(Received for publication, October 24, 1994; and in revised form, January 4, 1995)
From the
Synthetic peptides to selected sequences in human DNA polymerase
(pol
) were used to identify the region involved in the
interaction of pol
to proliferating cell nuclear antigen.
Peptides corresponding to sequences in five regions in the amino
terminus of human pol
and three in the carboxyl terminus, which
are conserved with the yeast homologs of pol
, were tested. These
studies showed that the peptide corresponding to the N2 region
(residues 129-149) selectively and specifically inhibited the
PCNA stimulation of pol
. This inhibition was relieved by
titration with excess PCNA. The identification of the N-2 region as
being involved in PCNA binding was supported by studies that
demonstrated that the N2 peptide could bind PCNA. Deletion mutants of
pol
expressed in Sf9 cells provided evidence that the binding
region for PCNA was located in the first 182 residues of the amino
terminus. These studies provide reasonable evidence that residues
within the region 129-149 of pol
are involved in the
binding site for PCNA.
DNA polymerase (pol
) (
)was discovered in
1976 as a new mammalian DNA polymerase that was different from the
known mammalian DNA polymerases in possessing an associated 3` to 5`
exonuclease activity (Byrnes et al., 1976). pol
has been
isolated and characterized from calf thymus and human placenta as a
heterodimer of a catalytic subunit of 125 kDa and a 50-kDa subunit of
unknown function (Lee et al., 1984, 1991a). The discovery of a
stimulating factor for pol
(Lee et al., 1984; Tan et
al., 1986) eventually led to its identification as proliferating
cell nuclear antigen, or PCNA (Prelich et al., 1987a, 1987b).
This stimulated a remarkable advance in our understanding of the
assembly of proteins required for DNA synthesis at the replication fork
through the exploitation of an in vitro SV40 DNA replication
assay (Li and Kelly, 1984). PCNA and pol
were shown to be
essential for in vitro SV40 DNA replication, and the effect of
PCNA was shown to be that of a processivity factor (Weinberg and Kelly,
1989; Lee et al., 1989; Tsurimoto et al., 1990). It
has been proposed that PCNA, the T4 gene 45 protein, and the
subunit of Escherichia coli DNA polymerase III all function as
sliding DNA clamps and exist as toroidal proteins that encircle the DNA
strand (Kong et al., 1992; Kuriyan and O'Donnell, 1993).
The assembly of the pol
PCNA complex at the primer terminus
also requires the actions of RFC, a 5-subunit protein complex whose
function is to load PCNA onto the primer-template terminus (Lee et
al., 1991b).
There is a significant amount of information on
the structural aspects of the DNA polymerase protein family to which
pol belongs (Wang, 1991; Yang et al., 1992). Multiple
sequence alignments have shown the presence of a core region that
contains six highly conserved regions. It is generally accepted that a
number of amino acid residues located in the core region of DNA
polymerase are involved in the catalytic function of the proteins
(Blasco et al., 1992, 1993; Dong et al., 1993). There
is some conservation in the carboxyl-terminal regions, which contain
two zinc-finger motifs (Wang et al., 1991), which are thought
to be involved in DNA binding. The sites involved in the 3` to 5`
exonuclease activity of DNA polymerase have also been identified (Simon et al., 1991; Morrison et al., 1991). The
amino-terminal regions of the pol
family, however, were found to
be highly dissimilar (Wang, 1991). We have cloned the cDNA for human
pol
and have performed multiple sequence alignments that
established that human pol
is closely related to yeast pol
and to members of the herpesvirus DNA polymerases and is a member of
the pol
protein family (Yang et al., 1992). These
alignments allowed us to show for the first time that while it had been
thought that the amino-terminal regions of the polymerases were very
different, within members of this group there are some significant
relationships. We had identified five regions of conservation which we
have labeled N1-N5 (Yang et al., 1992; Hao et
al., 1992). Our interests are directed toward structure-function
aspects that are unique to pol
, viz. the identification
of the protein-protein interaction sites.
In this study we report
the use of synthetic peptides to the sequences in the amino-terminal
region to probe for the pol region that is involved in its
binding to PCNA. Additional experimental approaches using deletion
mutants of pol
also support this hypothesis.
The peptides that were synthesized are shown in Table 1. In addition to the amino-terminal peptides, three
peptides covering the most carboxyl-terminal 61 residues of pol
were also synthesized. These peptides were then tested for their
ability to inhibit the PCNA stimulation of pol
activity using
sparsely primed poly (dA)
oligo(dT) as a template-primer. The
results are shown in Fig. 1. Of the peptides tested, only the
peptide N2 exhibited an ability to inhibit the PCNA stimulation of pol
. The N2 peptide inhibited 80% of the PCNA-stimulated DNA
polymerase activity at a concentration of 20 µM. At a
concentration of 50 µM, >95% of pol
enzymatic
activity was inhibited by the N2 peptide. The amino-terminal region
peptides N1, N3, N4, and N5 did not inhibit the PCNA stimulation of pol
(Fig. 1). The N2a and N2b peptides, which are the two
halves of the N2 peptide, also did not inhibit PCNA stimulation of pol
activity either singly or in combination (not shown). The
carboxyl-terminal synthetic peptides did not have any significant
effect on PCNA stimulation of pol
, although peptide C2 exhibited
a weak inhibition (35%) at a concentration of 100 µM (not
shown). These results indicated that the inhibition of pol
activity was due to interference with PCNA binding and was a specific
rather than a nonspecific effect. Analysis of the reaction products of
the pol
reaction by polyacrylamide gel electrophoresis was
performed (Fig. 2). These confirmed that only the N2 peptide
inhibited the reaction, as shown by the loss of processivity in its
presence.
Figure 1:
Effects of synthetic
peptides on the PCNA-stimulation of pol activity. Purified human
placental pol
was assayed using poly(dA)
oligo(dT) as the
template-primer in the presence of PCNA (see ``Experimental
Procedures'') and in the presence of increasing concentrations of
the peptides N1, N2, N3, N4, and N5 (Table 1). Data are shown as percent of
control activities in the absence of the
peptide.
Figure 2:
Effect of synthetic peptides on the pol
processivity. The effects of peptides on the processivity of the
pol
reaction were examined by polyacryalmide gel electrophoresis
of the reaction products as described under ``Experimental
Procedures.'' The lanes show the results in terms of additions to
the basic reaction mixture, which contains the template and labeled
nucleotides as follows: lane1, no addition; lane2, + PCNA; lane3, + pol
; lane4, + pol
+ PCNA; lane5, + pol
+ PCNA + N1; lane6, + pol
+ PCNA + N2a; lane7, + pol
+ PCNA + N2b; lane8, + pol
+ PCNA + N2; lane9, + pol
+ PCNA + N3; lane10, + pol
+ PCNA + N4; lane11, + pol
+ PCNA + N5; lane12, + pol
+ PCNA + C1; lane13, + pol
+ PCNA + C2; lane14, + pol
+ PCNA + C3; lane15, 1-kilobase ladder (Life Technologies, Inc.).
Peptides, where added, were at concentrations of 50
µM
Experiments to determine if the inhibition by peptide
could be competed against by titration with PCNA were performed (Fig. 3). Addition of increasing amounts of PCNA reversed the
inhibition. In the presence of 25 µM of peptide, 100% of
pol activity was recovered by as little as 1 µM PCNA. These results showed that PCNA readily competes against the
inhibitory effect of peptide N2 on PCNA-stimulated pol
activity.
These results support the view that N2 peptide competes with pol
for binding to PCNA.
Figure 3:
Reversibility of the N2 peptide inhibition
of pol by saturation with PCNA. Human pol
was assayed in
the presence of the indicated concentrations of N2, with increasing
amounts of PCNA. The data were plotted as percent of the control
PCNA-stimulated activity in the absence of added peptide
N2.
Figure 4: Dot blot analysis of the binding of PCNA to synthetic peptides. The synthetic peptides were dotted onto nitrocellulose membranes, which were then blocked and incubated with PCNA. The binding of PCNA to the membranes was then visualized by immunoblotting methods using an antibody to PCNA (see ``Experimental Procedures''). B refers to bovine serum albumin; P refers to PCNA.
The peptide N2 was immobilized on
CH-Sepharose, and the support was tested for its ability to bind PCNA.
An E. coli lysate expressing recombinant human PCNA was treated with Sepharose beads to which either N2 or N4 had
been attached, and the beads were washed, extracted with SDS buffer,
and then Western blotted (see ``Experimental Procedures'').
The results showed that the N2-beads were capable of binding PCNA as
shown by SDS-PAGE and silver staining (Fig. 5, leftpanel) or by Western blotting (Fig. 5, rightpanel). The binding was prevented by the addition of free
N2 peptide. The other peptides (Table 1) were also coupled to
CH-Sepharose, but all of these except N2 gave negative results as shown
for N4-CH-Sepharose in Fig. 5. Similar tests of bovine serum
albumin coupled to Sepharose were also negative. In other experiments,
similar results were obtained using a crude calf thymus extract in that
only the immobilized N2 peptide was able to bind PCNA.
Figure 5:
Binding of PCNA to immobilized N2 peptide.
N2 peptide was covalently coupled to CH-Sepharose. The Sepharose beads
(40 µl) were then used to bind PCNA from 0.8 ml of an E. coli lysate expressing the recombinant protein. After stringent washing
to remove nonspecifically bound material, the beads were extracted with
SDS-buffer and subjected to SDS-PAGE and Western blotting using an
antibody to PCNA (see ``Experimental Procedures''). Leftpanel, silver staining of SDS-PAGE. Lane1, material bound to N4-CH-Sepharose; lane2, material bound to N2-CH-Sepharose; lane3, material bound to N2-CH-Sepharose from a mixture of
the lysate with N2 peptide (1 mg); S, protein standards
(-macroglobulin, 180 kDa;
-galactosidase, 116
kDa; fructose 6-phosphate kinase, 84 kDa; pyruvate kinase, 58 kDa;
fumarase, 48 kDa; lactate dehydrogenase, 36 kDa; triosephosphate
isomerase, 26 kDa). Rightpanel, as in leftpanel but Western blotted with anti-PCNA antibodies.
(Authentic PCNA migrates between the 26- and 36-kDa
standards.)
Figure 6:
Deletion mutants of pol . The upperbar shows a diagram of the pol
sequence.
The boxes represent regions that are conserved with other DNA
polymerases as defined by Yang et al.(1992). Lowerbars show diagrammatically the deletions that were
made.
Figure 7:
Analysis of the ability of deletion
mutants of pol to bind to PCNA. Sf9 cells were co-infected with
recombinant baculovirus vectors for the full-length human pol
sequence (7) and the deletion mutants
2-249,
186-321,
336-715,
778-1047, and
674-1107 together with a baculovirus vector for human PCNA.
Cell lysates from each of the cultures were then immunoprecipitated
with an antibody to human PCNA. The immunoprecipitates were then
Western blotted with a mixture of two monoclonal antibodies to the
amino- and carboxyl-terminal regions of pol
. Control experiments
(not shown) indicated that levels of expression of the deletion mutants
were similar as determined by Western blotting of the Sf9 cell lysates. sfi
refers to the
674-1107
mutant.
These
mutants have not been fully characterized, but our preliminary studies
show that the possibility of interference by contaminating endogenous
baculovirus or insect cell DNA polymerases can be ruled out. The
full-length pol expressed in baculovirus is active and is
stimulated by PCNA, while the
2-249 deletion mutant is not
stimulated by PCNA.
Synthetic peptides were used to identify a site on pol
that is involved in its protein-protein interaction with PCNA. Our
results show that a 21-residue peptide containing the sequence of a
conserved region (N2) in the amino terminus of pol
is capable of
inhibiting the interaction of pol
with PCNA, presumably by
competitive binding to PCNA. The identification of this region as the
PCNA binding site is supported by the fact that this inhibition can be
competed out by further addition of PCNA and by the fact that the half
peptides of N2 (N2a, N2b) do not inhibit. Peptides have been usefully
employed for the study of protein interaction sites, e.g. for
epitope mapping and for structure-activity studies of peptide hormones
(Hruby et al., 1992). The use of synthetic peptides for
studies of protein-protein interaction sites in proteins has been more
limited, but have been successfully used in studies of the
autoinhibitory domains of the calcium/calmodulin protein kinases
(Soderling, 1990).
The peptide inhibition studies are supported by
the direct demonstration that the N2 peptide has the ability to bind to
PCNA, either by dot blotting or by binding of PCNA to immobilized N2
peptide. Analysis of deletion mutants of pol coexpressed with
PCNA in Sf9 cells by recombinant baculoviruses also confirm that the
PCNA binding region is located within the first 185 residues of the
amino terminus of pol
. The latter result is consistent with the
observations that the deletion of the first 220 amino acids of yeast
pol
(Brown and Campbell, 1993) result in loss of PCNA activation.
The successful use of the 21-residue N2 synthetic polypeptide also
indicates that the PCNA interaction site on pol
is limited to
this linear sequence of peptide, i.e. it does not require the
full tertiary structure of the protein, and is located within a
contiguous peptide sequence.
We have overexpressed human PCNA in E. coli and have shown that free PCNA at low concentration
exists in a dimer-trimer equilibrium and that discrete dimer and trimer
forms can be observed on gel permeation HPLC. These data
are consistent with the proposal that PCNA is a trimeric species that
forms a toroid by analogy with the E. coli DNA polymerase III
subunit and the T4 gene 45 protein based on sequence alignment of
the three proteins (Kong et al., 1992). In the
subunit,
each of the monomers possesses three structurally similar domains. If
PCNA is a trimeric species, then clearly the symmetry of the molecule
dictates that there are three potential binding sites for pol
, so
that the possibility that PCNA
pol
complexes involving more
than one pol
molecule exists.
The interaction of PCNA with the
peptides (and hence pol ) is independent of the presence of DNA.
We have shown that immobilized PCNA is able to bind pol
in the
absence of DNA. (
)Our studies show that the binding of pol
to PCNA is mediated by a region of peptide sequence within the
21-residue peptide in the amino terminus of pol
region. The N2
region can be characterized by the consensus
GX
GX
GX
YFY
between human, Saccharomyces cerevisiae, and Schizosaccharomyces pombe pol
. There are a number of
glycine-rich motifs known, including the nucleotide binding phosphate
or p-loop motif that is present in several protein families (Saraste et al., 1990) and the GXGXXG motif for the
binding of adenine nucleotides of the serine protein kinases (Knighton et al., 1991). However, since the crystal structure of pol
is unknown, the significance of this repeat in the N2 region is
uncertain. The question is raised as to whether this site is the only
site responsible for the binding of PCNA. This cannot be fully
ascertained, but the strong inhibition of binding by the peptide
suggests that this may indeed be the case.
These studies provide the
first identification of a protein-protein interaction site in human pol
in the form of its interaction site with PCNA. Given that the
assembly of pol
at the primer-template terminus requires a number
of accessory proteins, the location and nature of these
protein-interaction sites are therefore of some interest. It is now
clear that the formation of the replication complex, and possibly the
regulation of polymerase
in the cell cycle, may involve
protein-protein interactions with multiple protein partners, including
replication factor C and the cyclin-dependent kinase systems. It is
therefore of interest that recent studies have shown that PCNA can
interact with several cyclins, cyclin-dependent kinases, and p21 (Xiong et al., 1992, 1993; Brott et al., 1993). Recently,
p21 has been shown to inhibit SV40 DNA replication in vitro (Waga et al., 1994; Flores-Rozas et al., 1994)
in addition to its ability to inhibit cyclin-dependent kinase. Because
pol
activity requires the presence of PCNA, modulation of its
interaction with PCNA may be an important mechanism for the regulation
of pol
activity and hence of DNA replication, and a knowledge of
the structural basis for these protein-protein interactions may be of
significance. Further examination of the role of the N2 region in
mediating the interaction of pol
with PCNA is being performed by
site-directed mutagenesis.