(Received for publication, August 12, 1996, and in revised form, October 10, 1996)
From the Graduate Program in Molecular Biology,
Memorial Sloan-Kettering Cancer Center, New York, the
§ Department of Microbiology, Howard Hughes Medical
Institute, Cornell University Medical College, New York,
the¶ Laboratory of Molecular Biophysics, Howard Hughes Medical
Institute, The Rockefeller University, New York, NY 10021, and the
Department of Biochemistry and Molecular Biophysics, Washington
University School of Medicine, St. Louis, Missouri 63110
In eukaryotes, processive DNA synthesis catalyzed
by DNA polymerases and
(pol
and
) requires the
proliferating cell nuclear antigen (PCNA). It has recently been shown
that in humans (h), the PCNA function, required for both DNA
replication and nucleotide excision repair, can be inactivated by
p21CIP1 due to a specific interaction between hPCNA and the
carboxyl terminus of p21CIP1. In this report, we show that
Saccharomyces cerevisiae (S. cerevisiae) PCNA-dependent pol
-catalyzed DNA synthesis was
inhibited less efficiently than the human system by the intact
p21CIP1 protein and was unaffected by the p21CIP1
carboxyl-terminal peptide (codons 139-160). This species-specific response of PCNA to p21CIP1-mediated inhibition of DNA
synthesis results from a marked difference in the ability of h and
S. cerevisiae PCNA to interact with p21CIP1. As
shown by binding studies using the surface plasmon resonance technique,
hPCNA binds both full-length p21CIP1 and the p21CIP1
peptide-(139-160) stoichiometrically with a similar affinity (KD~ 2.5 nM) while S. cerevisiae PCNA binds p21CIP1 with ~10-fold less
affinity and does not interact with the p21CIP1
peptide-(139-160).
The DNA replication machinery is functionally conserved from
bacteria to mammals. The mechanism underlying DNA synthesis of primed
templates in T4 bacteriophage (1), Escherichia coli (2),
Saccharomyces cerevisiae (3), and humans (4, 5) depends on a
protein complex (the "clamp-loader") that binds to a
primer-template junction. In each case, the multisubunit clamp loader
complex loads a toroidal shaped protein (the "clamp") (6-8) onto a
primer end in an ATP-dependent reaction. The DNA
polymerases that catalyze deoxynucleotide incorporation are tethered to
DNA by a direct interaction with the clamp. This interaction, which occurs independently of the clamp loader, converts DNA polymerases from
dispersive into highly processive enzymes capable of catalyzing extensive DNA synthesis (9-12). Furthermore, chromosomal replicases of
E. coli (pol1 III core, complex, and
), phage T4 (T4gp43, T4gp44·62 complex, and T4gp45)
and eukaryotes (pol
or
, RF-C, and PCNA) not only possess
functional similarities but, in many cases, show marked sequence
conservation (13, 14).
In eukaryotes, insight into chromosomal DNA synthesis has been derived
from in vitro studies of simian virus 40 replication. With
the exception of one viral encoded protein, the SV40 T antigen, this
replication pathway is dependent on the cellular replication machinery
(15-17). The essential host proteins have been isolated, and in most
cases their detailed mechanisms have been elucidated (9, 10, 18, 19).
The DNA polymerase ·DNA primase complex is responsible for the
initiation of replication. DNA primase generates oligoribonucleotide
primers that are elongated by pol
for about 30 nucleotides
resulting in the accumulation of pre-Okazaki fragments. In the absence
of proteins that bind to 3
-hydroxyl primer ends, pol
is capable of
rebinding and elongating chains to mature Okazaki fragments on the
lagging strand and generating DNA chains that are approximately
one-half the length of the circular duplex DNA template on the leading
strand (20). This non-physiological mechanism, referred to as the
monopolymerase system, requires relatively high levels of the pol
·primase complex. When RFC and PCNA are present, they interfere
with pol
rebinding by binding to the 3
-hydroxyl primer ends where
they facilitate the tethering of pol
(or pol
) (3, 5, 21). As a
result, both leading strand synthesis and the completion of lagging
strand pre-Okazaki fragments depend on pol
(or pol
and pol
).
In eukaryotes, PCNA has been shown to be the target of a number of factors that control cell growth. One such factor, p21CIP1, is a checkpoint protein that acts as an antimitogenic signal by binding to and inhibiting cyclin-dependent kinases as well as by binding to PCNA and inhibiting in vitro PCNA-dependent DNA replication (22-29). These two disparate inhibitory functions have been shown to reside in separate domains of p21CIP1, a 164-amino acid protein. The cyclin-dependent kinase inhibitory activity of p21CIP1 is located within the amino-terminal domain while the PCNA binding region resides in the carboxyl-terminal domain. These distinct inhibitory activities have been demonstrated both in vitro and in vivo using the overexpressed amino- or carboxyl-terminal domains of p21CIP1 (30, 31).
A peptide derived from amino acids 141-160 within the
carboxyl-terminal domain of p21CIP1 that binds PCNA and
inhibits PCNA-dependent DNA synthesis in vitro
has previously been described (32). In this report, we have examined
the species specificity involved in the pol holoenzyme system using
enzymes isolated from S. cerevisiae and HeLa cells and have
shown that a chemically synthesized peptide that spans amino acid
residues 139-160 of p21CIP1 specifically inhibits reactions
dependent on human PCNA. This p21CIP1 peptide-(139-160) had no
effect on elongation reactions dependent on S. cerevisiae
PCNA, although both the full-length p21CIP1 protein and a
truncated p21CIP1 protein (truncated p21CIP1
protein-(70-164)) inhibited the S. cerevisiae pol
holoenzyme system albeit to a lesser extent than that observed with the
human pol
holoenzyme system.
Binding studies using surface plasmon resonance demonstrated that hPCNA binds p21CIP1 and the p21CIP1 peptide-(139-160) stoichiometrically (~three molecules bound per hPCNA trimer) with a similar affinity (KD~2.5 nM). The affinity between S. cerevisiae PCNA and p21CIP1 was 10-fold less than the affinity of hPCNA for p21CIP1, and there was no detectable interaction between the p21CIP1 peptide-(139-160) and S. cerevisiae PCNA. Recently the crystal structure of the p21CIP1 peptide-(139-160) complexed with hPCNA has been elucidated (33). Based on this structure and knowledge of the specific amino acid differences between S. cerevisiae and hPCNA, we propose a model explaining the differential effects of p21CIP1 and its derivatives on S. cerevisiae and hPCNA-dependent reactions.
The following proteins were
prepared as described previously: hpol , hRFC, hPCNA, HSSB, S. cerevisiae pol
, S. cerevisiae RFC, S. cerevisiae PCNA, p21CIP1,
p21CIP1peptide-(139-160), and hPCNA with an amino-terminal
cAMP-dependent protein kinase site (34-39).
The following proteins were commercially obtained: cAMP-dependent protein kinase (Sigma), T4gp32 protein, and E. coli SSB (Pharmacia Biotech, Inc.). The peptide acetyl-Gly-Arg-Lys-Arg-Arg-Gln-Thr-Arg-Leu-Ile-Phe-Ser-NH2 (molecular mass, 1.559 kDa) was obtained as a generous gift from Merck and is derived from amino acids 139-160 of p21CIP1 with a deletion of amino acids 146-155 (p21CIP1 peptide-(139-145+156-160) see Table II). The biotinylated p21CIP1 peptide-(139-160) was synthesized in the Microchemistry Laboratory of Sloan-Kettering Cancer Institute. These peptides were stored under similar conditions as those described for the p21CIP1 peptide-(139-160) (38).
|
PCNA containing a cAMP-protein kinase recognition site at its
amino-terminal domain was phosphorylated as follows. A reaction mixture
(40 µl) containing 20 mM Tris-HCl (pH 7.5), 12 mM Mg(OAc)2, 2 mM dithiothreitol,
0.1 M NaCl, 16.5 pmol of [-32P]ATP
(1.1 × 107 cpm/pmol), 65 pmol of amino-terminal
tagged PCNA, and 6.66 units of cAMP-protein kinase was incubated at
37 °C for 30 min. The reaction was halted by the addition of 2 µl
of 0.5 M EDTA, and aliquots, pre- and post-acid
precipitation (filtered through GFC glass filters in the latter case),
were counted by liquid scintillation. Approximately 60% of the
32P was recovered in PCNA, and its specific activity was
~1500 cpm/fmol. SDS-polyacrylamide gel electrophoresis analysis
indicated that PCNA was the only labeled protein formed (data not
shown).
Singly-primed DNA was isolated following hybridization of a 34-mer oligonucleotide to nucleotides 6300-6333 of circular M13 mp 7 (7.2 kilobases) DNA (containing ~10% linear molecules). The annealed product was 32P-labeled following the incorporation of a single dCMP residue (residue 6299) by Klenow fragment (Boehringer Mannheim). After phenol-CHCl3 extraction, the reaction mixture was filtered through a G-50 Sephadex column, and the excluded labeled singly-primed M13 DNA (1000-2000 cpm/fmol) was stored in buffer containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA at 4 °C.
Singly-nicked pBS DNA was prepared in a reaction mixture (1 ml) containing 300 µg/ml ethidium bromide, 5 mM Tris-HCl (pH 7.5), 125 mM NaCl, 0.1 mg/ml bovine serum albumin, 20 mM MgCl2, 400 µg of pBS DNA, and 12 µg of pancreatic DNase I that was incubated for 25 min at 37 °C. An equal volume of a solution of buffered-saturated phenol (Life Technologies, Inc.) was added followed by extraction of the aqueous phase with an equal volume of phenol-CHCl3/isoamyl alcohol (24:1, v/v). The aqueous phase was adjusted to 0.1 M NaCl, and 2 volumes of ethanol were added. The solution was centrifuged, dried in vacuo, and dissolved in 0.2 ml of 0.1 M NaCl in TE buffer (10 mM Tris-HCl (pH 8.0) + 1 mM EDTA (pH 8.0)). Electrophoresis of this DNA through a 1.2% alkaline agarose gel (30 mM NaOH, 1 mM EDTA) indicated the presence of an equal mixture of single-stranded circular DNA molecules and linear DNA fragments, reflecting the presence of a single nick in the duplex circular DNA. Consistent with this, agarose gel electrophoresis in TAE buffer (0.04 M Tris acetate + 1 mM EDTA (pH 8.0)) containing ethidium bromide indicated that the DNA had been quantitatively converted from a RF I structure to a RF II structure.
Protein Interaction Analyses and Determination of Kinetic ParametersThe immobilization of hPCNA, S. cerevisiae PCNA, p21CIP1, and the p21CIP1peptide-(139-160) to sensor chips was carried out using the carbodiimide covalent linkage protocol specified in the manufacturer's instructions (Pharmacia Biotech Inc.). The interaction between each immobilized ligand and these proteins in solution was then followed by monitoring changes in surface concentration on the sensor chip using the BIAcore 2000. The equilibrium dissociation constants (KD) for hPCNA binding to p21 and the p21CIP1 peptide-(139-160) were determined following passage of several concentrations of hPCNA over sensor chip surfaces to which each of these proteins had been covalently coupled. Both the BIAcore kinetic evaluation software version 1.2 and Scatchard analysis techniques were employed to determine KD values.
Singly-primed M13 Elongation and PCNA Loading AssaysThe
elongation of singly-primed M13 DNA and the subsequent analysis by
alkaline agarose gel electrophoresis were carried out as described (5)
with modifications as outlined in the figure legends. PCNA loading
experiments were carried out as described previously for (40).
The elongation
of a [32P]dCMP-labeled oligonucleotide primer (35 nucleotides) hybridized to circular single-stranded M13 mp 7 (7.2 kilobases) DNA by both the h and S. cerevisiae pol holoenzymes was compared (Fig. 1). Three different DNA
binding proteins, HSSB, E. coli SSB, and the T4 gp 32, were
compared in each system. HSSB was more effective than the other SSBs in
supporting elongation of the primed template by the human pol
holoenzyme (Fig. 1, lanes 1-3), whereas all three SSBs
supported elongation of the labeled primer by the yeast pol
holoenzyme to a similar extent (lanes 4-6). For this reason
all subsequent experiments were carried out using HSSB as the DNA
binding protein.
The six different permutations possible for reconstituting the pol holoenzyme from combinations of h and S. cerevisiae PCNA, RFC and pol
proteins were examined (Fig. 1, lanes
9-14). In all cases, the efficiency of full-length product
formation with interspecies mixtures of these proteins was reduced
compared with the full-length products formed in reactions containing
proteins from a single species. The most pronounced species specificity was observed with mixtures containing hpol
, hRFC, and S. cerevisiae PCNA (lane 10) and reactions carried out
with S. cerevisiae pol
, hRFC, and S. cerevisiae PCNA (lane 14). The finding that hRFC substituted poorly for S. cerevisiae RFC in the presence of
S. cerevisiae pol
is consistent with the data of Fien
and Stillman (37). The experiments described by Fien and Stillman (37) were carried out with S. cerevisiae SSB, whereas the
experiments described in Fig. 1 contained HSSB. Thus, the marked
specificity noted is most likely independent of the SSB used. Limited
elongation of primer chains was observed in reactions containing hPCNA,
S. cerevisiae RFC, and S. cerevisiae pol
(lane 9). The other combinations of human and S. cerevisiae proteins supported chain elongation (lanes
11-13), although a substantial number of products accumulated that had not been maximally extended. The formation of discrete shorter
products in reactions containing heterologous mixtures of proteins
indicated that the processive action of pol
was reduced compared
with reactions carried out with proteins from homologous species.
Primer extension assays were repeated with an unlabeled primed template
in the presence of [-32P]dNTPs (Table
I). Qualitatively, the level of nucleotide incorporation mirrored the results shown in Fig. 1, i.e. reactions
containing S. cerevisiae PCNA, hRFC, and hpol
were
virtually inactive. The combination of S. cerevisiae pol
, hRFC, and hPCNA resulted in substantial nucleotide incorporation
(Table I), but the resulting DNA chains were highly heterogeneous in
length (data not presented).
|
Elongation reactions catalyzed by either the h or S. cerevisiae pol holoenzyme were absolutely dependent on
addition of all three holoenzyme components (pol
, RFC, and PCNA) as
well as an SSB protein. Thus, in the absence of PCNA no detectable elongation of labeled primer was observed with either the human proteins (Fig. 1, lane 7) or the S. cerevisiae
proteins (Fig. 1, lane 8). Likewise, omission of S. cerevisiae pol
or S. cerevisiae RFC (Fig.
2A, lanes 15 and 16, respectively)
prevented DNA synthesis. Similar results were obtained following
omission of any one of the pol
holoenzyme components or SSB protein
(data not presented).
The p21CIP1 peptide-(139-160)
inhibits human but not yeast PCNA-dependent DNA synthesis.
A, reaction mixtures were as described in Fig. 1 except that
HSSB was used as the DNA binding protein in all cases. Where indicated,
12.5 pmol of p21CIP1 or 12.5 pmol of the p21CIP1 peptide-(139-160) was added. Reaction
mixtures were incubated for 10 min at 0 °C prior to the addition of
RFC and pol to permit an interaction between p21CIP1 or the
peptide and PCNA to take place. Following addition of RFC and pol
,
the incubation was continued at 37 °C for 20 min before being
processed as described in Fig. 1. Dried gels were exposed for
autoradiography for 4 h. B, influence of S. cerevisiae PCNA concentration on the effects of p21CIP1
and p21CIP1 peptide-(139-160). Reactions (10 µl) were as
described in Fig. 1 except that 4.6 fmol of singly-primed M13 DNA
(9.31 × 103cpm) was added. Reaction mixtures were
incubated at 37 °C for 5 min in the presence of 1.25 µM of p21CIP1 or p21CIP1
peptide-(139-160) prior to the addition of S. cerevisiae
pol
and RFC. Following addition of these proteins, reactions were incubated for a further 20 min at 37 °C before being processed as
described in Fig. 1. C, quantitative effects of the
p21CIP1 carboxyl-terminal-derived peptides on h and
sc (S. cerevisiae) pol
holoenzyme-catalyzed
deoxynucleotide incorporation as a function of PCNA concentration.
Reaction mixtures (10 µl) were as described in Fig. 1 except that 4.4 fmol of unlabeled primed M13 DNA was added with 20 µM
[
-32P]dCTP (1.2 × 104 cpm/pmol).
Reactions were incubated for 15 min at 37 °C after which time
aliquots were removed to measure the amount of labeled acid-insoluble
material formed. The amounts of p21CIP1 22-mer
peptide-(139-160) or 12-mer peptide-(139-145+156-160), hPCNA, or
S. cerevisiae PCNA (monomer) added were as indicated. The
total amount of nucleotide incorporated into an acid-insoluble form
(pmol) in the absence of p21CIP1 or peptides was 170 nM hPCNA, 27.8 pmol; 68 nM hPCNA, 24.3 pmol; 170 nM S. cerevisiae PCNA, 31 pmol.
The p21CIP1 Peptide-(139-160) Inhibits Human but Not Yeast PCNA-dependent DNA Synthesis
The interaction between
p21CIP1 or the carboxyl-terminal domain of p21CIP1 and
hPCNA inhibits in vitro SV40 DNA replication and
hPCNA-dependent elongation of primed DNA templates by the
pol holoenzyme (27, 28, 32, 38).
The influence of the p21CIP1 peptide-(139-160) on the
elongation of a 32P-labeled primed M13 single-stranded DNA
was examined (Fig. 2A). As previously observed, high levels
of full-length p21CIP1 blocked the elongation reaction
catalyzed by the hpol holoenzyme (Fig. 2A, lane 3).
Elongation catalyzed by the S. cerevisiae pol
holoenzyme
was also inhibited but, in contrast to the human system, the labeled
primer was elongated without the accumulation of full-length material
(Fig. 2A, lane 4). Interestingly, high levels of the
p21CIP1 peptide-(139-160) had no effect on DNA elongation by
the S. cerevisiae pol
holoenzyme (lane 6). As
previously noted, this p21CIP1 peptide-(139-160)
quantitatively inhibited DNA synthesis by the hpol
holoenzyme (Fig.
2A, lane 5). Elongation catalyzed by any combination of
human and S. cerevisiae proteins that included hPCNA was
also completely inhibited by the peptide (lanes 8, 10, and
12). The combination of hpol
, S. cerevisiae
RFC, and S. cerevisiae PCNA was the only heterologous pol
holoenzyme complex that included S. cerevisiae PCNA and
supported elongation (lane 13). This reaction was not
altered by addition of the p21CIP1 peptide-(139-160)
(lane 14).
In the presence of the hpol holoenzyme, the extent of inhibition of
DNA elongation by p21CIP1 and its carboxyl-terminal derivatives
was influenced by the ratio of PCNA to the inhibitor (27, 28, 38). For
this reason, the influence of the p21CIP1 peptide-(139-160) on
the S. cerevisiae pol
holoenzyme was examined at various
PCNA concentrations (Fig. 2B). DNA elongation became more
efficient with increasing levels of S. cerevisiae PCNA (Fig. 2B, lanes 1, 4, and 7). Addition of the
p21CIP1 peptide-(139-160) did not affect the elongation of the
labeled primer irrespective of the level of S. cerevisiae
PCNA present (Fig. 2B, compare lanes 1 and
3; lanes 4 and 6; lanes 7 and 9). In contrast, the influence of the p21CIP1
peptide-(139-160) on hpol
holoenzyme-catalyzed nucleotide
incorporation was dependent upon the level of PCNA added (Fig.
2C). In the presence of 170 and 68 nM hPCNA, 120 and 45 nM of the p21CIP1 peptide-(139-160) were
required to inhibit DNA elongation by 50%, respectively.
Using overlapping 20-amino acid peptides Warbrick et al.
(32) showed that the critical region of p21CIP1 required for
its interaction with PCNA spans amino acids 144-151 (see Table
II). Consistent with this finding high levels of a 12-mer peptide derived from amino acids 139-160 of p21CIP1
containing a deletion of amino acids 146-156 (see Table II) did not
affect DNA synthesis catalyzed by the hpol holoenzyme (Fig. 2C).
Full-length
p21CIP1 inhibited the S. cerevisiae pol holoenzyme reaction, and the length of DNA formed was dependent upon
the ratio between the inhibitor and PCNA. Thus, reducing the level of
PCNA in the presence of a fixed amount of p21CIP1 resulted in
the production of less full-length material (Fig. 2B,
compare lanes 2, 5, and 8).
The influence of p21CIP1 on DNA synthesis carried out in the
presence of higher levels of singly-primed M13 DNA substrate was examined in the presence of a low concentration of S. cerevisiae PCNA (Fig. 3A). Addition of a
high level of p21CIP1 to reactions containing a limiting amount
of S. cerevisiae PCNA resulted in marked inhibition of DNA
synthesis (lane 4). Addition of a lower level of
p21CIP1 in the presence of the same concentration of S. cerevisiae PCNA resulted in more extensive DNA synthesis
(lane 6).
Further experiments investigating the influence of p21CIP1 on
the incorporation of labeled nucleotides confirmed that the extent of
inhibition of hpol holoenzyme-catalyzed elongation by
p21CIP1 was dependent upon the level of hPCNA present (Fig.
3B). Nucleotide incorporation was reduced by 50% in the
presence of 0.37, 0.18, and 0.10 µM p21CIP1 in
reactions containing 170, 68, and 34 nM hPCNA (monomer),
respectively. In the presence of the S. cerevisiae pol
holoenzyme, 50% inhibition was observed with 1.2 µM
p21CIP1 in the presence of either 170 or 17 nM
S. cerevisiae PCNA, whereas reactions containing a low level
of S. cerevisiae PCNA (1.7 nM) required the
addition of 0.8 µM p21CIP1 to inhibit DNA
synthesis by 50%. Altering the amount of S. cerevisiae RFC
or S. cerevisiae pol
added to reactions containing a
limiting concentration of S. cerevisiae PCNA did not
significantly alter the level of p21CIP1 required to inhibit
DNA synthesis by 50% (data not shown).
The inhibition of the S. cerevisiae pol holoenzyme by
p21CIP1 and the lack of inhibition by the p21CIP1
peptide-(139-160) prompted us to examine the effects of the addition of a peptide that spans an additional 70 amino acids within the carboxyl terminus of p21CIP1 (Fig. 3C). Previous
studies have shown that a truncated p21CIP1 protein derived
from amino acids 70-164 within the carboxyl-terminal of
p21CIP1 was capable of binding to hPCNA as well as inhibiting
hpol
holoenzyme-catalyzed elongation of primed templates (31). In contrast to the effects of the p21CIP1 peptide-(139-160), the
truncated p21CIP1 protein-(70-164) inhibited the elongation
reaction catalyzed by the S. cerevisiae pol
holoenzyme
(Fig. 3C, lanes 7-9). These results suggest that amino
acids within codons 70-139 are capable of interaction with S. cerevisiae PCNA. Like the full-length p21CIP1 protein, the
effects of the truncated p21CIP1 protein-(70-164) were more
pronounced in the presence of low levels of PCNA than at higher PCNA
concentrations (compare lanes 9 and 7).
In order to
explain the different effects of the p21CIP1 and
p21CIP1 peptide-(139-160) on S. cerevisiae and
hPCNA-dependent reactions, we determined whether these
proteins exhibit differential binding affinities for PCNA.
p21CIP1 was shown previously to directly interact with hPCNA by
coimmunoprecipitation, gel filtration, and surface plasmon resonance
binding (27, 28). The interaction detected using the latter procedure
indicated that ~2.3 molecules of p21CIP1 monomer bound to
each PCNA trimer. Consistent with this, passage of solutions of
p21CIP1 over a sensor surface on which 110 fmol of hPCNA (3320 RU) was immobilized resulted in an immediate increase in mass (Fig.
4A). In this experiment, 140 fmol of
p21CIP1 (3009 RU) was retained on the hPCNA sensor chip,
corresponding to a stoichiometry of ~1 molecule of p21CIP1
monomer bound per molecule of PCNA monomer. Fourteen-fold less p21CIP1 (10 fmol, 222 RU) bound to a sensor surface to which 92 fmol (2748 RU) of S. cerevisiae PCNA had been covalently
coupled (Fig. 4B). The background values have been
subtracted from these data and were calculated following passage of
p21CIP1 solutions over a blank sensor chip surface (Fig.
4C). These data indicate that the affinity of S. cerevisiae PCNA for p21CIP1 is 14-fold less than the
affinity of hPCNA for p21CIP1. These results were confirmed
following passage of S. cerevisiae or hPCNA solutions over a
sensor surface chip to which p21CIP1 was immobilized (data not
shown).
A 100-fold difference in the association between S. cerevisiae and hPCNA with the p21CIP1 peptide-(139-160) was observed. In this experiment, 100 fmol (281.4 RU) and 1 fmol (30 RU) of the p21CIP1 peptide-(139-160) were retained by sensor chips to which hPCNA (110 fmol, 3320 RU) or S. cerevisiae PCNA (92 fmol, 2748 RU) had been covalently coupled, respectively (Fig. 4, D and E). As described above, background values have been subtracted and were calculated following passage of the p21CIP1 peptide-(139-160) over a blank sensor chip surface (Fig. 4F). These data indicate that approximately 1 molecule of the p21CIP1 peptide-(139-160) binds to each monomer molecule of hPCNA, similar to the stoichiometry observed with the full-length p21CIP1 protein. In contrast, no significant interaction was observed between the p21CIP1 peptide-(139-160) and S. cerevisiae PCNA. Identical results were obtained following p21CIP1 peptide-(139-160) immobilization on a sensor chip over which solutions of h or S. cerevisiae PCNA were continuously flowed (data not shown).
Sensorgrams recorded using different hPCNA concentrations passing over
the surface of chips to which p21CIP1 or the biotinylated
p21CIP1 peptide-(139-160) had been coupled are shown in Fig.
5, A and B, respectively. The
apparent equilibrium dissociation constants (KD) for
hPCNA binding to both p21 and the p21CIP1 peptide-(139-160)
are very similar (p21 KD = 2.55 × 109 M ± 1.05 × 10
9
M and p21CIP1 peptide-(139-160)
KD = 2.3 × 10
9 M ± 1.5 × 10
9 M) as calculated using the
BIAcore kinetics evaluation software version 1.2. Similarly, the slopes
of the Scatchard plots (Fig. 5C) of each of these proteins
are superimposable indicating that the KD values are
very similar (p21 KD = ~3.1 × 10
9 M and p21CIP1 peptide-(139-160)
KD = 2.8 × 10
9
M).
The Influence of p21CIP1 and the p21CIP1 Peptide-(139-160) on the RFC-dependent Loading of PCNA onto DNA
Previous experiments suggested that p21CIP1 inhibits DNA synthesis as a result of its effect on DNA chain elongation rather than RFC-catalyzed PCNA loading. At a fixed concentration of hPCNA, the addition of increasing levels of p21CIP1 resulted in the concomitant decrease in the length of DNA chains synthesized (27).
hPCNA containing a cAMP protein kinase recognition site at its amino
terminus was labeled with 32P. The direct loading of hPCNA
onto DNA by RFC could then be followed after A15m agarose gel
filtration which separated 32P-PCNA on DNA from free
32P. As shown in Fig. 6, incubation of hRFC
and 32P-labeled PCNA with singly-nicked pBR322 DNA and ATP
resulted in the elution of a peak of 32P-labeled hPCNA from
the sizing column in the excluded volume. When reactions were carried
out in the presence of p21CIP1 or the p21CIP1
peptide-(139-160) (in amounts that quantitatively inhibited DNA synthesis), the formation of an excluded peak of
32P-labeled PCNA complexed to DNA was inhibited by only
50%. This finding indicates that the inhibitory effects of
p21CIP1 and the p21CIP1 peptide-(139-160) on hpol holoenzyme-catalyzed DNA synthesis are not predominantly due to
inhibition of RFC-catalyzed PCNA loading but to inhibition of the
subsequent interaction between PCNA and pol
and/or the ability of
PCNA to slide along DNA.
The experiments presented here investigated the interactions
between p21CIP1 and a p21CIP1 carboxyl-terminal derived
peptide with PCNA and the subsequent effect on pol -catalyzed DNA
elongation. Full-length p21CIP1 inhibited DNA synthesis
catalyzed by h or S. cerevisiae pol
holoenzymes to an
extent dependent upon the ratio between p21CIP1 and PCNA. In
contrast, addition of the p21CIP1-derived peptide-(139-160)
only inhibited reactions dependent upon hPCNA, not S. cerevisiae PCNA. Thus, reactions containing S. cerevisiae pol
and S. cerevisiae RFC with hPCNA
were inhibited by the p21CIP1 peptide-(139-160), whereas
reactions containing h or S. cerevisiae pol
, S. cerevisiae RFC, and S. cerevisiae PCNA were unaffected by the peptide. The species origin of the pol
and RFC did not influence DNA synthesis inhibition by p21CIP1 and its
derivatives.
Real time interaction analysis presented here demonstrated that the affinity of the p21CIP1 for hPCNA was ~15-fold greater than its affinity for S. cerevisiae PCNA. This is consistent with the biochemical data presented indicating that 12 times more p21CIP1 was required to inhibit S. cerevisiae PCNA-dependent nucleotide incorporation by 50% than was required to inhibit hPCNA-dependent incorporation by 50%. Furthermore, the affinity of the p21CIP1 peptide-(139-160) for hPCNA was 100-fold greater than its affinity for S. cerevisiae PCNA. These data are also consistent with the singly-primed M13 elongation assays presented which indicated that addition of the p21CIP1 peptide-(139-160) in amounts stoichiometric with hPCNA inhibited nucleotide incorporation by 50%, whereas the addition of excess peptide did not affect reactions dependent upon S. cerevisiae PCNA.
The crystal structure of the p21CIP1 peptide-(139-160)
complexed with hPCNA has recently been determined (33). This analysis demonstrated that the p21CIP1 peptide-(139-160) is bound to
the inter-domain connector loop that links the amino- and
carboxyl-terminal domains of each PCNA monomer (residues 119-133).
Additional interactions are formed with both domains of PCNA. A
schematic model of the hPCNA-p21CIP1 peptide-(139-160) complex
derived from crystal structure analyses is presented in Fig.
7. Although the -carbon backbone traces of h and
S. cerevisiae PCNA are very similar (8, 41), significant differences in the backbone conformation are located in the regions where the p21CIP1 peptide-(139-160) interacts with hPCNA.
These differences may account for the distinct effects of both
p21CIP1 and the p21CIP1 peptide-(139-160) in these two
systems.
First, and of particular interest, the configuration of the hPCNA interconnector loop that forms part of the p21CIP1 peptide-(139-160) binding surface differs in S. cerevisiae PCNA. A number of residues in this interconnector loop contribute to the formation of two hydrophobic pockets in hPCNA that bind the Met-147, Phe-150, Tyr-151, and Ile-158 residues of the p21CIP1 peptide-(139-160). There is also an extensive array of hydrogen bonding-mediated contacts between hPCNA and the p21CIP1 peptide-(139-160) in this region. Thus both the conformation and sequence of the interconnector loop of hPCNA are important in forming the interface with the p21CIP1 peptide-(139-160). Specific differences in the amino acid sequence between the h and S. cerevisiae PCNA are likely to exclude some favorable contacts with the p21CIP1 peptide-(139-160) and S. cerevisiae PCNA. For example, Ile-158 of the p21CIP1 peptide-(139-160) fits snugly into a small hydrophobic pocket adjacent to the hPCNA connector loop. Substitution of a cysteine residue (Cys-27) for asparagine at this position in S. cerevisiae PCNA would significantly alter the nature of the binding site. Additionally, Arg-156 of the p21CIP1 peptide-(139-160) participates in hydrogen bonding interactions with Asp-29 and Gln-125 of hPCNA, whereas in the S. cerevisiae PCNA these residues are replaced by glutamine and phenylalanine, respectively, precluding these interactions.
In the carboxyl-terminal region of the hPCNA connector loop, residues
Leu-126, Gly-127, Ile-128, and Pro-129 form one edge of a binding cleft
into which the methionine and tyrosine side chains of the
p21CIP1 peptide-(139-160) are inserted. The conformation of
the S. cerevisiae PCNA connector loop in this region greatly
differs to that of the hPCNA and probably precludes insertion of these
p21CIP1 peptide side chains. The p21CIP1
peptide-(139-160) tyrosine residue also interacts with the carboxamide group of Gln-131 in hPCNA, which is replaced by a leucine (Leu-131) in
S. cerevisiae PCNA, although the backbone of the connector loop has shifted such that the carboxylate moiety of Glu-130 in S. cerevisiae PCNA occupies the same relative position. In
addition to these changes, subtle shifts in the position of a
-strand and a short loop which border this hydrophobic cavity
optimize the complementarity of the p21CIP1 peptide-(139-160)
and hPCNA surfaces. The presence of the Met-40 in hPCNA (instead of a
valine residue in S. cerevisiae PCNA) augments these
interactions in tailoring the shape of the binding site.
Thus, a number of the specific residues that dictate the conformation of the connector loop of hPCNA and its high affinity interaction with the p21CIP1 peptide-(139-160) are not conserved in S. cerevisiae PCNA, and these differences probably account for the inability of the p21CIP1 peptide-(139-160) to interact with or inhibit S. cerevisiae PCNA.
Results from Warbrick et al. (32) also suggest that a region
that includes the interdomain connector of hPCNA is essential for its
interaction with p21CIP1. Using the yeast 2-hybrid system, they
demonstrated that the interconnector loop of each PCNA monomer
interacts with p21CIP1, whereas PCNA with a deletion of amino
acids 100-150 could not form this complex. The connector loop of hPCNA
also appears to be critical for its interaction with pol (42). A
monoclonal antibody against hPCNA that blocks the interaction between
hpol
and hPCNA was competed by a peptide derived from amino acids 121-135 within the connector loop of PCNA. Further evidence for the
importance of this region of PCNA comes from cold-sensitive mutations
of the S. cerevisiae PCNA gene (POL30) that are
clustered in the interconnector domain region and exhibit a cell
division cycle phenotype at the restrictive temperature arresting with a 2C DNA content (43).
The effects of p21CIP1 and the p21CIP1
peptide-(139-160) on hPCNA-dependent DNA synthesis, the
complex stoichiometry, and dissociation constants between each of these
agents and hPCNA are all quantitatively similar. This suggests that the
biological consequence of the p21CIP1 interaction with hPCNA is
quantitatively due to the region contained within the 22-amino acid
p21CIP1 peptide-(139-160). In contrast, the effects of
full-length p21CIP1 and the p21CIP1 peptide-(139-160)
on S. cerevisiae PCNA are different. Furthermore, p21CIP1 was much less effective in blocking S. cerevisiae PCNA-dependent reactions than
hPCNA-dependent reactions. These findings indicate that
p21CIP1 may affect the S. cerevisiae pol
-holoenzyme by a mechanism distinct from that observed with the hpol
-holoenzyme. Since S. cerevisiae PCNA has a low affinity
for p21CIP1 and no detectable affinity for the p21CIP1
peptide-(139-160), other sites within p21CIP1 (present in the
p21CIP1 peptide-(70-164)) may contribute to its interaction
with S. cerevisiae PCNA and subsequent effects on the
S. cerevisiae pol
-holoenzyme activity.
The loading of PCNA onto DNA by RFC was maximally inhibited by 50%
following addition of concentrations of p21CIP1 that completely
blocked the elongation of primed DNA. Hübscher's laboratory (44)
has shown that PCNA loaded onto circular DNA can still slide off the
DNA in the presence of p21CIP1 after linearization of the DNA.
These results suggest that p21CIP1 may act to inhibit the
interaction between the polymerase and PCNA through its ability to bind
to the interconnector domain of PCNA. This would suggest that all
reactions dependent upon PCNA and pol (or pol
) would be
affected by p21CIP1 including DNA repair reactions in which
PCNA plays the same role as in replication, acting to tether the
polymerase to DNA (45, 46).
We thank Dr. Z.-Q. Pan for preparations of p21 protein, David Valentine for figure graphics, and Bill Farley from Pharmacia Biosensor for critical evaluation of biacore data. We are indebted to Dr. Alan Oliff of Merck for help in obtaining the p21CIP1 peptide-(139-145+156-160) as described in Table II.