Proliferating cell nuclear antigen
The DNA polymerase accessory factor PCNA has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, four mutants of human PCNA were constructed and they were analyzed in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, it was found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and it was shown that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis (Jonsson, 1998).
PCNA, which is recognized as a DNA polymerase processivity factor, has direct interactions with various proteins involved in the important genetic information processes in Eukarya. The crystal structure of PCNA from the hyperthermophilic archaeon, Pyrococcus furiosus (PfuPCNA) was determined at 2.1 Å resolution, and it was found that the toroidal ring-shaped structure, which consists of homotrimeric molecules, is highly conserved between the Eukarya and Archaea. This allowed its interaction with the loading factor to be examined at the atomic level. The replication factor C (RFC) is known as the loading factor of PCNA on to the DNA strand. P. furiosus RFC (PfuRFC) has a PCNA binding motif (PIP-box) at the C-terminus of the large subunit (RFCL). An 11 residue-peptide containing a PIP-box sequence of RFCL inhibits the PCNA-dependent primer extension ability of P. furiosusPolI in a concentration-dependent manner. To understand the molecular interaction mechanism of PCNA with PCNA binding proteins, the crystal structure of PfuPCNA complexed with the PIP-box peptide was solved. The interaction mode of the two molecules is remarkably similar to that of human PCNA and a peptide containing the PIP-box of p21(WAF1/CIP1). Moreover, the PIP-box binding may have some effect on the stability of the ring structure of PfuPCNA by some domain shift. Structural analysis on PfuPCNA suggests that the interaction mode of the PIP-box with PCNA is generally conserved among the PCNA interacting proteins and that the functional meaning of the interaction via the PIP-box possibly depends on each protein. A movement of the C-terminal region of the PCNA monomer by PIP-box binding may cause the PCNA ring to be more rigid, suitable for its functions (Matsumiya, 2002).
Flap endonuclease-1 (FEN1) is a key enzyme for maintaining genomic stability
and replication. Proliferating cell nuclear antigen (PCNA) binds FEN1 and
stimulates its endonuclease activity. The structural basis of the
FEN1-PCNA interaction was revealed by the crystal structure of the complex
between human FEN1 and PCNA. The main interface involves the C-terminal tail of
FEN1, which forms two ß-strands connected by a short helix, the ßA
-alphaA- ßB
motif, participating in ß/ß and hydrophobic interactions with PCNA. These
interactions are similar to those observed for the
p21CIP1/WAF1 peptide. However, this structure involving the
full-length enzyme has revealed additional interfaces that are involved in the
core domain. The interactions at the interfaces maintain the enzyme in an
inactive 'locked-down' orientation and might be utilized in rapid DNA-tracking
by preserving the central hole of PCNA for sliding along the DNA. A hinge region
present between the core domain and the C-terminal tail of FEN1 would play a
role in switching the FEN1 orientation from an inactive to an active
orientation (Sakurai, 2005 ).
Interaction of p21 family members with PCNA
The cyclin-dependent kinase inhibitor p21Cip1/Waf1 (Drosophila homolog: Dacapo) is responsible for the p53-dependent growth arrest of cells in G1 phase following DNA damage. This study investigates the regions of p21 involved in inhibition of the G1/S phase cyclin-dependent kinase, cyclin E/Cdk2, as well as regions of p21 important for binding to this kinase and recombinant PCNA. A series of overlapping peptides were synthesized spanning the entire p21 sequence. These were used in in vitro assays with cyclin E/Cdk2-immune complexes and with recombinant p21 and PCNA proteins. One amino-terminal p21 peptide spanning amino acids 15-40 antagonizes p21 binding and inhibition of cyclin E/Cdk2 kinase. Antagonism of p21 binding is lost in a similar peptide lacking amino acids 15-20, as well as in a peptide in which cysteine-18 is substituted for a serine (I. Chen, 1995a).
These results suggest that this peptide region is important for p21 interaction with cyclin E/Cdk2. A second peptide (amino acids 58-77) also antagonizes p21-activity, but this peptide does not affect the ability of p21 to interact with cyclin E/Cdk2. A region of p21 larger than 26 amino acids is presumably required for Cdk-inhibition because none of the peptides tested inhibit cyclin E/Cdk2. A peptide spanning amino acids 21-45 binds recombinant p21 in ELISA assays, and additional studies reveal a requirement for amino acids 26 through 45 for this interaction. A p21 peptide spanning amino acids 139-164 was found to bind PCNA; this peptide suppresses recombinant p21-PCNA interaction. Conformational analysis reveals that peptides spanning amino acids 21-45 and 139-164 tend toward an alpha-helical conformation in trifluoroethanol buffer, indicating that these regions are probably in a coiled conformation in the native protein. Taken together, these results provide an insight into domains of p21 that are involved in cyclin E/Cdk2 and PCNA interaction. These results also suggest that a potential p21 dimerization domain may lie in the amino-terminus of p21 (I. Chen, 1995a).
A 39 amino acid fragment of p21 is sufficient to bind PCNA with high affinity. This peptide can inhibit DNA replication in vitro; microinjection of a GST fusion protein containing this domain inhibits S phase in vivo. Despite its high affinity for PCNA, the free 39 amino acid peptide does not have a well-defined structure, to judge from circular dichroism and nuclear magnetic resonance measurements, suggesting an induced fit mechanism for the PCNA-p21 interaction. The association of the small peptide with PCNA is thermolabile, suggesting that portions of p21 adjoining the minimal region of contact stabilize the interaction. In addition, a domain containing 67 amino acids from the N-terminus of PCNA is defined as both necessary and sufficient for binding to p21 (J. Chen, 1996).
The protein p21 (WAF1, CIP1 or sdi1), induced by the tumour-suppressor protein p53, interacts with and inhibits two different targets essential for cell-cycle progression. One of these is the cyclin-Cdk family of kinases and the other is the essential DNA replication factor, proliferating-cell nuclear antigen (PCNA). Separate domains of p21 are responsible for interacting with and inhibiting the two targets. An amino-terminal domain inhibits cyclin-Cdk kinases; a carboxy-terminal domain inhibits PCNA. Using these separated domains, it has been determined that p21 inhibits different biological systems through different targets. The PCNA-binding domain is sufficient for inhibition of DNA replication based on simian virus 40, whereas the Cdk2-binding domain is sufficient for inhibition of DNA replication based on Xenopus egg extract and for growth suppression in transformed human cells (J. Chen, 1995).
Analysis of deletion mutants of p21, which cover the majority of the protein, reveals that deletion of either amino acids 142-147 or 149-154 results in loss of ability to bind PCNA. Site-directed mutagenesis in this region leads to the identification of the PCNA binding motif RQXXMTXFYXXXR and demonstrates that mutation of either amino acid Met-147 or Phe-150 results in almost complete ablation of PCNA binding. Interestingly, when a determination is made of DNA synthesis inhibitory activity of deletion mutants or point mutants that are unable to bind Cdk2 and/or PCNA, it is found that loss of binding to PCNA does not affect inhibitory activity, whereas lack of Cdk2 binding greatly reduces the same. This result suggests that the primary mechanism for inhibition of DNA synthesis by p21 occurs via inhibition of Cdk activity (Nakanishi, 1995).
Mammalian cell-cycle control by antimitogenic signals involves p21Cip1/WAF1, p27Kip1 and p57Kip2, a family of proteins that bind to and inhibit cyclin-dependent kinases (CDKs) required for initiation of S phase. The protein p21 also binds to the DNA polymerase delta processivity factor, proliferating-cell nuclear antigen (PCNA), and inhibits in vitro PCNA-dependent DNA replication. The CDK and PCNA inhibitory activities of p21 are functionally independent and reside in separate protein domains. The PCNA binding and inhibitory activities, which are not observed with p27 or p57, reside in the C-terminal domain of p21, whereas the CDK inhibitory activity resides in the conserved N-terminal domains of these proteins. When separately overexpressed in mammalian cells, the CDK and PCNA inhibitory domains prevent DNA replication, demonstrating a dual function of p21 as a cell-cycle inhibitor in vivo (Luo, 1995).
After exposure of normal human fibroblasts to UV-C light, nuclear binding of the proliferating cell nuclear antigen (PCNA) required for nucleotide excision repair, appears to be rapidly triggered in the G1 and G2 phases of the cell cycle. Association to repair sites of the detergent-insoluble form of PCNA reachs a peak 15-30 min after irradiation, and then decreases to basal levels within 24-48 h. In contrast, the nuclear association of p21 protein shows a slower kinetics, reaching maximal levels between 24 and 48 h; however, as with PCNA, this occurs only in G1 and G2 phases. Although the two proteins are known to be associated as detergent-soluble proteins, it is unknown whether they associate also in the detergent-insoluble form. To address this question, the chromatin-bound form of PCNA was released by using DNAse I. DNA digestion results in the almost complete release of PCNA from its binding sites, while only about 60% of nuclear-bound p21 could be solubilized. Immunoprecipitation of PCNA and p21 released by enzymatic digestion shows that p21 is associated with PCNA bound to late DNA repair sites. These results indicate that during nucleotide excision repair, nuclear binding of PCNA precedes that of p21 protein, and suggest that temporal association of p21 with the detergent-insoluble fraction is coincident with the disassembly of PCNA from DNA repair sites (Savio, 1996).
Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta; these three proteins constitute the pol delta holoenzyme. Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme (Flores-Rozas, 1994).
The p53 tumour-suppressor protein controls the expression of a gene encoding the p21 cyclin-dependent protein kinase (CDK) regulator. Levels of p21 protein are increased in senescent cells and p21 overexpression blocks the growth of tumour cells. In normal human cells, but not in many tumour cells, p21 exists in a quaternary complex with a cyclin, a CDK, and the proliferating-cell nuclear antigen (PCNA). p21 controls CDK activity, thereby affecting cell-cycle control, whereas PCNA functions in both DNA replication and repair. Simian virus 40 DNA replication in vitro was used to show that p21 directly inhibits PCNA-dependent DNA replication in the absence of a cyclin/CDK. p21 blocks the ability of PCNA to activate DNA polymerase delta, the principal replicative DNA polymerase. This regulation results from a direct interaction between p21 and PCNA. Thus, during p53-mediated suppression of cell proliferation, p21 and PCNA may be important for coordinating cell-cycle progression, DNA replication and repair of damaged DNA (Waga, 1994).
Proper control of the mammalian cell cycle requires the function of cyclin-dependent kinase (CDK) inhibitors. The p21 family currently includes three distinct genes, p21, p27(Kip1), and p57(Kip2), that share a common N-terminal domain for binding to and inhibiting the kinase activity of CDK-cyclin complexes. The p21 protein also binds to proliferating cell nuclear antigen (PCNA) through a separate C-terminal domain affecting DNA replication and repair. The p27 and p57 proteins also each contain unique C-terminal domains whose functions are unknown. The human p57 protein, like p21, contains a PCNA-binding domain within its C terminus. When its N-terminal CDK-cyclin binding domain is removed, the altered p57 can prevent DNA replication in vitro and S phase entry in vivo. Disruption of either CDK/cyclin or PCNA binding partially reduces p57's ability to suppress myc/RAS-mediated transformation in primary cells, while loss of both inhibitory functions completely eliminates p57's suppressive activity. Thus, control of cell cycle and suppression of cell transformation by p57 requires both CDK and PCNA inhibitory activity, and disruption of either or both functions may lead to uncontrolled cell growth (Watanabe, 1998).
The crystal structure of the human DNA polymerase delta processivity factor PCNA, complexed with a 22 residue peptide derived from the C-terminus of the cell-cycle checkpoint protein p21(WAF1/CIP1), has been determined at 2.6 angstrom resolution. p21 binds to PCNA in a 1:1 stoichiometry with an extensive array of interactions that include the formation of a beta sheet with the interdomain connector loop of PCNA. An intact trimeric ring is maintained in the structure of the p21-PCNA complex, with a central hole available for DNA interaction. The ability of p21 to inhibit the action of PCNA is therefore likely to be due to its masking of elements on PCNA that are required for the binding of other components of the polymerase assembly (Gulbis, 1996).
Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. There is marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells are cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remains at a low level and is accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells is only transient. At early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells is p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF can be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction is a consequence of receptor tyrosine kinase activation by EGF. The increased p21Cip1/WAF1 is associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, these results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells (Fan, 1995).
The p21 protein binds to both cyclin-dependent kinases (Cdks) and the proliferating cell nuclear antigen (PCNA). In mammalian cells, DNA damage results in an increase in the level of p53 protein, which stimulates expression of the gene encoding p21, which in turn leads to an inhibition of Cdk activity. Biochemical studies have shown that the direct interaction between p21 and PCNA blocks the latter's function in DNA replication but not in DNA repair. In addition to the p53-dependent damage response, the stimulation of quiescent cells with serum can also cause a p53-independent elevation in p21 gene expression. It is not clear, however, whether the induction of p21 protein under these two circumstances serves the same purpose. The kinetics of p21 induction by DNA damage and serum stimulation was investigated as well as the consequent effects on cell-cycle progression. Using both normal and repair-deficient human cells, the nuclear distribution of p21 in relation to that of PCNA was examined. In vivo immunofluorescence staining experiments indicate that, following UV damage, DNA repair is not inhibited by the presence of a large amount of p21 protein in the nucleus; in contrast, cells undergoing DNA replication during S phase contain very low amounts of p21. The addition of serum induces a transitory elevation of p21 levels, whereas UV damage to cells results in a sustained, high level of p21 that is more tightly associated with the nuclear structure. Interestingly, cells deficient in global nucleotide excision-repair display a distinct pattern of detergent-insoluble p21 that co-localizes with PCNA. The in vivo studies presented here indicate that p21 has a differential effect on DNA replication and DNA repair, and that the induction of p21 by serum and DNA damage may have different consequences. The co-localization of p21 and PCNA in the nucleus of normal and repair-deficient human cells indicates that p21 and PCNA interact during post-damage events (Li, 1996).
Cyclin D1 is a key regulator of the G1 phase of the cell cycle. Inhibition of cyclin D1 function results in cell cycle arrest, whereas unregulated expression of the protein accelerates G1. Cyclin D1 is localized to the nucleus during G1. During repair DNA synthesis, subsequent to UV-induced DNA damage, G1 cells readily lose their cyclin D1 while the proliferating cell nuclear antigen (PCNA) tightly associates with nuclear structures. Microinjection of cyclin D1 antisense accelerates DNA repair, whereas overexpression of cyclin D1 prevents DNA repair and the relocation of PCNA after DNA damage. Coexpression of cyclin D1 with its primary catalytic subunit, Cdk4, or with Cdk2, also prevents repair. In contrast, coexpression of PCNA, which is also a cyclin D1-associated protein, restores the ability of cells to repair their DNA. Acute overexpression of cyclin D1 in fibroblasts prevents them from entering S phase. Again, these effects are abolished by coexpression of cyclin D1 together with PCNA, but not with Cdk4 or Cdk2. Altogether, these results indicate that down-regulation of cyclin D1 is necessary for PCNA relocation and repair-related DNA synthesis, as well as for the start of DNA replication. Cyclin D1 appears to be an essential component of a G1-checkpoint (Pagano, 1994).
DNA-(cytosine-5) methyltransferase (MCMT) methylates newly replicated mammalian DNA, but the factors regulating this activity are unknown. MCMT is shown to bind proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA replication and repair. Binding of PCNA requires amino acids 163 to 174 of MCMT; it occurs in intact cells at foci of newly replicated DNA and does not alter MCMT activity. A peptide derived from the cell cycle regulator p21(WAF1) can disrupt the MCMT-PCNA interaction, which suggests that p21(WAF1) may regulate methylation by blocking access of MCMT to PCNA. MCMT and p21(WAF1) may be linked in a regulatory pathway, because the extents of their expression are inversely related in both SV40-transformed and nontransformed cells (Chuang, 1997).
The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is capable of binding to both cyclin-CDK and the proliferating cell nuclear antigen (PCNA). Through its binding to PCNA, p21 can regulate the function of PCNA differentially in replication and repair. To gain an understanding of the precise mechanism by which p21 affects PCNA function, a new assay was devised for replication factor C (RFC)-catalyzed loading of PCNA onto DNA, a method that utilizes a primer-template DNA attached to agarose beads via biotin-streptavidin. RFC is shown to remain transiently associated with PCNA on the DNA after the loading reaction. Addition of p21 does not inhibit RFC-dependent PCNA loading; rather, p21 forms a stable complex with PCNA on the DNA. In contrast, the formation of a p21-PCNA complex on the DNA results in the displacement of RFC from the DNA. The nonhydrolyzable analogs of ATP, adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS) and adenyl-imidodiphosphate, each stabilizes the primer recognition complex containing RFC and PCNA in the absence of p21. RFC in the ATPgammaS-activated complex is no longer displaced from the DNA by p21. It is proposed that p21 stimulates the dissociation of the RFC from the PCNA-DNA complex in a process that requires ATP hydrolysis and then inhibits subsequent PCNA-dependent events in DNA replication. The data suggest that the conformation of RFC in the primer recognition complex might change on hydrolysis of ATP. It is also suggested that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome (Waga, 1998).
The faithful replication of genomic DNA is crucial for maintaining genome stability. In eukaryotes, DNA rereplication is prevented by the temporal regulation of replication licensing. Replication-licensing factors are required to form prereplicative complexes during G1 phase, but are inactivated in S phase to prevent rereplication. A vertebrate CUL4 CRL ubiquitin ligase (CRL4) complex containing Cdt2 as the substrate recognition subunit promotes proper DNA replication, in part, by degrading the replication-licensing factor Cdt1 during S phase. This study shows that the C. elegans CRL4(Cdt2) complex has a conserved role in degrading Cdt1. Furthermore, CRL4(Cdt2) restrains replication licensing in both C. elegans and humans by targeting the degradation of the cyclin-dependent kinase (CDK) inhibitors CKI-1 and p21(Cip1), respectively. Human CRL4(Cdt2) targets the degradation of p21 in S phase, with the in vivo ubiquitylation of p21 by CRL4(Cdt2) dependent on p21 binding to PCNA. Inactivation of Cdt2 induces rereplication, which requires the presence of the CDK inhibitor p21. Strikingly, coinactivation of CRL4(Cdt2) and SCF(Skp2) (which redundantly targets p21 degradation) prevents the nuclear export of the replication-licensing factor Cdc6 during S phase, and the block on nuclear export is dependent on p21. This work defines the degradation of p21 as a critical aspect of replication licensing in human cells (Kim, 2008).
The CDK inhibitor p21 has a central role as an effector of cell cycle arrest when it is transactivated by the p53 tumor suppressor protein in response to DNA damage (Besson, 2008). While p21 is primarily considered an inhibitor of CDK/cyclin complexes, it also has CDK-independent functions involving the regulation of the cytoskeleton, transcription, and apoptosis (Besson, 2008). During the cell cycle, p21 levels are high in G1 phase, decrease significantly during S phase, and increase again during G2 phase. p21 has been shown to undergo proteasome-mediated degradation through the actions of a number of E3s: SCFSkp2, MDM2, MDMX, and APC/CCdc20. APC/CCdc20 targets the degradation of p21 during prometaphase (Amador, 2007). MDM2 and MDMX promote the proteasomal turnover of p21 predominantly in G1 and early S phases (Jin, 2008). SCFSkp2 has been shown to target p21 degradation in S-phase cells; however, the current study suggests that this degradation is not restricted to S phase. In contrast, CRL4Cdt2-mediated degradation of p21 occurs primarily in S phase (Kim, 2008).
This study shows that the in vivo ubiquitylation of p21 by CRL4Cdt2 is dependent on p21 binding to PCNA. The binding of Cdt1 to chromatin-associated PCNA has been proposed as a mechanism to ensure that Cdt1 degradation is S-phase-specific, and a similar mechanism may explain the S-phase specificity of the CRL4Cdt2-mediated p21 degradation. The significance of the PCNA-p21 interaction is underlined by the independent discovery that CRL4Cdt2 targets p21 degradation in response to DNA damage in a PCNA-dependent manner (Kim, 2008).
Interaction of PCNA with the repair endonucleases FEN and Xeroderma pigmentosum G and with mismatch repair proteins
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