(Received for publication, June 19, 1995)
From the
p21, a p53-induced gene product that blocks cell cycle
progression at the G phase, interacts with both
cyclindependent kinases and proliferating cell nuclear antigen (PCNA).
PCNA functions as a processivity factor for DNA polymerases
and
and is required for both DNA replication and nucleotide excision
repair. Previous studies have shown that p21 inhibits simian virus 40
(SV40) DNA replication in HeLa cell extracts by interacting with PCNA.
In this report we show that p21 blocks nucleotide excision repair of
DNA that has been damaged by either ultraviolet radiation or alkylating
agents, and that this inhibition can be reversed following addition of
PCNA. We have determined that p21 is more effective in blocking DNA
resynthesis than in inhibiting the excision step.
We further show
that a peptide derived from the carboxyl terminus of p21, which
specifically interacts with PCNA, inhibits polymerase -catalyzed
elongation of DNA chains almost stoichiometrically relative to the
concentration of PCNA. When added at higher levels, this peptide also
blocks both SV40 DNA replication and nucleotide excision repair in HeLa
cell extracts. These results indicate that p21 interferes with the
function of PCNA in both in vitro DNA replication and
nucleotide excision repair.
In response to DNA damage, higher eukaryotic cells elevate the
level of the tumor suppressor protein p53 (Maltzman and Czyzyk, 1984;
Kastan et al., 1991; Fritsche et al., 1993; Hall et al., 1993). This protein functions as a sequence-specific
transcription factor that activates the synthesis of a number of
proteins that act as cell cycle regulators. One such protein, p21 (also
known as Cip1, WAF1, and Sdi1), binds to cyclin-dependent kinases
(CDKs) ()and inhibits their activity resulting in cell cycle
arrest at the G
phase, presumably to allow for the repair
of damaged DNA (El-Deiry et al., 1993; Harper et al.,
1993; Gu et al., 1993; Xiong et al., 1993; Noda et al., 1994). However, p53 is not the only transcriptional
inducer of p21. It has recently been shown by several laboratories that
p21 expression can be regulated independently of p53 during several
situations including normal tissue development and cellular
differentiation (Halevy et al., 1995; Parker et al.,
1995; Macleod et al., 1995). Thus, p21 appears to function as
a general growth inhibitor in response to DNA damage and cellular
differentiation.
Subsequent studies have shown that, in addition to
its ability to bind to CDKs, p21 also directly interacts with
proliferating cell nuclear antigen (PCNA) (Flores-Rozas et
al., 1994; Waga et al., 1994). Chen et al.(1995)
and Luo et al.(1995) have shown that these two distinct
inhibitory activities of p21 reside in different domains of the
protein. The NH-terminal domain of p21 contains the CDK
inhibitory activity, while the COOH-terminal domain contains the PCNA
binding and the DNA synthesis inhibitory activities. Luo et
al.(1995) have shown that when separately expressed in R-1B/L17
cells, each of these domains is able to block DNA synthesis. This
finding suggests that p21 may function to block entry into S phase by
two different mechanisms: inactivating CDKs or neutralizing the
function of PCNA.
PCNA acts as a processivity factor for both DNA
polymerases and
(pol
and
), and is required for
DNA replication (for reviews, see Chalberg and Kelly(1989),
Stillman(1989), and Hurwitz et al.(1990)). In the presence of
a primer-binding protein, replication factor C (RF-C; also known as
activator 1, A1; for reviews see Tsurimoto et al.(1990) and
Hurwitz et al. (1990)), PCNA is loaded onto the DNA primer
junction allowing the binding of pol
or
, which can then
catalyze the elongation of DNA chains (Lee and Hurwitz, 1990; Burgers,
1991). These three proteins (PCNA, RF-C, and pol
) constitute the
pol
holoenzyme, which is essential for the replication of simian
virus 40 (SV40) DNA in vitro. p21 has been shown to inhibit
both in vitro SV40 DNA replication and the elongation of
primed DNA templates catalyzed by the pol
holoenzyme
(Flores-Rozas et al., 1994; Waga et al., 1994). In
each system, the inhibition by p21 was found to be reversed by the
addition of excess PCNA. Quantitative analysis of the binding of p21 to
PCNA indicates that 1 mol of p21 binds to each monomer of PCNA. The
active form of PCNA in DNA replication has been shown to be a trimer
(Krishna et al., 1994). The inhibition of the processive
action of the pol
holoenzyme by p21 has been shown to depend on
both the molar ratio between p21 and PCNA and on the length of DNA
replicated. Higher ratios of p21 to PCNA were found to be more
inhibitory and the replication of longer chains was more sensitive to
p21 than the extension of DNA chains over short lengths (Flores-Rozas et al., 1994).
In addition to its role in replication, PCNA
is also essential for DNA nucleotide excision repair (Shivji et
al., 1992; Nichols and Sancar, 1992). Nucleotide excision occurs
through the introduction of nicks 3-5 nucleotides 3` and
22-24 nucleotides 5` to the site of damage, resulting in the
removal of fragments 25-29 nucleotides in length (Huang et
al., 1992; Svoboda et al., 1993). This gap is then filled
in by pol or
with the undamaged strand acting as the
template (Zeng et al., 1994; Aboussekhra et al.,
1995). PCNA is thought to act in conjunction with the DNA polymerase
during nucleotide excision repair, undertaking a role similar to that
observed in replication.
We have investigated the effect of p21 on PCNA-dependent DNA nucleotide excision repair in vitro. In this report, we present evidence that both the p21 protein and a peptide derived from the carboxyl end of p21 that binds PCNA specifically inhibit the repair of DNA that has been damaged by either alkylating agents or by ultraviolet (UV) radiation treatment. We further determined that this inhibition results from the ability of p21 to block the DNA resynthesis reaction following the excision of the damaged nucleotides. These findings are contradictory to previous reports by Li et al. (1994) and Shivji et al.(1994), who found that high levels of p21, which blocked DNA replication, did not affect nucleotide excision repair.
Figure 1: Influence of both time and the concentration of HeLa extracts on the repair of UV-damaged DNA. A, reaction mixtures contained both UV-damaged and undamaged DNA and HeLa extracts as indicated. After incubation at 37 °C for times as indicated, the two plasmids were purified, linearized, and separated by agarose gel electrophoresis. The gel stained with ethidium bromide is shown at the top, and the autoradiogram is shown at the bottom. B, quantitation of dCMP incorporated into each plasmid is shown. Top, 50 µg of extract protein was used. Bottom, reaction was incubated for 60 min.
Figure 2:
p21 inhibits repair of UV-damaged DNA. A, SDS-PAGE analysis of the fractions isolated after
S-Sepharose chromatography of p21. The p21 protein bound to the
S-Sepharose column was eluted with a linear salt gradient as described
under ``Materials and Methods.'' The S-Sepharose eluted
fractions (3 µl) were subjected to SDS-PAGE and stained with 0.1%
Coomassie Brilliant Blue. The numbers at the top of
the gel indicate the fractions analyzed. The position of the p21
protein is indicated. B, effect of p21 fractions eluted from
the S-Sepharose column on the repair of UV-damaged DNA. Reactions
contained none (lane1) or 0.75 µl of the
S-Sepharose fractions (lanes 2-8, as indicated), HeLa
extract (1.26 mg of protein/ml), and other components as described
under ``Materials and Methods.'' After incubation at 37
°C for 30 min, reactions were stopped and analyzed as described
under ``Materials and Methods.'' Both the ethidium bromide
staining and autoradiogram of the gel are shown. C,
quantitation and comparison of the concentration of p21 in the various
reactions and the inhibition of repair indicated as a percentage. In
the absence of p21, 0.5 pmol of [P]dCMP was
incorporated into the UV-damaged plasmid (representing 100%
activity).
Figure 3:
PCNA addition reversed the inhibitory
effects of p21 on the repair of UV-damaged DNA. A, all
reactions contained HeLa extracts (1.25 mg of protein/ml) and other
components as described under ``Materials and Methods.'' The
addition of p21 was as follows: none (lanes1 and 10-12), 0.42 µM (lanes2-5), or 0.84 µM (lanes
6-9). In experiments in which the effects of the addition of
exogenous PCNA were examined, the amount of PCNA (as a monomer) added
was 0.17 µM (lanes3, 7, and 10), 0.54 µM (lanes4, 8, and 11), or 1.08 µM (lanes5, 9, and 12). After incubation at 37
°C for 30 min, reactions were stopped and analyzed as described
under ``Materials and Methods.'' Both the ethidium bromide
staining and autoradiogram of the gel are shown. B,
quantitation of the experiment shown in A. In the absence of
p21, 0.5 pmol of [P]dCMP was incorporated into
the UV-damaged plasmid (representing 100%
activity).
Figure 4:
The influence of the p21 carboxyl-terminal
peptide on the elongation of primed DNA templates and SV40 DNA
replication. A, the p21 carboxyl-terminal peptide inhibits
elongation of labeled singly primed DNA by the pol holoenzyme.
Reaction mixtures (10 µl) contained 30 mM Tris-HCl, pH
7.8, 2 mM DTT, 1.5 µg of bovine serum albumin, 33.3
µM dNTPs, 2 mM ATP, 7 mM
MgCl
, 5.1 fmol of singly primed M13 DNA (2600 cpm/fmol),
0.25 µg of HSSB, 0.075 unit of RF-C, 0.05 unit of pol
, and
PCNA and p21 carboxyl-terminal peptide in amounts indicated. Reaction
mixtures lacking RF-C and pol
were incubated for 5 min at 37
°C and cooled to 0 °C, and then RF-C and pol
were added.
After 20 min at 37 °C, reaction mixtures were adjusted to 10 mM EDTA, loading dye containing SDS was added, and the mixtures were
subjected to alkaline agarose gel (1.5%) electrophoresis, dried, and
autoradiographed. B, the p21 carboxyl-terminal peptide
inhibits SV40 DNA replication by crude extracts of HeLa cells.
Replication of SV40 DNA by crude extracts was carried out in reaction
mixtures (10 µl) containing 75 ng of pSV01
EP, 0.32 µg of
SV40 T antigen, 70 ng of HSSB, crude extract from HeLa cells and the
p21 carboxyl-terminal peptide as indicated, and other reagents as
described previously (Wobbe et al., 1985). Reactions were
incubated for 60 min at 37 °C, and the amount of acid-insoluble
radioactivity formed (using [
-
P]dCTP) was
measured. In the absence of p21 peptide, 9.22, 7.74, and 3.29 pmol of
dCMP were incorporated with 10, 5, and 2 mg of protein/ml,
respectively.
Figure 5:
The p21 carboxyl-terminal peptide inhibits
the repair of UV-damaged DNA. A, reactions contained HeLa
cytosolic extracts at 0.63 mg of protein/ml (lanes 1-4)
or 1.26 mg of protein/ml (lanes 5-12) and other
components, as described under ``Materials and Methods.'' The
p21 carboxyl-terminal peptide was added at 2.4 µM (lane2), 9.6 µM (lanes3 and 6), 24 µM (lanes4 and 7), or 48 µM (lane8). A
control peptide C1 (NLFCSEEMPSSDDC) was added at 24 µM (lane9) or 48 µM (lane10). Another control peptide C2 (EPPLSQEAFADLWKK) was
added to 24 µM (lane11) or 48
µM (lane12). After incubation at 37
°C for 60 min, reactions were stopped and analyzed as described
under ``Materials and Methods.'' Both the ethidium bromide
staining and autoradiogram of the gel are shown. B,
quantitation of the experiment shown in A. In the absence of
the p21 peptide, 0.5 and 1.14 pmol of [P]dCMP
were incorporated into the UV-damaged plasmid with 0.63 and 1.26 mg/ml
HeLa extracts, respectively (each representing 100%
activity).
Figure 6: p21 inhibits repair of AAAF-treated DNA in WI-L2 whole cell extracts. A, DNA repair reactions were carried out in the absence (lane1) or in the presence of increasing amounts of p21 (lanes 2-6) under conditions described under ``Materials and Methods.'' After incubation at 30 °C for 3 h, reactions were stopped and analyzed as described (Wood et al., 1988). Both the ethidium bromide staining and autoradiogram of the gel are shown. B, quantitation of the results shown in A.
Figure 7: p21 protein inhibits both the excision and resynthesis steps of nucleotide excision repair. A, schematic drawing of the substrate and relevant restriction enzyme sites used to examine excision and damage-specific DNA synthesis. B and C, effect of p21 on the repair synthesis (B) and excision activity (C). Assay conditions were as described under ``Materials and Methods.'' D, the remaining activity (%) was determined relative to control reactions that lacked p21. The activities observed in the absence of the inhibitors represented 100% activity. The data points indicated represent the average of two to four experiments, and the standard error is shown. In the figure presented, the excision activity, assayed by release of radiolabeled fragments containing the damaged site, is represented by circles, whereas the repair synthesis, assayed by the incorporation of radiolabel into the specific restriction fragment containing the repaired region, is represented by squares.
Figure 8: Effect of the p21 carboxyl-terminal peptide on the repair synthesis (A) and excision activity (B). Assay conditions were as described under ``Materials and Methods.'' C, the remaining activity (%) was determined relative to control reactions that lacked the p21 peptide. The activities observed in the absence of the inhibitors represented 100% activity. The data points indicated represent the average of two to four experiments, and the standard error is shown. In the figure presented, the excision activity was assayed by the release of radiolabeled fragments containing the damaged site and is represented by circles. The repair synthesis, assayed by the incorporation of radiolabel into the specific restriction fragment containing the repaired region, is represented by squares.
Figure 9: Influence of the addition of exogenous PCNA on the repair synthesis of the 140-mer oligonucleotide substrate carried out in the presence or absence of p21. Assay conditions were as described under ``Materials and Methods.'' A, an autoradiograph illustrating the repair synthesis assay with HeLa CFE, p21 and PCNA. Numbers to the left indicate the size in nucleotides of the DNA fragments. The 28-mer is the fragment containing the repair patch, whereas the 20-mer is the internal control DNA. The other radiolabeled fragments result from nonspecific incorporation. B, quantitation of the results shown in A. The value 100% (shown in lane1) was obtained from the amount of radioactivity recovered in the 28-mer.
PCNA,
RF-C, HSSB, and pol were prepared as described previously (Kenny et al., 1990; Lee et al., 1991a). In all experiments,
where indicated, the concentration of PCNA refers to the monomer form.
The p21 peptide (GRKRRQTSMTDFYHSKRRLIFS) derived from the COOH terminus
of p21 spans amino acids 139-160 and was synthesized in the
Microchemistry Laboratory of Sloan-Kettering Cancer Institute and
stored either as the solid or dissolved in 10 mM Tris-HCl, pH
8.0, 1 mM EDTA, and 0.1 M NaCl.
Escherichia coli UC6444 was transformed with plasmid pHIT5 in order to express the Nth protein (both were kindly provided by R. Cunningham, State University of New York, Albany), which was then isolated based on a procedure provided by R. Cunningham (Cunningham and Weiss, 1985).
The p21 protein that bound to the DEAE-Sepharose column was eluted with buffer D containing 0.5 M NaCl, yielding 35 mg of protein. This fraction could be solubilized and further purified using the procedure described above.
The preparation of N-acetoxy-2-acetylaminofluorine (AAAF)-treated pBSIIKS plasmid DNA (used in experiments described in Fig. 6) was as described previously (Legerski et al., 1977; Landegent et al., 1984). pGEX2T was used as a control plasmid.
The DNA substrate used to assay both damage-specific DNA
synthesis and excision (described in Fig. 7Fig. 8Fig. 9) was a double-stranded 140-mer
containing a cholesterol ``lesion'' in place of a normal base
at position 70 of one strand. The oligomers (Matsunaga et al.,
1995) used to prepare this substrate were purchased from Operon
Technologies, Inc. or Midland Certified Reagent Co., T4 polynucleotide
kinase and T4 DNA ligase were purchased from New England Biolabs, Inc.,
and [-
P]ATP was from ICN. The substrate
used to measure repair synthesis was prepared by mixing equimolar
amounts of the two complementary 140-mers, one containing the
cholesterol lesion, in a solution containing 20 mM Tris-HCl,
pH 7.5, 50 mM NaCl, and 2 mM MgCl
; after
heating at 90 °C for 5 min, the DNA was annealed by slow cooling to
25 °C over a period of several hours. The substrate was stored at
-20 °C in annealing buffer and diluted prior to use in a
buffer containing 1 mM Tris-HCl, pH 7.4, 1 mM NaCl,
and 0.1 mM EDTA. The substrate used in the excision assay
contains a
P-radiolabel 5` to the lesion and was prepared
as described previously (Matsunaga et al., 1995). This
substrate was stored and diluted as described above for the assay of
repair synthesis.
Repair assays with AAAF-treated DNA (see Fig. 6) were carried out as described previously (Wood et al., 1988). Reaction mixtures (50 µl) contained 192 µg of WI-L2 whole cell extract protein, 0.25 µg each of damaged and undamaged DNAs, and other components as described (Wood et al., 1988). Histidine-tagged p21 (a gift of Dr. J. W. Harper; see Harper et al.(1993)) was added to the reaction mixture in the absence of DNA, and the mixture was incubated on ice for 30 min. After the addition of DNA, the reaction mixture was incubated at 30 °C for another 3 h.
In the
repair assays described in Fig. 7Fig. 8Fig. 9,
HeLa CFE (50 µg, 2 mg/ml) was mixed with substrate DNA (2 pmol) in
reactions (25 µl) containing 35 mM Hepes (pH 7.9), 10
mM Tris-HCl (pH 7.5), 60 mM KCl, 40 mM NaCl,
5.6 mM MgCl, 0.4 mM EDTA, 0.8 mM DTT, 2 mM ATP, 3.2% glycerol, 0.2 mg/ml bovine serum
albumin, 200 µM each of dCTP, dGTP, and dTTP, 8 µM dATP, and 4 µCi of [
-
P]dATP
(DuPont NEN) and incubated at 30 °C for 60 min. After the repair
reaction,
P-radiolabeled 20-mer was added to each reaction
as an internal control to monitor the recovery of substrate DNA. The
mixture was deproteinized with proteinase K (0.2 mg/ml) followed by
phenol, phenol:chloroform, and ether extractions, and the DNA was
precipitated with ethanol in the presence of 40 µg of oyster
glycogen. Recovered DNA was sequentially digested with PvuII
and HinPI (New England Biolabs, Inc.), deproteinized, and
precipitated. DNA was resuspended in formamide/dye mixture and resolved
on a 12% denaturing polyacrylamide gel. Following autoradiography, the
level of damage-specific repair synthesis was determined by scanning
the autoradiographs using a Molecular Dynamics Computing Densitometer
Series 300 instrument. Damage-specific repair synthesis was quantitated
by determining the amount of labeled nucleotide incorporated into the
28-mer generated after PvuII/HinPI digestion.
Nonspecific nicking and resynthesis of the undamaged strand was not
observed (a labeled 30-mer would be generated as a product formed by
the asymmetric PvuII/HinPI restriction pattern). The
amount of repair synthesis was normalized for DNA recovery by
quantitating the amount of 20-mer added as an internal control. When
the influence of p21 protein or the p21 carboxyl-terminal peptide on
repair synthesis was examined, reactions containing the inhibitors
(diluted with a solution containing 50 mM Tris-HCl, pH 7.5,
0.2 M NaCl, and 1 mM EDTA) were preincubated for 15
min at 30 °C with HeLa CFE (50 µg). This was followed by the
addition of the other components, and the DNA was processed as
described for the basic repair synthesis reaction.
During experiments in which the inhibitory effect of p21 was reversed by PCNA, p21 was incubated with the indicated amounts of PCNA at 30 °C for 15 min and then HeLa CFE was added to a final concentration of 2 mg/ml. After a second 15-min incubation at 30 °C, substrate DNA, and other reaction components (as above, except that reactions contained 78 mM NaCl) were added to a final volume of 25 µl and, following a 60-min incubation at 30 °C, the DNA was processed as described for the basic repair synthesis reaction.
The influence of p21 on the repair of UV-damaged DNA was examined (Fig. 2). For this purpose, the bacterial-expressed p21 protein was chromatographed through an S-Sepharose column and eluted using a salt gradient (see ``Materials and Methods''). The elution profile of p21, measured by SDS-PAGE analysis (Fig. 2, panelA), and the inhibition of repair activity by aliquots of the eluted fractions (Fig. 2, panelB) were compared. These results were quantitated as shown in Fig. 2(panelC). The peak of p21 eluted in fractions 12 and 14, coincident with the peak of inhibition of the repair reaction.
If the p21-mediated inhibition is due to its direct interaction with PCNA, addition of excess PCNA to the repair reaction should overcome this inhibition. An example of such an experiment is shown in Fig. 3A. In the presence of 0.42 µM p21, the repair reaction was inhibited 65% (Fig. 3A, lane2), while at 0.84 µM p21, the reaction was reduced by 90% (lane6). Addition of 0.17 µM PCNA (in its monomer form) to reactions containing 0.42 µM p21 almost completely reversed the inhibition (lane3). In the presence of the higher level of p21 (0.84 µM), inhibition of repair was only partially overcome by addition of PCNA even at PCNA concentrations approaching 1 µM. The addition of PCNA in the absence of p21 did not significantly affect the repair reaction (Fig. 3A, lanes 10-12). These results indicate that p21 inhibits the repair of UV-damaged DNA and that this effect can be overcome following the addition of excess PCNA. However, the extent of the reversal depends on the p21 concentration used.
More recently, Warbrick et al.(1995) have shown that
a peptide derived from the carboxyl domain of p21 interacts with PCNA
and inhibits SV40 DNA replication catalyzed by crude cytosolic
extracts. We have confirmed these observations using a peptide of 22
amino acid spanning codons 139-160 of p21. As shown, this highly
basic peptide markedly inhibited the pol -holoenzyme system at
concentrations even lower than those observed with p21 (Fig. 4A, see Table 1). In the presence of 0.17
µM PCNA (monomer), 0.17 µM peptide markedly
inhibited the elongation of singly primed M13 DNA template (lane5). This inhibition was completely reversed by the
addition of excess PCNA (0.85 µM, lane7). In contrast to the near stoichiometric action of the
peptide with the purified system, inhibition of the SV40 replication
reaction with crude extracts required substantially more peptide and
the extent of inhibition depended on the concentration of crude HeLa
cytosolic extract added. In the presence of 2, 5, and 10 mg of extract
protein/ml, 50% inhibition of SV40 replication was observed with 25,
47, and 86 µM peptide, respectively (Fig. 4B). These levels were substantially higher than
the level of intact p21 required to inhibit SV40 replication in crude
extracts (see Table 1for a comparison). The reasons for this
discrepancy are unclear. The possibility that the peptide was degraded
by proteolysis in crude extracts was addressed. However, the addition
of protease inhibitors did not decrease the amount of peptide needed to
inhibit replication. Similar observations with crude extracts were made
with the carboxyl terminus of p21 (codons 76-164). (
)
The 22-amino acid peptide derived from the carboxyl domain of p21 inhibited the repair of UV-damaged DNA by HeLa extracts in a manner analogous to that observed with the SV40 DNA replication system (Fig. 5A). In the presence of 0.63 and 1.26 mg of extract protein/ml, the repair reaction was inhibited 50% by 7.7 and 29 µM of the peptide, respectively, levels slightly higher than those required to inhibit the replication of SV40 DNA. Nonspecific peptides, C1 and C2 (described in the legend to Fig. 5A), at comparable levels had no effect on the repair reaction. These observations indicate that the PCNA-interacting region of p21 alone is capable of inhibiting the repair reaction in the absence of the CDK-inhibiting domain, albeit at higher levels. At the high levels of peptide used, no attempts was made to reverse the inhibition with additional PCNA.
The repair of AAAF-DNA can also be carried out by HeLa cytosolic extracts, and this reaction was inhibited by p21 in a manner analogous to that observed with WI-L2 whole cell extracts (data not shown). Excess PCNA reversed the inhibition (data not presented).
As shown in Fig. 7(B and C), 0.27 µM of p21 inhibited damage-specific DNA synthesis by 50%, while approximately 2 µM of p21 was required to inhibit the excision step by 50%. The effect of the p21 carboxyl-terminal peptide on both resynthesis and excision steps was also determined (Fig. 8). Although the p21 peptide inhibited resynthesis 50% at 20 µM, no effect was observed in the excision assay at 40 µM, a concentration that almost totally inhibited the resynthesis step.
The inhibition of resynthesis by p21 was reversed by exogenous PCNA addition (Fig. 9). The slight stimulation of resynthesis observed at low concentrations of PCNA (Fig. 9A, lane4) is most likely an experimental artifact. Reversal of the inhibition of the excision step was not attempted because of the high concentration of p21 required for 50% inhibition. It has been previously shown with partially purified fractions that PCNA is required for both the excision and resynthesis steps (Nichols and Sancar, 1992). The results presented here indicate that the resynthesis step is more sensitive to p21 than the excision step. In keeping with these observations, recent studies with highly purified fractions isolated from higher eukaryotes (Mu et al., 1995) and from Saccharomyces cerevisiae (Guzder et al., 1995) indicate that PCNA plays no role in the excision steps. It is interesting to note that with the highly purified excinuclease systems examined, the release of the excised oligonucleotide was effected by denaturing conditions. This raises the possibility that the recycling of the excision process may be stimulated by the PCNA-dependent repair synthesis observed with partially purified fractions, as previously suggested (Nichols and Sancar, 1992).
The results presented here (summarized in Table 1)
demonstrate that both p21 and a peptide derived from the carboxyl end
of p21 that binds PCNA specifically inhibit the repair of DNA damaged
by either alkylating agents or UV radiation treatment. We further
determined that this inhibition is due to the blockage of the
resynthesis reaction following the excision step. These observations,
along with those reported previously, indicate that p21 inhibits two
PCNA-dependent reactions, one leading to the repair of damaged DNA and
the other leading to the elongation of primed DNA templates by pol
and/or pol
(Flores-Rozas et al., 1994; Waga et
al., 1994; Li et al., 1994). At present it is not clear
whether it is pol
and/or pol
that participates in
nucleotide excision repair (Zeng et al., 1994; Aboussekhra et al., 1995), and there is also some controversy concerning
which of these two polymerases participates in lagging strand
maturation and in leading strand synthesis during DNA replication
(Morrison et al., 1990; Burgers, 1991; Lee et al.,
1991b; Araki et al., 1992; Budd and Campbell, 1993). Although
it is difficult to compare the SV40 replication reaction and the
nucleotide excision repair assay carried out by crude extracts, the
results presented here indicate that there may be a quantitative
difference in the inhibitory effects of p21 in these reactions (see Table 1for a comparison). As expected from previous observations
regarding the elongation of primed templates over short lengths (10
nucleotides) and over long regions (7 kilobases), the replication
reaction is more sensitive to inhibition by p21 than the repair
reaction (Flores-Rozas et al., 1994). These studies carried
out using the highly purified pol
holoenzyme concluded that the
molar ratio of p21 to PCNA (as a monomer) required to block short
length extensions was nearly 100-fold greater than the molar ratio
required for inhibition of elongation over a 7-kilobase length. From
the measurements of PCNA present in crude extracts, a much lower ratio
of p21 to PCNA inhibited the repair reactions (a molar ratio of p21 to
PCNA of
3, inhibited repair about 50%).
Since nucleotide
excision repair requires relatively short-patch synthesis by pol
or pol
, we anticipated that this PCNA-dependent reaction would be
as insensitive to p21 inhibition as that found in the elongation over a
10-nucleotide stretch. However, this was not the case, suggesting that
the role of PCNA in nucleotide excision repair may prove more complex
than the role played in the pol
or pol
holoenzyme reaction.
At present, it is not clear how PCNA participates in the repair of
damaged DNA. It is clear that RF-C is essential for the loading of PCNA
in replication reactions, and an involvement of RF-C in the repair of
damaged DNA has been suggested by Aboussekhra et al.(1995). In
contrast to pol
, pol
can extend DNA chains in the absence
of RF-C and PCNA (Nishida et al., 1988; Burgers, 1991; Lee et al., 1991b). However, in the presence of salt
concentrations more closely resembling physiological conditions, pol
activity becomes completely dependent on both RF-C and PCNA
(Burgers, 1991; Lee et al., 1991b). Although PCNA can be
loaded onto primed DNA templates in the absence of RF-C, such reactions
required extremely high levels of PCNA (Burgers and Yoder, 1993).
The results presented here are in contrast to the data presented by Li et al.(1994) and by Shivji et al.(1994), who have reported that high levels of p21 had no effect on the repair of UV damaged DNA. The levels of p21 used in their experiments (up to 40 µg/ml, equivalent to 1.9 µM), are well within the range that we found inhibitory. The reasons for the discrepancy are unclear. One contributing factor that we have noticed concerns the degree of aggregation of p21. We have observed that, with time, the inhibitory effects of p21 in both replication and repair decreased, and this was correlated with increased aggregation. This was observed with both free p21 and the histidine-tagged p21.
Li et al.(1994)
have proposed that pol or pol
acts to completely fill in
the 30-nucleotide stretch essential for the repair of the excised
damaged region of DNA. In their model, they proposed that PCNA,
complexed with p21, is effective in fixing the polymerase to the primer
end but PCNA needs not act as a processivity factor since the
polymerase acts over a short region. This provocative model suggests
that the p21-PCNA complex governs how the polymerase interacts with
PCNA. In contrast, our findings suggest that the formation of the
p21-PCNA complex, which requires high levels of p21, inhibits repair of
damaged DNA. The availability of purified excision-repair systems (Mu et al., 1995; Aboussekhra et al., 1995) should permit
a better evaluation of the role of PCNA and the effect of p21.
Chen et al.(1995) and Luo et al.(1995) have recently shown
that while the NH-terminal domain of p21 contains the CDK
inhibitory activity, the CH
-terminal domain contains the
PCNA binding and DNA synthesis inhibitory activities. Luo et
al.(1995) have further demonstrated that each of these domains
(codons 1-75 as the NH
terminus, and codons
76-164 as the carboxyl domain), when expressed in R-1B/L17 cells,
was sufficient to block DNA synthesis, although the carboxyl domain of
p21 was less efficient. There is some controversy concerning the
growth-inhibitory effect of the carboxyl terminus of p21 in
vivo. Chen et al.(1995) have reported that overexpression
of the NH
-terminal domain of p21 (codons 1-90), but
not the carboxyl end (codons 87-164) in human SaOs2 cells,
blocked cell growth. However, the expression of the carboxyl end of p21
and its interaction with PCNA in transfected SaOs2 cells was not
examined.
It appears that CDKs are the prime targets of p21 in
blocking entry into S phase. What is the physiological significance of
inhibition of PCNA function by p21? We postulate that there is a
quantitative difference in the level of p21 induced by p53 in response
to DNA damage. Relatively low levels of p21 are sufficient to inhibit
CDK activity but not to inhibit PCNA. As a result, cells are arrested
in G to repair DNA damage. However, when the level of p21
induced becomes higher, perhaps due to severe DNA damage, it acts to
inhibit PCNA function in both DNA replication and repair. The
PCNA-inhibitory function of p21 is perhaps related to p53-induced
apoptosis. However, it remains to be determined whether p21 is required
for p53-mediated apoptosis and whether DNA replication and repair
functions are impaired by p21 in cells committed to apoptosis.