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First published online 9 September 2008
doi: 10.1242/jcs.027730
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Research Article |
1 Cell Cycle and Genomic Stability Laboratory, Fundación Instituto Leloir-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina
2 Laboratory of Molecular and Cellular Therapy, Fundación Instituto Leloir-CONICET, Universidad de Buenos Aires, Buenos Aires, Argentina
3 Department of Biological Sciences, Columbia University, New York, NY 10027, USA
* Author for correspondence (e-mail: vgottifredi{at}leloir.org.ar)
Accepted 3 July 2008
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(pol eta), with PCNA and impairs the assembly of pol foci after UV. Moreover, this obstruction correlates with accumulation of phosphorylated H2AX and increased apoptosis. By showing that p21 is a negative regulator of PCNA-pol interaction, our data unveil a link between efficient TLS and UV-induced degradation of p21.
Key words: Cell death, p21 (CDKN1A), PCNA, pol (pol eta), UV irradiation
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; Moldovan et al., 2007; Warbrick, 2000). PCNA forms a sliding platform required for the processivity of DNA polymerases 
and 
during DNA replication (Burgers, 1991). PCNA also participates in several forms of DNA repair [including nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR)] and in various aspects of post-replicative processing (Moldovan et al., 2007). Recently, a role of PCNA in the activation of translesion DNA synthesis (TLS), a process that avoids replication blockage during S phase, was revealed. TLS was initially linked to PCNA ubiquitylation, a modification of PCNA that is essential for post-UV cell survival in S. cerevisiae (Hoege et al., 2002; Stelter and Ulrich, 2003). In mammals, ubiquitylated PCNA has been reported to have a much higher affinity than unmodified PCNA for the TLS-specific DNA polymerase (pol ) (Bienko et al., 2005; Kannouche et al., 2004; Parker et al., 2007; Plosky et al., 2006), an enzyme that replicates past cyclobutane pyrimidine dimers (CPDs). More recently, others have proposed that PCNA ubiquitylation promotes the disassembly of factors that prevent pol recruitment to replication foci (Haracska et al., 2006). Together, these results suggest a central role of PCNA ubiquitylation in the switch from replicative to TLS polymerases at sites of stalled replication.
Although PCNA is clearly a master regulator of DNA synthesis-associated processes, much less is known about potential modulators of its functions. The p21 protein (also known as CDKN1A and p21Cip1/Waf1), a member of the family of cyclin-dependent kinase (CDK) inhibitors (CKIs), has been shown to interact with PCNA and to inhibit PCNA functions (Bruning and Shamoo, 2004; Warbrick, 1998). In vitro, p21 interferes with the interaction of PCNA with replication factor C (RFC) (Oku et al., 1998), DNA polymerase (Podust et al., 1995; Waga et al., 1994) and FEN1 (Chen et al., 1996). p21 also obstructs the interaction of PCNA with DNA-repair factors required for NER (Gary et al., 1997). Taken together, these data argue that, in vitro, p21 inhibits the resynthesis step of the repair process (Pan et al., 1995; Shivji et al., 1998). In vivo, however, although some groups have found an inhibitory role of p21 in NER-related unscheduled DNA synthesis (UDS) outside of S phase (Bendjennat, 2003; Cooper et al., 1999), others have reported a positive or null role of p21 in NER (McDonald et al., 1996; Perucca et al., 2006; Sheikh et al., 1997; Smith et al., 2000). The role of p21 in TLS is also under investigation and a recent report has demonstrated that p21 reduces TLS efficiency and TLS-associated mutagenic load (Avkin et al., 2006). In all cases, the effect of p21 on a given UV-associated process is difficult to assess because p21 is promptly degraded after UV irradiation (Kaur et al., 2007; Lee et al., 2006; Lee et al., 2007; Soria et al., 2006). To overcome such a limitation, it was imperative to utilize a non-degradable p21 protein. Using this approach, we have recently shown that PCNA ubiquitylation is impaired when p21 is stabilized (Soria et al., 2006), which suggests a negative effect of p21 on some UV-stimulated process(es).
Using various non-degradable mutants of p21 we addressed, in parallel, the role of p21 in DNA replication, NER and TLS. Our data indicate that only the CDK-binding domain of p21 is essential for the inhibition of DNA replication. In agreement, the p21-PCNA interaction was not sufficient to displace replicative polymerases such as pol . This was also in line with the inability of p21 to inhibit the NER-dependent DNA synthesis attributed to replicative polymerases. The PCNA-p21 interaction efficiently impairs pol association with PCNA and inhibits pol foci assembly. This correlates with increased histone H2AX phosphorylation and cell death. Thus, in contrast to the current proposed model that links the negative effect of p21 on TLS with a positive effect of p21 on the ubiquitylated PCNA-pol axis (Livneh, 2006), our data identify p21 as a selective negative regulator of PCNA partners in TLS. Moreover, the increased levels of PCNA-pol interaction and pol foci formation in unstressed p21–/– cells suggest that during unstressed DNA replication, p21 might prevent the mutagenesis that results from uncontrolled activity of permissive polymerases. In turn, after UV irradiation, the progressive reduction in p21 levels might allow gradual loading of TLS polymerases onto damaged DNA.
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). Independently of p53 status, p21 is promptly degraded after UV exposure (Kaur et al., 2007; Lee et al., 2006; Lee et al., 2007; Soria et al., 2006) and its impact on DNA replication and repair remains obscure. To study the involvement of p21 in such processes after UV irradiation we used p21 mutants that resist UV-induced proteolysis (Fig. 2) (Soria et al., 2006). First, we established the amount of exogenous p21 expression that resembles physiological p21 upregulation. A comparison between the upregulation of p21 observed after well-characterized genotoxic treatments such as daunorubicin and actinomycin D (Gottifredi et al., 2004; Soria et al., 2006) and after transfection of various amounts of p21 expression vector is shown in supplementary material Fig. S1B. In addition to wild-type p21 (p21wt), we used a non-degradable p21 (6Mycp21) and mutants lacking the binding domains for PCNA [6Mycp21 (PCNA–)] and CDK [6Mycp21 (CDK–)]. The point mutations in the CDK and PCNA binding sites were sufficient to disrupt the targeted interactions (Soria et al., 2006). Additionally, the mutation in 6Mycp21 (PCNA–) also impaired the interaction of p21 with exogenously expressed GFP-PCNA (supplementary material Fig. S1C). Cell-cycle analysis revealed that both the p21wt and the stable mutant, 6Mycp21, efficiently block cell-cycle progression (Fig. 1A). Interestingly, the 6Mycp21 (PCNA–) construct, which retains the capacity to interact with CDK, was as efficient as the wild-type protein in promoting the accumulation of cells at G1/G2. However, the 6Mycp21 (CDK–) mutant, despite its ability to interact with PCNA, was clearly unable to modify the cell-cycle distribution (Fig. 1A). This is not dependent on GFP-PCNA expression, as similar results were obtained using only GFP as a transfection marker (Soria et al., 2006). In agreement, short pulses of BrdU incorporation that identify S-phase cells showed that replicative DNA synthesis was completely abolished in almost every cell transfected with p21wt, 6Mycp21 and 6Mycp21 (PCNA–), but not with 6Mycp21 (CDK–) (Fig. 1B). Finally, because a correlation between PCNA foci formation and S phase has been clearly established (Essers et al., 2005; Leonhardt et al., 2000; Sporbert et al., 2002), we tested whether the different p21 constructs impaired GFP-PCNA foci formation (Fig. 1C). A significant percentage (
40%) of control cells (empty vector, EV) showed a focal distribution of GFP-PCNA. The remaining 60% of the cells showed pan-nuclear GFP-PCNA localization, which corresponds to cells outside S phase (Essers et al., 2005; Leonhardt et al., 2000). As expected, when p21wt was cotransfected the number of cells with GFP-PCNA foci was drastically reduced (to less than 5%). Similar results were obtained when 6Mycp21 or 6Mycp21 (PCNA–) was expressed. However, the number of cells with GFP-PCNA foci was similar to that of control cells when 6Mycp21 (CDK–) was transfected (see Fig. 1C and Fig. 3B). Moreover, a clear colocalization of 6Mycp21 (CDK–) and PCNA (Fig. 1C and supplementary material Fig. S2A) and between 6Mycp21 (CDK–) and BrdU-positive foci (supplementary material Fig. S2A) was observed. Taken together, these data demonstrate that 6Mycp21 (CDK–) is unable to block cell cycle progression and, in agreement with previous findings (Chen et al., 1995), suggest that p21 binding to PCNA does not prevent the correct function of replicative polymerases.
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The CDK-binding and PCNA-binding motifs of p21 do not inhibit DNA synthesis associated with NER
Since the p21-PCNA interaction is insufficient to halt DNA replication in cells, we wondered whether it could affect the participation of PCNA in NER. This is of interest because we and others have reported a strong p21 downregulation induced by UV irradiation (Fig. 2A) (Kaur et al., 2007; Lee et al., 2006; Lee et al., 2007; Soria et al., 2006) that might be linked to DNA-repair-associated processes. NER is activated within minutes after UV exposure and is characterized by pan-nuclear relocalization of NER factors, including PCNA, to damaged DNA (Volker et al., 2001). This fast reorganization of NER factors neither requires nor affects PCNA foci assembly at these early time points. In fact, no changes in the number of cells with PCNA foci were detected at this time, or even at later times such as 1 hour (see Fig. 3A). To check whether the recruitment of NER-specific factors was affected by p21, human U2OS cells were UV irradiated through polycarbonate filters, which are porous and expose only discrete areas of the nucleus. This technique allows visualization of the sub-nuclear recruitment of NER factors to the irradiated spots (Essers et al., 2005; Green and Almouzni, 2003; Volker et al., 2001). As previously reported, spots with elevated levels of the helicase XPB (also known as ERCC3) were detected 30 minutes after UV irradiation in a high percentage of control cells (see Fig. 2B). None of the p21 constructs was able to alter this XPB accumulation into irradiated spots (Fig. 2B), suggesting that p21 does not affect early steps of NER. In line with this, XPB+ spots were observed in all phases of the cell cycle and PCNA relocalization to XPB+ spots was evident in cells outside S phase (diffused PCNA) in the presence of all the p21 mutants (supplementary material Fig. S3A). Also, all p21 constructs relocalized to XPB+ spots, with the exception of 6Mycp21 (PCNA–) (Fig. 2A). This suggests that p21 is recruited to NER sites by its interaction with PCNA.
The effect of the different p21 constructs on UV-induced DNA synthesis was then tested on U2OS (Fig. 2C) and human WI38 VA (data not shown) cells, with GFP-PCNA as a marker for transfected cells. After UV irradiation, the cells were incubated in high concentrations of BrdU for 4 hours. Non-irradiated (NI) cells transiting through S phase exhibited intense BrdU incorporation, whereas cells in G1/G2 presented no detectable BrdU incorporation (Fig. 2C). Perinuclear cytoplasmic BrdU was attributed to mitochondrial DNA synthesis as previously described (Davis and Clayton, 1996). By contrast, detectable accumulation of nuclear BrdU was observed in all cells outside of S phase after UV irradiation (see Fig. 2B). As expected, this UDS outside of S phase, previously associated with NER (Li et al., 1994; Perucca et al., 2006), was not observed in cells with deficient expression of the NER essential factor, XPA (supplementary material Fig. S3C). Regardless of the capacity of p21, 6Mycp21 and 6Mycp21 (CDK–) to relocalize into irradiated spots, no significant effect of p21 on UDS was observed (Fig. 2C and supplementary material Fig. S3B). Thus, in spite of its ability to form a complex with PCNA, p21 is incapable of blocking NER in vivo. Importantly, taken together, the data shown in Figs 1 and 2 suggest that p21-PCNA interaction does not affect the activity of replicative polymerases, neither during DNA replication nor NER.
PCNA, but not CDK, binding by p21 impairs assembly of new PCNA foci after UV irradiation
GFP-PCNA reorganizes into well-defined sub-nuclear foci after treatment with cis-diamminedichloroplatinum or UV irradiation, which suggests the involvement of these structures in DNA repair-associated activities (Solomon et al., 2004). We tested the effect of the different p21 constructs on GFP-PCNA foci formation after UV irradiation. In line with previous observations (Solomon et al., 2004), in control samples, cells with GFP-PCNA foci increased from 40% to almost 80% in 6 hours following UV exposure (EV, Fig. 3B). Organic solvent extraction is necessary to immunodetect endogenous PCNA (Kannouche et al., 2001); by contrast, GFP-PCNA detection is not limited by such a procedure (Essers et al., 2005; Leonhardt et al., 2000). In fact, we have obtained similar results using three different extraction procedures (pre-extraction with detergents before PFA fixation; PFA fixation followed by Triton extraction; or methanol/acetone fixation) or direct counts on living cells (not shown). The increase in the number of cells with detectable PCNA foci was much slower than the activation of pan-nuclear NER (2 hours versus 15-30 minutes) and it is unlikely to be directly linked to classic NER. However, it is worth mentioning that differences were observed in the architecture of GFP-PCNA foci before and after UV irradiation. Although the distribution of GFP-PCNA foci in some irradiated cells was indistinguishable from that of replication foci in unstressed cells, others displayed a greater number of smaller GFP-PCNA foci (compare the two cells shown for EV in Fig. 3A).
When p21wt was expressed we observed delayed, but not blocked, GFP-PCNA redistribution into foci after UV irradiation (Fig. 3B,C) that correlated with p21 degradation (see Fig. 2A). This also suggested that PCNA foci formation occurs outside S phase when cells are exposed to UV light. In line with this, the UV irradiation of cells accumulated in G1/G2 by the ectopic expression of non-degradable 6Mycp21 (PCNA–) also resulted in delayed, but yet efficient, reorganization of GFP-PCNA into foci structures (Fig. 3B,C). Significantly, these GFP-PCNA foci were also greater in number and smaller in size [Fig. 3A, p21wt and 6Mycp21 (PCNA–)], which suggests a different composition to PCNA foci outside of S phase. By contrast, 6Mycp21 strongly impaired GFP-PCNA foci formation at all times (Fig. 3A,C). Since both 6Mycp21 and 6Mycp21 (PCNA–) provoke the accumulation of cells in G1 phase (even after UV irradiation), these observations indicate that the PCNA-binding domain of p21 impairs GFP-PCNA foci formation outside of S phase. Taken together, these data also demonstrate that the p21 interaction with CDK blocks replication-associated, but not UV-induced, GFP-PCNA foci formation.
A completely different scenario was observed when cells expressing 6Mycp21 (CDK–) were subjected to UV irradiation. In this case, the number of cells with GFP-PCNA foci was unaffected at all times after UV irradiation (Fig. 3B,C; Table 1). Thus, PCNA binding by p21 might prevent PCNA foci formation in G1/G2, but fails to disrupt replication-associated PCNA foci in S phase. Interestingly, some cells were characterized by larger and fewer GFP-PCNA foci [6Mycp21 (CDK–), Fig. 3A]. Intriguingly, three-dimensional reconstructions of these structures indicate that they might derive from fusion/collapse of smaller, single foci (supplementary material Fig. S2B).
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Taken together, these observations indicate that different domains of p21 regulate PCNA foci formation before and after UV irradiation. Whereas the CDK-p21 interaction inhibits replication-associated PCNA foci formation in non-irradiated cells [Fig. 1, see DNA-replication inhibition by the 6Mycp21 (PCNA–) mutant], it does not impair PCNA foci formation after UV irradiation [Fig. 3B,C, delayed but efficient PCNA foci formation with 6Mycp21 (PCNA–)]. By contrast, the PCNA-p21 interaction does not affect PCNA recruitment to replication foci [Fig. 1, 6Mycp21 (CDK–)], but impairs PCNA foci formation outside of S phase after UV irradiation [Fig. 3B,C, no increase in PCNA foci formation after UV with 6Mycp21 (CDK–)].
We also performed a FLIP/FRAP analysis (fluorescence lost in photobleaching/fluorescence recovery after photobleaching) to establish the effect of p21 mutants on the intranuclear mobility of PCNA (supplementary material Fig. S4A). Cells with a pan-nuclear distribution of GPF-PCNA were characterized by high PCNA dynamics, independent of p21 status (supplementary material Fig. S4B, left panel). As expected (Essers et al., 2005), cells with PCNA foci were characterized by a major decrease in PCNA mobility after UV irradiation (see EV in supplementary material Fig. S4B, right panel). However, the mobility of GFP-PCNA was not strongly affected by 6Mycp21 (CDK–). Interestingly, only subtle changes in PCNA mobility were revealed in those few cells in which 6Mycp21 allowed GFP-PCNA to reorganize into foci (see supplementary material Fig. S4B, right-hand panel). These data suggest that other factors that modulate the consolidation of PCNA foci are regulated by the p21-PCNA interaction.
The PCNA-binding but not CDK-binding domain of p21 inhibits pol association with PCNA and its assembly into nuclear foci
Previous work from our group and others had suggested a role for p21 as a regulator of TLS (Avkin et al., 2006; Soria et al., 2006). Since pol recruitment to stalled replication forks has been linked to the accumulation of pol in nuclear foci that colocalize with PCNA (Kannouche et al., 2004; Plosky et al., 2006; Watanabe et al., 2004), we decided to test the effect of the various p21 constructs on pol foci formation using a previously described GFP-tagged construct of pol (Kannouche et al., 2001). As expected, a low percentage of unstressed cells showed GFP-pol foci (Fig. 4A, upper panel and Fig. 4B, NI), and this number increased steeply after UV irradiation in control (EV) cells. Similar results were obtained when cells with pol foci were quantified after detergent-extraction and PFA fixation (not shown). When p21wt and 6Mycp21 (PCNA–) were transfected, a delayed but otherwise unimpaired induction of pol foci formation was observed. This correlates with the retardation in PCNA foci formation observed when these constructs were expressed (Fig. 3C; Table 2). In agreement with GFP-PCNA foci organization (Fig. 3), a percentage of mock-transfected cells (EV) equivalent to the proportion of cells outside S phase and almost all cells transfected with p21wt or 6Mycp21 (PCNA–) were characterized by a smaller GFP-pol foci size. 6Mycp21 impaired GFP-pol foci formation after UV irradiation, which correlates with its effect on GFP-PCNA foci. Intriguingly, however, 6Mycp21 (CDK–) strongly impaired pol recruitment to foci at all times, despite the constant proportion of cells (40%) with PCNA foci [Fig. 4B,C, 6Mycp21 (CDK–)]. In fact, the strong colocalization of PCNA and 6Mycp21 (CDK–) indicates that p21-PCNA interaction prevents GFP-pol foci formation and chromatin association (see merged panels in supplementary material Fig. S5). Thus, whereas p21 recruitment to replication foci depends on PCNA foci formation, after UV irradiation the persistence of p21 at the replication sites by means of its interaction with PCNA interferes with pol recruitment to PCNA foci.
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To determine the role of endogenous p21 in GFP-pol foci formation after UV irradiation, we used isogenic human HCT116 p21+/+ and p21–/– cells as previously described by B. Vogelstein and colleagues (Bunz et al., 1998). A significant number of p21–/– cells with detectable pol focal organization were observed even before UV irradiation (Fig. 5). These pol foci were not as abundant as in UV-treated cells (see p21–/– samples in Fig. 5C). After UV irradiation, pol foci increased in both cells lines, but the number of cells with pol foci was higher in p21–/– up to 8 hours after UV (Fig. 5A). This correlated with the reduction in the levels of p21 in the p21+/+ cells (Fig. 5B). Similar results were obtained when p21wt was transiently transfected into the p21–/– cells (Fig. 5D,E), indicating that p21wt expression was indeed associated with the retardation in pol foci formation in p21+/+ cells.
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increases after UV irradiation (Kannouche et al., 2004). We therefore determined the effect of p21 on pol extractability and its interaction with PCNA in the chromatin-bound fraction. Whereas p21 expression did not alter the amount of Triton-insoluble GFP-pol , either before or after UV (Fig. 6A), we observed a clear inhibition of GFP-pol -PCNA interaction after UV, but only when stable p21 with an intact PCNA binding site was expressed (Fig. 6B). Pol -PCNA interaction was not affected by p21, before or after UV. Importantly, endogenous p21 also modulated endogenous pol -PCNA interaction without affecting pol -PCNA (Fig. 6C). These data are completely in line with results described in Figs 1, 2 and 4, and demonstrate that under equivalent experimental conditions, the PCNA-permissive polymerases interaction might be more efficiently impaired by p21 than is the PCNA-replicative polymerases interaction.
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The PCNA-binding motif of p21 increases cell death after UV irradiation
Together, our data suggest that p21 downregulation after UV irradiation might promote efficient TLS. To establish whether the deficient recruitment of pol to chromatin was associated with defective processing of DNA lesions and/or decreased cell viability, we first determined the levels of histone H2AX phosphorylation (
H2AX). This marker tightly associates with DNA damage, including that resulting from UV irradiation (Marti et al., 2006). In control cells, a substantial increase in H2AX at 4 hours after UV was followed by a return to basal levels at 24 hours (Fig. 7A, EV). None of the p21 constructs significantly altered the number of H2AX+ cells at 4 hours. However, 6Mycp21 (CDK–) promoted the accumulation of pan-nuclear H2AX to elevated levels, an event previously associated with S phase (Marti et al., 2006), that remained high even after 24 hours [Fig. 7A, 6Mycp21 (CDK–) and Fig. 7B]. Such increased levels of H2AX were only observed with the 6Mycp21 (CDK–) mutant (supplementary material Fig. S6). Moreover, by performing local irradiation experiments, we observed that pol failed to be recruited to damaged H2AX+ spots when 6Mycp21 (CDK–) was present, emphasizing the link between impaired pol recruitment and defects in DNA damage processing (Fig. 7C). Finally, a marked increase in cell death was observed when 6Mycp21 (CDK–) was transfected (Fig. 7D). Interestingly, 6Mycp21, which also inhibited pol foci formation and pol -PCNA interaction, promoted the maintenance of higher levels of H2AX and upregulated cell death. We believe that its effect on cell viability might be less evident than that of 6Mycp21 (CDK–) because 6Mycp21-expressing cells accumulate in G1 and pol function might be less crucial for survival outside of S phase. Taken together, these data suggest that the disruption of p21-PCNA interaction might be crucial to allow efficient TLS, which is necessary to prevent cell death associated with stalled forks.
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; Podust et al., 1995; Waga et al., 1994) and NER (Cooper et al., 1999; Luo et al., 1995; Pan et al., 1995; Shivji et al., 1998). In vivo, however, although some studies suggest that the PCNA-interacting domain of p21 blocks DNA replication (Cayrol et al., 1998; Cazzalini et al., 2003) and NER (Bendjennat, 2003; Cooper et al., 1999), many others report little or no effect on DNA replication (Chen et al., 1995; Lin et al., 1996; Luo et al., 1995; Medema et al., 1998; Nakanishi et al., 1995; Ogryzko et al., 1997) and NER (Li et al., 1994; Perucca et al., 2006). The landmark consideration that arose is that the p21:PCNA ratio is critical for the inhibition of DNA synthesis. PCNA is a highly abundant protein, especially during S phase, and even the highest physiological levels of p21 upregulation might be insufficient to titrate PCNA as the p21:PCNA ratio might never exceed 1:1 in vivo (Gottifredi et al., 2004; Luo et al., 1995). Conversely, in vitro, the inhibitory effect of the PCNA-interacting domain of p21 on DNA synthesis requires p21:PCNA ratios of 10:1 or higher (Cooper et al., 1999; Gottifredi et al., 2004; Shivji et al., 1998). In addition, the amount of p21 available could also depend on other events, such as p21 sequestration by CDK/cyclins and modifications to chromatin accessibility. In this work, by integrating various single-cell analysis approaches we demonstrate that the CDK-p21 interaction is pivotal for p21-dependent cell-cycle arrest. In fact, disruption of the CDK-binding domain of p21 is sufficient to allow cell-cycle progression, which also suggests that the p21-PCNA interaction does not efficiently contribute to cell-cycle arrest (Fig. 1). In line with this, the p21-PCNA interaction is also incapable of blocking the resynthesis step of NER, an event that also depends on replicative polymerases (Fig. 2). Therefore, we suggest that the effect of p21 on PCNA function might not relate to the inhibition of the loading/processivity of replicative polymerases, as discussed below.
p21 effects on PCNA, pol and replicative polymerases recruitment
The DNA polymerases of the B family (
, and ) function in DNA replication. The function of pol is independent of PCNA and is associated with the priming of DNA replication. Subsequently, DNA polymerases and , assisted by PCNA, take over DNA synthesis. Pol is responsible for the synthesis of the leading strand and pol associates with the synthesis of the lagging strand (Garg and Burgers, 2005). Pol belongs to a second group of polymerases (the Y family) that is involved in DNA damage tolerance and which is indispensable for translesion synthesis (Lehmann, 2006). Pol , and , interact with PCNA and all contain conserved PCNA-interacting protein motifs (PIP boxes) that allows binding to the interdomain connecting loop (IDCL) of the PCNA monomer (Moldovan et al., 2007; Warbrick, 1998). Importantly, p21 also interacts with the IDCL, and it does so with much higher affinity than any of the other known PCNA-interacting proteins (Bruning and Shamoo, 2004). This has led to the general belief that p21 blocks DNA polymerase recruitment by competing for the same PCNA binding site. This might represent an oversimplification for the multi-subunit replicative polymerases (pol and ) because their interactions with PCNA involve different PCNA-interacting motifs and occur at multiple sites (Eissenberg et al., 1997; Johansson et al., 2004; Maga et al., 1999; Xu et al., 2001; Zhang et al., 1999). For detailed reviews on multi-domain interactions between PCNA and its partners see Moldovan et al. (Moldovan et al., 2007) and Prosperi (Prosperi, 2006). The single-domain interaction model does however appear to be applicable to the structurally simpler single-subunit TLS polymerases. In fact, a single PIP box motif on human pol 
and pol is largely responsible for their interaction with PCNA (Haracska et al., 2005; Haracska et al., 2001).
Our data are consistent with the observations mentioned immediately above. p21 does not directly block the recruitment of PCNA to S-phase replication foci, PCNA-associated DNA synthesis (Fig. 1 and supplementary material Fig. S2) or the PCNA-pol interaction (Fig. 6B). Conversely, p21 obstructs pol recruitment to the replication foci after UV irradiation and, remarkably, also in unstressed cells (Fig. 4B; Fig. 5; Table 2). Moreover, p21 binding to PCNA is also a crucial modulator of pol -PCNA interaction after UV exposure (Fig. 6B,C). In agreement with previous reports (Li et al., 1994; Medema et al., 1998; Perucca et al., 2006), our data reinforce the inability of p21 to displace replicative polymerases from DNA synthesis factories. Nevertheless, the ability of p21 to block pol recruitment to stalled replication sites may impair and/or delay polymerase switching during TLS.
p21 effects on TLS
In a previous report (Soria et al., 2006), we showed that p21 downregulation is required for efficient PCNA ubiquitylation after UV irradiation. Non-degradable p21 (6Mycp21) impairs PCNA ubiquitylation and, surprisingly, this effect depends on the CDK-binding domain of p21. PCNA ubiquitylation modulates the function of TLS polymerases (Hoege et al., 2002; Kannouche et al., 2004; Plosky et al., 2006; Stelter and Ulrich, 2003; Watanabe et al., 2004). Moreover, the Y family polymerases contain ubiquitin-binding domains, termed UBM and UBZ, responsible for the increased affinity of these polymerases for ubiquitylated PCNA (Bienko et al., 2005; Parker et al., 2007). In this context, it seems contradictory that the 6Mycp21 (CDK–) mutant, which inhibits pol recruitment, fails to impair PCNA ubiquitylation. However, both domains of p21 could collaborate to modulate the polymerase switch at the replication fork. Moreover, the real contribution of PCNA ubiquitylation to TLS is a field of continuing controversy (Haracska et al., 2006; Lehmann et al., 2007; Parker et al., 2007; Prakash et al., 2005) and more work will be necessary to shed light on the role of PCNA ubiquitylation in vivo.
It has been proposed that p21 acts as a positive regulator of TLS because the transient downregulation of p21 positively modulates PCNA ubiquitylation after UV irradiation (Avkin et al., 2006; Livneh, 2006). Here, by contrast, we show that p21, via its PCNA-interacting domain, impairs PCNA-pol interaction and pol recruitment to stalled replication foci after UV irradiation. These data indicate that p21 might act as a negative regulator of TLS, controlling both the loading of pol to PCNA and the PCNA ubiquitylation status (Soria et al., 2006). This model provides an alternative scenario in line with the finding that p21–/– cell lines show increased TLS efficiency and associated mutagenesis (Avkin et al., 2006).
Taken together, these data highlight the importance of appropriate cellular levels of p21, which might play a crucial role in the management of pol loading. In the absence of DNA damage, p21 might impede the accidental loading of pol and the consequential mutagenesis (see Fig. 5; Fig. 6C; Table 2, ANOVA for 4C). After UV, when TLS plays a decisive role, progressive p21 degradation might allow pol gradual access to replication forks, thus averting the replication fork blockage that could trigger cell death.
Future perspectives
Our findings raise a wide range of questions regarding the impact of p21 on PCNA-dependent DNA synthesis and regarding the significance of PCNA and pol foci formation throughout the cell cycle. The data in Figs 3 and 4 suggest that PCNA and pol also organize into foci in the G1/G2 phases of the cell cycle after UV irradiation. Since no TLS events are expected to take place outside of S phase, the biological significance of these PCNA/pol foci remains to be determined. Furthermore, recent papers suggest the involvement of pol and other Y polymerases in additional processes, such as gene conversion, homologous recombination and cell death (Kawamoto et al., 2005; Liu and Chen, 2006; McIlwraith et al., 2005), suggesting the existence of as yet unknown functions of Y polymerases. Our findings provide new insights into the potential role of p21 as a regulator of TLS polymerases, which merits further exploration.
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). GFP-PCNA was kindly provided by Dr M. C. Cardoso (Max Delbrück Center for Molecular Medicine, Berlin, Germany) and has been described previously (Leonhardt et al., 2000). GFP-pol was a gift of Dr A. Lehmann and is described elsewhere (Kannouche et al., 2001). UVC irradiation was delivered with a CL-1000 ultraviolet cross-linker equipped with 254 nm tubes (UVP). For full-cell irradiation, doses from 10 to 40 J/m2 were delivered after removal of the culture medium. For local irradiation, polycarbonate filters containing multiple 5 µm pores (Millipore, TMTP01300) were positioned in direct contact with cells and subjected to 80 J/m2 [equivalent to a lower dose as reported in Green and Almouzni (Green and Almouzni, 2003)]. Genotoxic agents used were daunorubicin, 0.22 µM (Oncogene Research Products) and actinomycin D, 5 nM (Calbiochem).
Cell-cycle analysis
Cells were fixed with ice-cold ethanol and samples resuspended in PBS containing RNase I (50 mg/ml) and propidium iodide (PI) (25 mg/ml, Sigma). Stained samples were subjected to FACS (FACScalibur, Becton Dickinson) and data were analyzed using Summit 4.3 software (DakoCytomation). The profiles shown were obtained by gating the GFP-PCNA-positive cells by dual-channel FACS analysis.
BrdU-incorporation assays
For detection of replicative DNA synthesis, cells were incubated for 30 minutes in DMEM/10% FBS containing 10 µM BrdU (Sigma). For detection of repair-associated DNA synthesis, 100 µM BrdU was added to the culture medium and incubated for 4 hours post-UV irradiation. Prior to immunofluorescence, cells were subjected to a denaturing step with 1.5 M HCl for 4 minutes in order to expose the BrdU epitope for antibody detection.
Immunostaining and microscopy
Cells were plated on 10-mm diameter coverslips, transfected and fixed. For imaging, cells were fixed in 4% paraformaldehyde/sucrose for 15 minutes at room temperature, followed by a 10-minute incubation with 0.1% Triton X-100. This fixation method does not alter the GFP-PCNA/pol distribution when compared with that observed in vivo (data not shown) and enables colocalization analysis with p21, which is highly extractable and frequently lost after other fixation protocols. For quantifying the percentage of cells with GFP-PCNA/pol foci, cells were incubated in ice-cold methanol for 20 minutes at –20°C followed by a 30-second pulse of ice-cold acetone (Ogi et al., 2005). This method allows detection of only well-assembled GFP-PCNA/pol foci. Blocking was performed overnight in PBS/2% donkey serum (Sigma). Coverslips were incubated for 1 hour in primary antibodies: anti-p21 AB1 (Oncogene Research Products), anti-p21 C19 (Santa Cruz), anti-BrdU (Amersham), anti-H2AX (Upstate) and anti-XPB (Santa Cruz). Secondary anti-mouse Cy2/Cy3-conjugated antibodies were from Jackson ImmunoResearch. GFP-PCNA and GFP-pol were detected by GFP autofluorescence. DAPI (Sigma) staining was used to visualize nuclei. Images were obtained with a Zeiss Axioplan confocal microscope. Live cell imaging and FLIP/FRAP experiments were performed at 37°C using a Zeiss Axiovert 100M confocal microscope as described (Essers et al., 2002; Essers et al., 2005).
Protein analysis
For Triton extractability experiments, cells were incubated for 60 seconds in PBS containing 1% Triton X-100. The Triton-soluble fraction was collected and the remaining insoluble fraction was solubilized by resuspension in an equal volume of sample buffer. For immunoprecipitations, cells were grown on 10-cm plates, transfected and lysed as described (Soria et al., 2006). For immunoprecipitations of chromatin-associated PCNA, a previously described protocol was used (Bi et al., 2006). Immunoprecipitations were performed using anti-p21 C19 and anti-PCNA PC10 (Santa Cruz). For direct western blot analysis, samples were lysed in Laemmli buffer. Western blots were performed using a combination of anti-p21 antibodies (C19 and AB1); polyclonal Ab 1801 against human p53; SMP14, 2A10, 3F3 against MDM2 (generously provided by A. Levine, Rockefeller University, New York, NY); a rabbit polyclonal against PIG3 (kindly provided by D. Hill, Oncogene Research Products, Cambridge, MA); a monoclonal Ab against GFP (Santa Cruz); a polyclonal Ab against pol (Santa Cruz); a polyclonal Ab against pol (Abcam); and a polyclonal Ab against actin (sigma). Incubation with secondary antibodies (Sigma) and detection (ECL, Amersham) were performed according to manufacturers' instructions.
Statistical analysis
Student's t-test and analysis of variance (ANOVA) were performed using the GraphPad InStat software. Other calculations and graphics were performed using Microsoft Excel 2003.
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Acknowledgments |
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and GFP-PCNA plasmids; T. Zimmermann for generous advice regarding the FLIP/FRAP set-ups; and Kristine McKinney for helpful discussion. Funding was provided by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) PICT22052/PICT01322, CONICET (National Council of Sciences) and The Academy of Sciences for the Developing World to V.G., Fundación Barón to O.L.P. and the National Institute of Health (RO3-TW007440) to C.P. V.G. and O.L.P. are researchers from CONICET. G.S. and J.S. are supported by fellowships from CONICET and ANPCyT, respectively. |
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