent with this explanation.
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The loss of the ability of the PCNA mutants to stimulate in
vitro DNA synthesis catalyzed by pol could be due to (i) the disruption of the trimer formation of hPCNA, (ii) inhibition of the
RF-C-catalyzed loading of PCNA onto DNA, (iii) prevention of the
binding of pol
to the hPCNA, or (iv) interference in the
translocation of the PCNA-pol
complex along the DNA template. Chemical cross-linking assays showed that the first possibility is
unlikely, and in addition, the CD spectrum of the hPCNA mutants essentially showed no secondary structural changes due to mutations. The PCNA mutants showed similar functional behavior with respect to the
RF-C-catalyzed loading of PCNA onto DNA, with either the poly(dA)·oligo(dT) assays or the M13 template-primers, indicating that the inability of the four PCNA mutants to stimulate pol
was
not due to a loss in the interaction between RF-C and PCNA, because
RF-C is not required in the poly(dA)·oligo(dT) assays. It is also
unlikely that the loss in the ability to stimulate DNA synthesis is the
result of interference in the translocation of PCNA clamp or pol
along the DNA template because EMSA and ELISA assays showed
unequivocally that the defect of these four PCNA mutants were a
consequence of the loss of the binding of pol
to PCNA.
The formation of ternary pol -PCNA-template-primer complexes was not
detected using the hPCNA mutants, V123A, L126S, G127A, I128A, P129A,
Q131E, and E132Q (Fig. 7). The stability of the pol
-PCNA-template-primer complex is determined by two factors: the
interaction between pol
and PCNA, and the interaction between the
pol
-PCNA complex and the DNA template-primer. Disruption of either
interaction could lead to the dissociation of the pol
-PCNA-template-primer complex. As expected, the four PCNA mutants (V123A, L126S, G127A, and I128A) that displayed a severe loss in their
ability to stimulate pol
activity fell within this group,
indicating that the loss of these four PCNA mutants in the stimulation
of pol
activity was the result of an altered interaction with pol
. However, mutants P129A, Q131E, and E132Q, which are still
functional in the stimulation of pol
activity, showed loss of
function in this assay. It is possible that even if the interactions
between the pol
-PCNA complex and the DNA template-primer were
weakened such that they were not detected by EMSA, they might still be
sufficient to stimulate DNA synthesis effectively.
The crystal structure of hPCNA complexed with a 22-residue peptide
derived from the C terminus of cell-cycle checkpoint protein p21
(residues 139-160) has been solved recently (17). This reveals that
the p21 peptide forms an extended contact with the interdomain connecting loop (17, 31). There are three major structural elements of
PCNA that are involved in the interaction with p21. First, the
formation of an anti-parallel sheet structure between residues
152-160 of p21 and residues 119-127 of PCNA. Following this region of
interaction, there is a second major interaction involving residues
Leu-126, Ile-128, and Tyr-133 of the PCNA loop. These three residues,
together with Met-40, Val-45, Leu-47, Pro-234, Tyr-250, Ala-252 ,and
Pro-253, form a large hydrophobic pocket that interacts with residues
147-151 (MTDFY) of p21. Gly-127, Ile-128, and Pro-129 form one edge of
a binding cleft in the large hydrophobic pocket into which the side
chains of Met-147, Phe-150, and Tyr-151 of p21 are inserted. There is a
small hydrophobic pocket, formed by residues Leu-121 and Val-123 of the
PCNA loop, which interacts with Ile-158 of the p21 peptide (17). When
the interaction sites of p21 are listed against the mutants of PCNA that affect its interaction with pol
(Table I), it is seen that
there is a very close correlation between the residues whose mutation
are strongly affected and those involved in the PCNA/p21 peptide
interaction. There is strong evidence that the effects of p21 on the
inhibition of DNA pol
activity is mediated by a competition with
PCNA (12, 13). The current information of the p21-PCNA structure is
thus highly relevant, as it leads to a reasonable proposition that the
binding of pol
to PCNA may involve similar interactions as with p21
to PCNA, namely interaction of an extended region of peptide sequence
with the interdomain loop via formation of an anti-parallel
sheet
structure and possibly interaction with the two hydrophobic pockets.
The proposal that a short peptide sequence of pol
is involved in
the interaction with PCNA is supported by experiments showing that a
synthetic peptide corresponding to residues 129-149 of pol
is able
to inhibit PCNA activation of pol
(32). This sequence is part of a
conserved region in the N terminus between yeast, mammalian, Epstein-Barr, and herpes viral DNA polymerases (33).
There have been several other mutational studies of PCNA, which,
however, have not provided information on the role of the interdomain
connector loop in the interaction with pol . A systematic analysis
based on the mutation of 29 charged residues of hPCNA to alanine on the
basis that alteration of charged residues might have the least effect
on the structure of PCNA has been reported (28). Eight of the mutants
(Lys-13, Lys-14, Lys-20, Lys-77, Lys-110, Arg-146, Arg-149, and
Lys-217) showed defects in the stimulation of pol
; these residues
are located in the helices on the inner surface of the PCNA toroid. Of
the residues mutated that lie on the outer surface of PCNA, effects on
pol
activity were observed with Asp-41, Arg-64, and Asp-122. The
activities reported for the stimulation of pol
were <50% for the
Asp-41 and between 50 and 80% for the Arg-64 and Asp-122 mutants. The latter is one of the residues we mutated; however, we did not observe
significant effects in any of the functional assays. A mutational
analysis of Saccharomyces cerevisiae PCNA in which 21 pairs
of proximal charged residues were mutated has also been reported (34).
In this study, the D41A/D42A mutation exhibited a reduced activity with
pol
. Two double mutants in the loop region, K127A/E129A and
E129A/E130A, displayed no phenotypic defects. Arroyo et al.
(35) have mutated Schizosaccharomyces pombe PCNA, choosing 7 residues in the loop regions of PCNA that are conserved in S. pombe, S. cerevisiae, rice, and human PCNA. Their results showed
that mutation of S. pombe Leu-2 and Arg-64 resulted in reduced abilities to stimulate pol
, and they concluded that these
residues may play a role in interaction with pol
. The agreement
between the fission yeast and the study of hPCNA that Arg-64 is
involved is of interest, as mutation of Asp-63 of hPCNA had no effect
(28). These studies show some agreement regarding the effects of Asp-41
and Arg-64 on PCNA stimulation of pol
. Asp-41 is located in the
loop between
sheets
C1 and
D1 of hPCNA, which forms one edge
of the large hydrophobic pocket of hPCNA. Arg-64 is located in the loop
between
sheets
E1 and
F1 of hPCNA, which provides residues
that form one edge of the small hydrophobic pocket of hPCNA. Mutation
of D41A could cause local structure perturbations of the large
hydrophobic pocket, whereas mutation of R64A could affect the local
structure of the small hydrophobic pocket, which includes Ala-67 and
Gly-69. While a direct interaction between residues Asp-41 and Arg-64
with pol
is not excluded, these findings provide provocative
evidence that the hydrophobic pockets that are involved in p21 binding may also be important in the binding of pol
. Leu-2 in S. pombe and S. cerevisiae PCNA is not conserved in hPCNA
where it is a phenylalanine residue. In both S. cerevisiae and human PCNA, this residue is buried but is adjacent
to residues that form the small hydrophobic pocket (7, 17).
Recently, a sequence motif has been identified in p21 (36), Fen1 (37),
Fen1 homologues in yeast (RTH1, RAD2, rad2, and rad13), and human
XP-G.2 All these proteins
bind to the same site in PCNA and have the consensus sequence
QGRLDXFF (37). Cdc27, a putative third subunit of DNA
polymerase in the fission yeast (38, 39), also contains a similar
sequence motif. Deletion or mutation of this motif in Cdc27 abolishes
PCNA binding in vitro and Cdc27 also binds to the
interdomain connector loop region of
PCNA.3 In p21, the cognate
sequence QTSMTDFY (residues 144-151) is contained within the binding
site with PCNA (17), and as discussed above, the Met, Phe, and Tyr
residues interact with the large hydrophobic pocket. The question
raised in relation to our studies is whether the N2 region of pol
also contains a sequence that can be recognized as a member of the
consensus. There is no obvious strong relationship; however, the N2
region (33) contains the highly conserved feature of 3 adjacent
aromatic residues, IHGFAPYFY (residues 141-149), which could
potentially serve the same role of providing a hydrophobic interaction
with PCNA.
Current research now provides a structural basis for the findings that
PCNA interacts with a number of other proteins in the form of the
identification of a putative peptide motif for PCNA binding (36-39),
and the description of the binding of p21 to PCNA at the atomic level
(17). Interestingly, the existence of a putative third subunit (Cdc27)
of yeast pol that binds to PCNA3 raises the issue of
redundancy in the binding of the pol
holoenzyme to PCNA, since our
studies indicate that the catalytic subunit of pol
(p125) interacts
directly with PCNA. An additional issue is whether it is p125 or the
third subunit (or both) that is responsible for the functional
interaction of pol
with PCNA. The enzyme we used in this study was
purified by the immunoaffinity chromatography and gel filtration, and
may contain other components besides the two subunit form that is
isolated after stringent conventional chromatography
(19).4 In our hands, the pol
p125 subunit overexpressed in Sf9 cells could be stimulated
only 5-fold with PCNA in the presence of recombinant p50 expressed in
E. coli (40). Polymerase
obtained by co-expression of
the p125 and p50 subunits (pol
core) in the baculovirus system could be stimulated by PCNA at most 8-fold, far less than the stimulation obtained by the holoenzyme prepared from the immunoaffinity column.4 Thus, it is possible that the full functional
effects of PCNA on pol
may involve multi-point interactions. That
these interactions may involve the same site on PCNA is feasible, since
its trimeric structure should contain three binding sites.
In summary, we have obtained mutational evidence for the localization
of the region of PCNA that is involved in binding to pol to the
interdomain connector loop of PCNA. This evidence is consistent with,
and strengthened by, a previous analysis of the epitope of an
inhibitory antibody. In addition, the recent molecular analysis of the
p21-PCNA interaction site, which is also localized to the interdomain
connector loop, provides a basis for the prediction that pol
may
interact in a similar way, and a rational mechanism for the ability of
p21 to inhibit DNA synthesis.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 31973.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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Current address: Dept. of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595. Tel.: 914-594-4070; Fax: 914-594-4058.
1
The abbreviations used are: pol , polymerase
; PCNA, proliferating cell nuclear antigen; RF-C, replication factor
C; RPA, replication protein A; PAGE, polyacrylamide gel
electrophoresis; PBS, phosphate-buffered saline; EMSA, electrophoretic
mobility shift assay; Bis-Tris,
bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane.
2 L. S. Cox, personal communication.
3 S. A. MacNeill, personal communication.
4 P. Zhang and M. Y. W. T. Lee, manuscript in preparation.
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