PCNA-binding Domain Research Articles

Proliferating Cell Nuclear Antigen Recruits Cyclin-dependent Kinase Inhibitor Xic1 to DNA and Couples Its Proteolysis to DNA Polymerase Switching

The Xenopus cyclin-dependent kinase (CDK) inhibitor, p27(Xic1) (Xic1), binds to CDK2-cyclins and proliferating cell nuclear antigen (PCNA), inhibits DNA synthesis in Xenopus extracts, and is targeted for ubiquitin-mediated proteolysis. Previous studies suggest that Xic1 ubiquitination and degradation are coupled to the initiation of DNA replication, but the precise timing and molecular mechanism of Xic1 proteolysis has not been determined. Here we demonstrate that Xic1 proteolysis is temporally restricted to late replication initiation following the requirements for DNA polymerase alpha-primase, replication factor C, and PCNA. Our studies also indicate that Xic1 degradation is absolutely dependent upon the binding of Xic1 to PCNA in both Xenopus egg and gastrulation stage extracts. Additionally, extracts depleted of PCNA do not support Xic1 proteolysis. Importantly, while the addition of recombinant wild-type PCNA alone restores Xic1 degradation, the addition of a PCNA mutant defective for trimer formation does not restore Xic1 proteolysis in PCNA-depleted extracts, suggesting Xic1 proteolysis requires both PCNA binding to Xic1 and the ability of PCNA to be loaded onto primed DNA by replication factor C. Taken together, our studies suggest that Xic1 is targeted for ubiquitination and degradation during DNA polymerase switching through its interaction with PCNA at a site of initiation.

Recruitment of DNA methyltransferase I to DNA repair sites.

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair.

P21WAF1/CIP1 Regulates the p300 Sumoylation Motif CRD1 through a C-Terminal Domain Independently of Cyclin/CDK Binding

Although best known for its ability to inhibit Cyclin/Cdk complexes and the replicationprotein PCNA, p21WAF1/CIP1 is a multifunctional protein that interacts with many cellularbinding partners, including a number of transcriptional regulators. Previously, wecharacterised p21 derepression of the p300 sumoylation-dependent transcriptionalrepression domain, CRD1. Such repression domains are at least partially dependent uponrecruitment of histone deacetylase (HDAC) complexes but the mechanism through whichp21 selectively disrupts CRD1 activity remains unknown. Here, we demonstrate thatdistinct motifs in the C-terminus of p21 are required for regulation of p300 CRD1function and that this effect does not correlate with Cyclin or PCNA binding. Throughthe creation of N-terminal glutathione-s-transferase fusion proteins, which also overcomethe problems of instability that result from many p21 mutations, we investigated p21binding to HDACs. Although p21 binds both Class I and Class II HDACs in vitro, onlyweak association with HDAC1 and 2 is seen in cells. Mutation of the p21 PCNA bindingdomain significantly increases this interaction suggesting that binding is mutuallyexclusive and only naturally occurs under certain conditions. Binding of HDACs alsofailed to correlate with CRD1 inducibility, suggesting that p21 targets othertranscriptional repression complexes to mediate this effect.

Suppression of clonogenicity by mammalian Dnmt1 mediated by the PCNA-binding domain

Overexpression of the major DNA methyltransferase Dnmt1 is cytotoxic and has been hypothesized to result in aberrant hypermethylation of genes required for cell survival. Indeed, overexpression of mouse or human Dnmt1 in murine and human cell lines decreased clonogenicity. By frame-shift and deletion constructs, this effect of mouse Dnmt1 was localized at the N-terminal 124 amino acid domain, which mediates interaction with proliferating cell nuclear antigen (PCNA). Mutation of the PCNA-binding site restored normal cloning efficiencies. Overexpression of Dnmt3A or Dnmt3B, which do not interact with PCNA, yielded weaker effects on clonogenicity. Following introduction of the toxic domain, no significant effects on apoptosis, replication, or overall DNA methylation were observed for up to 3 d. Suppression of clonogenicity by Dnmt1 was also observed in cell lines lacking wild-type p53, p21(CIP1), or p16(INK4A). Suppression of clonogenicity by Dnmt1 overexpression may act as a fail-safe mechanism against carcinogenicity of sustained Dnmt1 overexpression.

P21Waf1/Cip1 and the prevention of oxidative stress.

pulmonary dysfunction resulting from acute oxygen toxicity is due at least in part to the death of lung epithelial cells. Pulmonary epithelial cells are essential to the maintenance of the alveolar-capillary barrier, and compromise may lead to leakage of fluid and proteins in the airways and alveoli

Decreased p21 Levels Are Required for Efficient Restart of DNA Synthesis after S Phase Block

The cyclin-dependent kinase inhibitor p21, a major transcriptional target of the tumor suppressor p53, plays a critical role in cell cycle arrest in G1 and G2 after DNA damage. It was previously shown that in some human cell lines when S phase is arrested, p53 is transcriptionally impaired such that some p53 targets including p21 are only weakly induced. We show here that during S phase arrest proteasome-mediated turnover of p21 is significantly increased in a manner that is independent of p53. It is well established that p21 can interact both with cyclin-dependent kinase complexes and with proliferating cell nuclear antigen (PCNA). Interestingly, the scant amount of p21 detected during S phase block cannot fully saturate cyclin A-cyclin-dependent kinase 2 complexes and does not interact detectably with PCNA. Importantly, DNA elongation assays in isolated nuclei show that the C terminus of p21 containing the PCNA-binding domain effectively blocks this process. This implies that p21 down-regulation could be an essential requirement for efficient restart of DNA synthesis. In line with this, only cells expressing low levels of p21 immediately progress through the cell cycle upon release from S phase arrest, whereas the remaining few high p21 producing cells move much more slowly through S. Thus, p21 down-regulation is multiply determined and is required for the reversibility of the arrest in S phase.

The Pol32 Subunit of DNA Polymerase δ Contains Separable Domains for Processive Replication and Proliferating Cell Nuclear Antigen (PCNA) Binding

We have carried out a domain analysis of POL32, the third subunit of Saccharomyces cerevisiae DNA polymerase delta (Pol delta). Interactions with POL31, the second subunit of Pol delta, are specified by the amino-terminal 92 amino acids, whereas interactions with the replication clamp proliferating cell nuclear antigen (PCNA, POL30) reside at the extreme carboxyl-terminal region. Pol32 binding, in vivo and in vitro, to the large subunit of DNA polymerase alpha, POL1, requires the carboxyl-proximal region of Pol32. The amino-terminal region of Pol32 is essential for damage-induced mutagenesis. However, the presence of its carboxyl-terminal PCNA-binding domain enhances the efficiency of mutagenesis, particularly at high loads of DNA damage. In vitro, in the absence of effector DNA, the PCNA-binding domain of Pol32 is essential for PCNA-Pol delta interactions. However, this domain has minimal importance for processive DNA synthesis by the ternary DNA-PCNA-Pol delta complex. Rather, processivity is determined by PCNA-binding domains located in the Pol3 and/or Pol31 subunits. Using diagnostic PCNA mutants, we show that during DNA synthesis the carboxyl-terminal domain of Pol32 interacts with the carboxyl-terminal region of PCNA, whereas interactions of the other subunit(s) of Pol delta localize largely to a hydrophobic pocket at the interdomain connector loop region of PCNA.

The PCNA–RFC Families of DNA Clamps and Clamp Loaders

The proliferating cell nuclear antigen PCNA functions at multiple levels in directing DNA metabolic pathways. Unbound to DNA, PCNA promotes localization of replication factors with a consensus PCNA-binding domain to replication factories. When bound to DNA, PCNA organizes various proteins involved in DNA replication, DNA repair, DNA modification, and chromatin modeling. Its modification by ubiquitin directs the cellular response to DNA damage. The ring-like PCNA homotrimer encircles double-stranded DNA and slides spontaneously across it. Loading of PCNA onto DNA at template-primer junctions is performed in an ATP-dependent process by replication factor C (RFC), a heteropentameric AAA+ protein complex consisting of the Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 subunits. Loading of yeast PCNA (POL30) is mechanistically distinct from analogous processes in E. coli (beta subunit by the gamma complex) and bacteriophage T4 (gp45 by gp44/62). Multiple stepwise ATP-binding events to RFC are required to load PCNA onto primed DNA. This stepwise mechanism should permit editing of this process at individual steps and allow for divergence of the default process into more specialized modes. Indeed, alternative RFC complexes consisting of the small RFC subunits together with an alternative Rfc1-like subunit have been identified. A complex required for the DNA damage checkpoint contains the Rad24 subunit, a complex required for sister chromatid cohesion contains the Ctf18 subunit, and a complex that aids in genome stability contains the Elg1 subunit. Only the RFC-Rad24 complex has a known associated clamp, a heterotrimeric complex consisting of Rad17, Mec3, and Ddc1. The other putative clamp loaders could either act on clamps yet to be identified or act on the two known clamps.

Phosphorylation of the PCNA binding domain of the large subunit of replication factor C on Thr506 by cyclin-dependent kinases regulates binding to PCNA.

Replication factor C (RF-C) complex binds to DNA primers and loads PCNA onto DNA, thereby increasing the processivity of DNA polymerases. We have previously identified a distinct region, domain B, in the large subunit of human RF-C (RF-Cp145) which binds to PCNA. We show here that the functional interaction of RF-Cp145 with PCNA is regulated by cdk-cyclin kinases. Phosphorylation of either RF-Cp145 as a part of the RF-C complex or RF-Cp145 domain B by cdk-cyclin kinases inhibits their ability to bind PCNA. A cdk-cyclin phosphorylation site, Thr506 in RF-Cp145, identified by mass spectrometry, is also phosphorylated in vivo. A Thr506-->Ala RF-Cp145 domain B mutant is a poor in vitro substrate for cdk-cyclin kinase and, consequently, the ability of this mutant to bind PCNA was not suppressed by phosphorylation. By generating an antibody directed against phospho-Thr506 in RF-Cp145, we demonstrate that phosphorylation of endogenous RF-Cp145 at Thr506 is mediated by CDKs since it is abolished by treatment of cells with the cdk-cyclin inhibitor roscovitine. We have thus mapped an in vivo cdk-cyclin phosphorylation site within the PCNA binding domain of RF-Cp145.

The Cdk and PCNA domains on p21Cip1both function to inhibit G1/S progression during hyperoxia

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G(1) when exposed to 95% oxygen. Instead, cells arrested in S and G(2). Stable expression of p53 restored induction of p21 and G(1) arrest without affecting mRNA expression of the other Cip or INK4 G(1) kinase inhibitors. To confirm the role of p21 in G(1) arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G(1) arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G(1) during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G(1) arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.

Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.

The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.

Elucidation of an Archaeal Replication Protein Network to Generate Enhanced PCR Enzymes

Thermostable DNA polymerases are an important tool in molecular biology. To exploit the archaeal repertoire of proteins involved in DNA replication for use in PCR, we elucidated the network of proteins implicated in this process in Archaeoglobus fulgidus. To this end, we performed extensive yeast two-hybrid screens using putative archaeal replication factors as starting points. This approach yielded a protein network involving 30 proteins potentially implicated in archaeal DNA replication including several novel factors. Based on these results, we were able to improve PCR reactions catalyzed by archaeal DNA polymerases by supplementing the reaction with predicted polymerase co-factors. In this approach we concentrated on the archaeal proliferating cell nuclear antigen (PCNA) homologue. This protein is known to encircle DNA as a ring in eukaryotes, tethering other proteins to DNA. Indeed, addition of A. fulgidus PCNA resulted in marked stimulation of PCR product generation. The PCNA-binding domain was determined, and a hybrid DNA polymerase was constructed by grafting this domain onto the classical PCR enzyme from Thermus aquaticus, Taq DNA polymerase. Addition of PCNA to PCR reactions catalyzed by the fusion protein greatly stimulated product generation, most likely by tethering the enzyme to DNA. This sliding clamp-induced increase of PCR performance implies a promising novel micromechanical principle for the development of PCR enzymes with enhanced processivity.

C-terminal deletion mutant p21(WAF1/CIP1) enhances E2F-1-mediated apoptosis in colon adenocarcinoma cells.

The present study was designed to investigate the efficacy of combination gene therapy using adenoviral vectors expressing gene products shown to possess apoptotic activity: E2F-1 (Ad-E2F-1) and a C-terminal deletion mutant of p21(WAF1/cIP1) (Ad-p21(-PCNA)), on growth inhibition and apoptosis of human colon cancer cells in vitro and in vivo. Marked E2F-1 and p21(-PCNA) overexpression in response to adenovirus infection was evident by Western blot analysis. IC(25) concentrations of each virus were used for each treatment in vitro to detect cooperative effects on cell death. Coexpression of E2F-1 and p21(-PCNA) resulted in an additive effect on cell death compared to infection with either virus alone. Cell cycle analysis, poly(ADP-ribose) polymerase (PARP) cleavage and analysis of cell morphology also revealed that coinfection with both Ad-E2F-1 and Ad-p21(-PCNA) enhanced cellular apoptosis compared to either virus alone. Interestingly, E2F-1 protein expression was markedly enhanced in the E2F-1/p21(-PCNA) adenovirus combination compared to Ad-E2F-1 infection alone. However, these same effects were not evident in cells coinfected with Ad-E2F-1 and an adenovirus expressing wild-type human p21(WAF1/CIP1) (Ad-p21(WT)). The increase in E2F-1 expression with coexpression of E2F-1 and p21(-PCNA) was not a result of increased E2F-1 protein stability, but was related to increased transcriptional activity from the CMV promoter. Cell cycle analysis revealed G1 arrest 72 hours following single-gene therapy with either the wild-type or mutant p21, whereas increased accumulation of cells in G2/M phase was demonstrated in the E2F-1-overexpressing cells. In the combined therapies, E2F-1/p21(-PCNA) treatment still resulted in G1 arrest, but E2F-1 was able to counteract the G1 arrest when coinfected with p21(WT). These results provide further evidence of the importance of the p21:PCNA-binding domain in mediating the complex cell cycle interaction between E2F-1 and p21. Simultaneous intratumoral injection of Ad-E2F-1 and Ad-p21(-PCNA) dramatically reduced tumor burden of SW620 xenografts compared to either treatment alone in our in vivo model but not in HT-29 colon cancer xenografts. When combined with Ad-p21(-PCNA), E2F-1 adenovirus therapy resulted in approximately 95% decrease in tumor volume of SW620 tumor xenografts compared with controls (P<.05). In conclusion, although simultaneous delivery of E2F-1 and p21(-PCNA) transgenes results in increased E2F-1 expression and enhanced apoptosis of both SW620 and HT-29 colon cancer cells in vitro, this combination was only effective in the treatment of SW620 metastatic colon cancer in vivo. This may represent a potentially useful combination gene therapy strategy for metastatic colon cancer.

Mediation of Proliferating Cell Nuclear Antigen (PCNA)-dependent DNA Replication through a Conserved p21Cip1-like PCNA-binding Motif Present in the Third Subunit of Human DNA Polymerase δ

The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.

Structure of DNA polymerase delta from Saccharomyces cerevisiae.

DNA polymerase delta (Pol delta) from Saccharomyces cerevisiae consists of three subunits, Pol3 (125 kDa), Pol31 (55 kDa), and Pol32 (40 kDa), present at a 1:1:1 stoichiometry in purified preparations. Previously, based on gel filtration studies of Pol delta, we suggested that the enzyme may be a dimer of catalytic cores, with dimerization mediated by the Pol32 subunit (Burgers, P. M., and Gerik, K. J. (1998) J. Biol. Chem. 273, 19756-19762). We now report on extensive gel filtration, glycerol gradient sedimentation, and analytical equilibrium centrifugation studies of Pol delta and of several subassemblies of Pol delta. The hydrodynamic parameters of these assemblies indicate that (i) Pol32 is a rod-shaped protein with a frictional ratio f/f(0) = 2.22; (ii) any complex containing Pol32 also has an extremely asymmetric shape; (iii) the results of these studies are independent of concentration (varied between 0.1-20 microm); (iv) all complexes are monomeric under the conditions studied (up to 20 microm). Moreover, a two-hybrid analysis of the Pol32 subunit did not detect a Pol32-Pol32 interaction in vivo. Therefore, we conclude that the assembly structure of Pol delta is that of a monomer.

Inhibition of Cell Proliferation by the PCNA-Binding Region of p21 Expressed as a GFP Miniprotein

p21 (WAF1/Cip1) is the only member of the CIP/KIP family which has a well-characterized PCNA-binding domain. p21 is known to have an important function in the coordination of the cellular pathways which are activated in response to DNA damage, though the significance of the p21–PCNA interaction is not completely clear. We have analyzed the effects of expressing a miniprotein containing the PCNA-binding domain of p21 upon the cell cycle and upon the proliferation of various cell types. We have compared this with the effect of expressing a mutant form which is defective in PCNA-binding, but which retains the secondary cyclin–CDK-inhibitory site. No PCNA-dependent effects were seen in the short term upon cell cycle distribution. However, clonogenic assays show that the GFP-peptide miniprotein can significantly suppress proliferation in a PCNA-dependent manner. In some cell types, however, the suppression of proliferation was not PCNA-dependent, suggesting that cellular environment is a contributory factor to the effect of this miniprotein. The capacity of this peptide sequence to suppress cell proliferation in vivo is of interest as the basis for the design of potential antiproliferative therapeutic agents.

The human homologue of fission Yeast cdc27, p66, is a component of active human DNA polymerase delta.

An essential eukaryotic DNA polymerase, DNA polymerase delta (pol delta), synthesizes DNA processively in the presence of proliferating cell nuclear antigen (PCNA). Recently, a 66 kDa polypeptide (p66) that displays significant homology within its PCNA binding domain to that of fission yeast cdc27 was identified as a component of mouse and calf thymus pol delta. Our studies show that p66 interacts tightly with other subunits of pol delta during size fractionation of human cell extracts, and co-immunoprecipitates with these subunits along with PCNA-dependent polymerase activity. Active human pol delta could be reconstituted by co-expressing p125, p50, and p66 recombinant baculoviruses, but not by co-expressing p125 and p50 alone. Interaction studies demonstrated that p66 stabilizes the association between p125 and p50. Pull-down assays with PCNA-linked beads demonstrated that p66 increases the overall affinity of pol delta for PCNA. These results indicate that p66 is a functionally important subunit of human pol delta that stabilizes the pol delta complex and increases the affinity of pol delta for PCNA.

Functional analysis of human FEN1 in Saccharomyces cerevisiae and its role in genome stability.

The flap endonuclease, FEN1, is an evolutionarily conserved component of DNA replication from archaebacteria to humans. Based on in vitro results, it processes Okazaki fragments during replication and is involved in base excision repair. FEN1 removes the last primer ribonucleotide on the lagging strand and it cleaves a 5' flap that may result from strand displacement during replication or during base excision repair. Its biological importance has been revealed largely through studies in the yeast Saccharomyces cerevisiae where deletion of the homologous gene RAD27 results in genome instability and mutagen sensitivity. While the in vivo function of Rad27 has been well characterized through genetic and biochemical approaches, little is understood about the in vivo functions of human FEN1. Guided by our recent results with yeast RAD27, we explored the function of human FEN1 in yeast. We found that the human FEN1 protein complements a yeast rad27 null mutant for a variety of defects including mutagen sensitivity, genetic instability and the synthetic lethal interactions of a rad27 rad51 and a rad27 pol3-01 mutant. Furthermore, a mutant form of FEN1 lacking nuclease function exhibits dominant-negative effects on cell growth and genome instability similar to those seen with the homologous yeast rad27 mutation. This genetic impact is stronger when the human and yeast PCNA-binding domains are exchanged. These data indicate that the human FEN1 and yeast Rad27 proteins act on the same substrate in vivo. Our study defines a sensitive yeast system for the identification and characterization of mutations in FEN1.

Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway.

The CDK inhibitor, p21WAF1/Cip1 blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, whereas the C-terminus binds and inhibits PCNA (proliferating cell nuclear antigen) function. PCNA is essential for processivity of both DNA polymerase delta and epsilon. We have performed a detailed analysis of growth inhibition by the N- and C-terminal regions of p21, and determined whether the N- and C-terminal regions mediate this effect by different mechanisms. Expression of either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21. The effectiveness of the two p21 domains is dependent on their stability which is determined by the ubiquitin-proteasome pathway. The stabilization of the N- and C-terminal region of p21 increases their effectiveness as inhibitors of DNA synthesis to levels comparable to full length p21. Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In contrast, inhibition by the C-terminal region of p21 is not accompanied by suppression of E2F activity, but is mediated via PCNA binding. The C-terminal region of p21 therefore inhibits cell growth by a mechanism distinct from that of the N-terminal region containing the CDK-cyclin inhibitory domain.

The C-terminal domain of p21 inhibits nucleotide excision repair In vitro and In vivo.

The protein p21(Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase delta. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.