Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.
Eukaryotic proliferating cell nuclear antigen (PCNA) is a replication accessory protein that functions in DNA replication, repair, and recombination. The various functions of PCNA are regulated by post-translational modifications including mono-ubiquitylation, which promotes translesion synthesis, and sumoylation, which inhibits recombination. To understand how the SUMO modification regulates PCNA, we generated a split SUMO-modified PCNA protein and showed that it supports cell viability and stimulates DNA polymerase δ activity. We then determined its X-ray crystal structure and found that SUMO occupies a position on the back face of the PCNA ring, which is distinct from the position occupied by ubiquitin in the structure of ubiquitin-modified PCNA. We propose that the back of PCNA has evolved to be a site of regulation that can be easily modified without disrupting ongoing reactions on the front of PCNA, such as normal DNA replication. Moreover, these modifications likely allow PCNA to function as a tool belt, whereby proteins can be recruited to the replication machinery via the back of PCNA and be held in reserve until needed.
DNA replication; DNA recombination; DNA repair; protein-DNA interactions; translesion synthesis
The crystal structure of PCNA from the halophilic archaeon H. volcanii reveals specific features of the charge distribution on the protein surface that reflect adaptation to a high-salt environment and suggests a different type of interaction with DNA in halophilic PCNAs.
The sliding clamp proliferating cell nuclear antigen (PCNA) plays vital roles in many aspects of DNA replication and repair in eukaryotic cells and in archaea. Realising the full potential of archaea as a model for PCNA function requires a combination of biochemical and genetic approaches. In order to provide a platform for subsequent reverse genetic analysis, PCNA from the halophilic archaeon Haloferax volcanii was subjected to crystallographic analysis. The gene was cloned and expressed in Escherichia coli and the protein was purified by affinity chromatography and crystallized by the vapour-diffusion technique. The structure was determined by molecular replacement and refined at 3.5 Å resolution to a final R factor of 23.7% (R
free = 25%). PCNA from H. volcanii was found to be homotrimeric and to resemble other homotrimeric PCNA clamps but with several differences that appear to be associated with adaptation of the protein to the high intracellular salt concentrations found in H. volcanii cells.
PCNA–DNA interactions; sliding clamps; halophilic environment
Proliferating cell nuclear antigen (PCNA) is a critical player in cell proliferation. It interacts with a myriad of cellular proteins in genomic DNA replication and cell cycle control. This makes PCNA an attractive target for developing antiproliferative therapeutics. Indeed the binding of a human tumor suppressor protein, p21, to PCNA contributes to its antiproliferative effect in cells. In this work we reported a fluorescence polarization-based binding assay for determining the affinity between the p21 peptide and human PCNA. In order to improve the potency of the p21-based PCNA antagonist, we exploited the homotrimeric structure of PCNA and developed multivalent peptide-based PCNA antagonists. The di- and trivalent p21-based antagonists bind to PCNA with low nanomolar dissociation constant. Moreover, we showed that the multivalent PCNA antagonists inhibited PCNA-dependent DNA synthesis in a human cell extract with improved avidity when compared to the monovalent p21 peptide. The fluorescence polarization assay holds promise for the discovery of potent small-molecule PCNA inhibitors given its ready adaptability to a high-throughput screening format.
PCNA; multivalent binding; fluorescence assay; DNA synthesis; antagonist
New therapeutic approaches that can accelerate neutrophil apoptosis under inflammatory conditions to enhance the resolution of inflammation are now under study. Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells is a key regulator of neutrophil survival. The nuclear-to-cytoplasmic relocalization occurred during granulocytic differentiation and is dependent on a nuclear export sequence thus strongly suggesting that PCNA has physiologic cytoplasmic functions. In this review, we will try to put into perspective the physiologic relevance of PCNA in neutrophils. We will discuss key issues such as molecular structure, post-translational modifications, based on our knowledge of nuclear PCNA, assuming that similar principles governing its function are conserved between nuclear and cytosolic PCNA. The example of cystic fibrosis that features one of the most intense neutrophil-dominated pulmonary inflammation will be discussed. We believe that through an intimate comprehension of the cytosolic PCNA scaffold based on nuclear PCNA knowledge, novel pathways regulating neutrophil survival can be unraveled and innovative agents can be developed to dampen inflammation where it proves detrimental.
inflammation; neutrophil; apoptosis; PCNA; cystic fibrosis
Ubiquitination of proliferating cell nuclear antigen (PCNA) plays a crucial role in regulating replication past DNA damage in eukaryotes, but the detailed mechanisms appear to vary in different organisms. We have examined the modification of PCNA in Schizosaccharomyces pombe. We find that, in response to UV irradiation, PCNA is mono- and poly-ubiquitinated in a manner similar to that in Saccharomyces cerevisiae. However in undamaged Schizosaccharomyces pombe cells, PCNA is ubiquitinated in S phase, whereas in S. cerevisiae it is sumoylated. Furthermore we find that, unlike in S. cerevisiae, mutants defective in ubiquitination of PCNA are also sensitive to ionizing radiation, and PCNA is ubiquitinated after exposure of cells to ionizing radiation, in a manner similar to the response to UV-irradiation. We show that PCNA modification and cell cycle checkpoints represent two independent signals in response to DNA damage. Finally, we unexpectedly find that PCNA is ubiquitinated in response to DNA damage when cells are arrested in G2.
The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a “sliding clamp” that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair.
There is compelling evidence that proliferating cell nuclear antigen (PCNA), a DNA sliding clamp, co-ordinates the processing and joining of Okazaki fragments during eukaryotic DNA replication. However, a detailed mechanistic understanding of functional PCNA:ligase I interactions has been incomplete. Here we present the co-crystal structure of yeast PCNA with a peptide encompassing the conserved PCNA interaction motif of Cdc9, yeast DNA ligase I. The Cdc9 peptide contacts both the inter-domain connector loop (IDCL) and residues near the C-terminus of PCNA. Complementary mutational and biochemical results demonstrate that these two interaction interfaces are required for complex formation both in the absence of DNA and when PCNA is topologically linked to DNA. Similar to the functionally homologous human proteins, yeast RFC interacts with and inhibits Cdc9 DNA ligase whereas the addition of PCNA alleviates inhibition by RFC. Here we show that the ability of PCNA to overcome RFC-mediated inhibition of Cdc9 is dependent upon both the IDCL and the C-terminal interaction interfaces of PCNA. Together these results demonstrate the functional significance of the β-zipper structure formed between the C-terminal domain of PCNA and Cdc9 and reveal differences in the interactions of FEN-1 and Cdc9 with the two PCNA interfaces that may contribute to the co-ordinated, sequential action of these enzymes.
Proliferating cell nuclear antigen (PCNA), a processivity factor
for DNA polymerases δ and ɛ,
is involved in DNA replication as well as in diverse DNA repair pathways.
In quiescent cells, UV light-induced bulky DNA damage triggers the
transition of PCNA from a soluble to an insoluble chromatin-bound
form, which is intimately associated with the repair synthesis by polymerases δ and ɛ.
In this study, we investigated the efficiency of PCNA complex formation
in response to ionizing radiation-induced DNA strand breaks in normal
and radiation-sensitive Ataxia telangiectasia (AT) cells by immunofluorescence
and western blot techniques. Exposure of normal cells to γ-rays
rapidly triggered the formation of PCNA foci in a dose-dependent
manner in the nuclei and the PCNA foci (40–45%)
co-localized with sites of repair synthesis detected by bromodeoxyuridine
labeling. The chromatin-bound PCNA gradually declined with increasing
post-irradiation times and almost reached the level of unirradiated
cells by 6 h. The PCNA foci formed after γ-irradiation
was resistant to high salt extraction and the chromatin association
of PCNA was lost after DNase I digestion. Interestingly, two radiosensitive
primary fibroblast cell lines, derived from AT patients harboring
homozygous mutations in the ATM gene, displayed an efficient PCNA
redistribution after γ-irradiation.
We also analyzed the PCNA complex induced by a radiomimetic agent, Bleomycin
(BLM), which produces predominantly single- and double-strand DNA
breaks. The efficiency and the time course of PCNA complex induced
by BLM were identical in both normal and AT cells. Our study demonstrates
for the first time that the ATM gene product is not required for
PCNA complex assembly in response to DNA strand breaks. Additionally,
we observed an increased interaction of PCNA with the Ku70 and Ku80
heterodimer after DNA damage, suggestive of a role for PCNA in the
non-homologous end-joining repair pathway of DNA strand breaks.
AlkB PCNA-interacting motif (APIM) is present in >200 proteins and may mediate PCNA binding during genotoxic stress.
Numerous proteins, many essential for the DNA replication machinery, interact with proliferating cell nuclear antigen (PCNA) through the PCNA-interacting peptide (PIP) sequence called the PIP box. We have previously shown that the oxidative demethylase human AlkB homologue 2 (hABH2) colocalizes with PCNA in replication foci. In this study, we show that hABH2 interacts with a posttranslationally modified PCNA via a novel PCNA-interacting motif, which we term AlkB homologue 2 PCNA-interacting motif (APIM). We identify APIM in >200 other proteins involved in DNA maintenance, transcription, and cell cycle regulation, and verify a functional APIM in five of these. Expression of an APIM peptide increases the cellular sensitivity to several cytostatic agents not accounted for by perturbing only the hABH2–PCNA interaction. Thus, APIM is likely to mediate PCNA binding in many proteins involved in DNA repair and cell cycle control during genotoxic stress.
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, we have designed a new assay 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. Using this assay, we showed that RFC remains transiently associated with PCNA on the DNA after the loading reaction. Addition of p21 did not inhibit RFC-dependent PCNA loading; rather, p21 formed a stable complex with PCNA on the DNA. In contrast, the formation of a p21-PCNA complex on the DNA resulted in the displacement of RFC from the DNA. The nonhydrolyzable analogs of ATP, adenosine-5′-O-(3-thiotriphosphate) (ATPγS) and adenyl-imidodiphosphate, each stabilized the primer recognition complex containing RFC and PCNA in the absence of p21. RFC in the ATPγS-activated complex was no longer displaced from the DNA by p21. We propose 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. We also suggest that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome.
Proliferating cell nuclear antigen (PCNA, sliding clamp) is a toroidal-shaped protein that encircles DNA and plays a pivotal role in DNA replication, modification and repair. To perform its vital functions, the clamp has to be opened and resealed at primer−template junctions by a clamp loader molecular machine, replication factor C (RFC). The mechanism of this process constitutes a significant piece in the puzzle of processive DNA replication. We show that upon clamp opening the RFC/PCNA complex undergoes a large conformational rearrangement, leading to the formation of an extended interface between the clamp and RFC. Binding of ring-open PCNA to all five RFC subunits transforms the free-energy landscape underlying the closed- to open state transition, trapping PCNA in an open conformation. Careful comparison of free-energy profiles for clamp opening in the presence and absence of RFC allowed us to substantiate the role of RFC in the initial stage of the clamp-loading cycle. RFC does not appreciably destabilize the closed state of PCNA. Instead, the function of the clamp loader is dependent on the selective stabilization of the open conformation of the clamp.
Sliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. In an ATP-dependent reaction, clamp loader complexes, such as the Replication Factor-C (RFC) complex in eukaryotes, open the clamp and load it around primer-template DNA.
We built a model of RFC bound to PCNA and DNA based on existing crystal structures of clamp loaders. This model suggests that DNA would enter the clamp at an angle during clamp loading, thereby interacting with positively charged residues in the center of PCNA. We show that simultaneous mutation of Lys 20, Lys 77, Arg 80, and Arg 149, which interact with DNA in the RFC-PCNA-DNA model, compromises the ability of yeast PCNA to stimulate the DNA-dependent ATPase activity of RFC when the DNA is long enough to extend through the clamp. Fluorescence anisotropy binding experiments show that the inability of the mutant clamp proteins to stimulate RFC ATPase activity is likely caused by reduction in the affinity of the RFC-PCNA complex for DNA. We obtained several crystal forms of yeast PCNA-DNA complexes, measuring X-ray diffraction data to 3.0 Å resolution for one such complex. The resulting electron density maps show that DNA is bound in a tilted orientation relative to PCNA, but makes different contacts than those implicated in clamp loading. Because of apparent partial disorder in the DNA, we restricted refinement of the DNA to a rigid body model. This result contrasts with previous analysis of a bacterial clamp bound to DNA, where the DNA was well resolved.
Mutational analysis of PCNA suggests that positively charged residues in the center of the clamp create a binding surface that makes contact with DNA. Disruption of this positive surface, which had not previously been implicated in clamp loading function, reduces RFC ATPase activity in the presence of DNA, most likely by reducing the affinity of RFC and PCNA for DNA. The interaction of DNA is not, however, restricted to one orientation, as indicated by analysis of the PCNA-DNA co-crystals.
The archaeal/eukaryotic proliferating cell nuclear antigen (PCNA) toroidal clamp interacts with a host of DNA modifying enzymes, providing a stable anchorage and enhancing their respective processivities. Given the broad range of enzymes with which PCNA has been shown to interact, relatively little is known about the mode of assembly of functionally meaningful combinations of enzymes on the PCNA clamp. We have determined the X-ray crystal structure of the Sulfolobus solfataricus PCNA1–PCNA2 heterodimer, bound to a single copy of the flap endonuclease FEN1 at 2.9 Å resolution. We demonstrate the specificity of interaction of the PCNA subunits to form the PCNA1–PCNA2–PCNA3 heterotrimer, as well as providing a rationale for the specific interaction of the C-terminal PIP-box motif of FEN1 for the PCNA1 subunit. The structure explains the specificity of the individual archaeal PCNA subunits for selected repair enzyme ‘clients’, and provides insights into the co-ordinated assembly of sequential enzymatic steps in PCNA-scaffolded DNA repair cascades.
The high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA) to a resolution of 2.0 Å.
The overall architecture of HvPCNA is very similar to other known PCNAs, which are highly structurally conserved. Three commonly observed adaptations in halophilic proteins are higher surface acidity, bound ions and increased numbers of intermolecular ion pairs (in oligomeric proteins). HvPCNA possesses the former two adaptations but not the latter, despite functioning as a homotrimer. Strikingly, the positive surface charge considered key to PCNA's role as a sliding clamp is dramatically reduced in the halophilic protein. Instead, bound cations within the solvation shell of HvPCNA may permit sliding along negatively charged DNA by reducing electrostatic repulsion effects.
The extent to which individual proteins adapt to halophilic conditions varies, presumably due to their diverse characteristics and roles within the cell. The number of ion pairs observed in the HvPCNA monomer-monomer interface was unexpectedly low. This may reflect the fact that the trimer is intrinsically stable over a wide range of salt concentrations and therefore additional modifications for trimer maintenance in high salt conditions are not required. Halophilic proteins frequently bind anions and cations and in HvPCNA cation binding may compensate for the remarkable reduction in positive charge in the pore region, to facilitate functional interactions with DNA. In this way, HvPCNA may harness its environment as opposed to simply surviving in extreme halophilic conditions.
A stable stoichiometric complex of archaeal DNA polymerase with proliferating cell nuclear antigen (PCNA) was formed using a PCNA monomer mutant and the complex was successfully crystallized.
Replicative DNA polymerase interacts with processivity factors, the β-subunit of DNA polymerase III or proliferating cell nuclear antigen (PCNA), in order to function with a long template DNA. The archaeal replicative DNA polymerase from Pyrococcus furiosus interacts with PCNA via its PCNA-interacting protein (PIP) motif at the C-terminus. The PCNA homotrimeric ring contains one PIP interacting site on each monomer and since the ring can accommodate up to three molecules simultaneously, formation of a stable stoichiometric complex of PCNA with its interacting protein has been difficult to control in vitro. A stable complex of the DNA polymerase with PCNA, using a PCNA monomer mutant, has been purified and crystallized. The best ordered crystal diffracted to 3.0 Å resolution using synchrotron radiation. The crystals belong to space group P21212, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 Å.
DNA polymerase; proliferating cell nuclear antigen
The sliding clamp Proliferating Cell Nuclear Antigen (PCNA) functions as a recruiter and organizer of a wide variety of DNA modifying enzymes including nucleases, helicases, polymerases and glycosylases. The 5′-flap endonuclease Fen-1 is essential for Okazaki fragment processing in eukaryotes and archaea, and is targeted to the replication fork by PCNA. Crenarchaeal XPF, a 3′-flap endonuclease, is also stimulated by PCNA in vitro. Using a novel continuous fluorimetric assay, we demonstrate that PCNA activates these two nucleases by fundamentally different mechanisms. PCNA stimulates Fen-1 by increasing the enzyme's binding affinity for substrates, as suggested previously. However, PCNA activates XPF by increasing the catalytic rate constant by four orders of magnitude without affecting the KM. PCNA may function as a platform upon which XPF exerts force to distort DNA substrates, destabilizing the substrate and/or stabilizing the transition state structure. This suggests that PCNA can function directly in supporting catalysis as an essential cofactor in some circumstances, a new role for a protein that is generally assumed to perform a passive targeting and organizing function in molecular biology. This could provide a mechanism for the exquisite control of nuclease activity targeted to specific circumstances, such as replication forks or damaged DNA with pre-loaded PCNA.
Proliferating cell nuclear antigen (PCNA) is a homotrimeric protein that functions as a sliding clamp during DNA replication. Several mutant forms of PCNA that block translesion DNA synthesis have been identified in genetic studies in yeast. One such mutant protein (encoded by the rev6-1 allele) is a glycine to serine substitution at residue 178, located at the subunit interface of PCNA. To better understand how this substitution interferes with translesion synthesis, we have determined the X-ray crystal structure of the G178S PCNA mutant protein. This substitution has little effect on the structure of the domain in which the substitution occurs. Instead, significant, local structural changes are observed in the adjacent subunit. The most notable difference between mutant and wild-type structures is in a single, extended loop (comprising amino acid residues 105-110), which we call loop J. In the mutant protein structure, loop J adopts a very different conformation in which the atoms of the protein backbone have moved by as much as 6.5 Å from their positions in the wild-type structure. To better understand the functional consequences of this structural change, we have examined the ability of this mutant protein to stimulate nucleotide incorporation by DNA polymerase eta (pol η). Steady state kinetic studies show that while wild-type PCNA stimulates incorporation by pol η opposite an abasic site, the mutant PCNA protein actually inhibits incorporation opposite this DNA lesion. These results show that the position of loop J in PCNA plays an essential role in facilitating translesion synthesis.
Proliferating cell nuclear antigen (PCNA) is a ubiquitous protein that interacts with multiple partners and regulates nuclear activities, including chromatin assembly, histone modifications, replication, and DNA damage repair. The role of specific partners in regulating PCNA activities is not fully understood. Here we identify the nucleosome binding protein HMGN1 as a new PCNA-interacting protein that enhances the binding of PCNA to chromatin but not to purified DNA. Two tetrapeptides in the conservative domain of HMGN1 contain amino acids necessary for the binding of HMGN1 to PCNA. Deletion of both tetrapeptides abolishes the HMGN1-PCNA interaction. PCNA preferentially binds to the linker DNA adjacent to an HMGN-containing nucleosome. In living cells, loss of HMGN1 decreases the rate of PCNA recruitment to damaged DNA sites. Our study identifies a new factor that facilitates the interaction of PCNA with chromatin and provides insights into mechanisms whereby nucleosome binding architectural proteins affect the cellular phenotype.
Mammalian DNA polymerase δ (pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and various DNA repair processes. Its function as a chromosomal DNA polymerase is dependent on the association with proliferating cell nuclear antigen (PCNA) which functions as a molecular sliding clamp. All four of the pol δ subunits (p125, p50, p68, and p12) have been reported to bind to PCNA. However, the identity of the subunit of pol δ that directly interacts with PCNA and is therefore primarily responsible for the processivity of the enzyme still remains controversial. Previous model for the network of protein-protein interactions of the pol δ-PCNA complex showed that pol δ might be able to interact with a single molecule of PCNA homotrimer through its three subunits, p125, p68, and p12 in which the p50 was not included in. Here, we have confirmed that the small subunit p50 of human pol δ truthfully interacts with PCNA by the use of far-Western analysis, quantitative ELISA assay, and subcellular co-localization. P50 is required for mediation of the interaction between pol δ subassemblies and PCNA homotrimer. Thus, pol δ interacts with PCNA via its four subunits.
Proliferative cell nuclear antigen (PCNA), a conserved plant protein as well as an important replication factor, is induced in response to geminivirus infection in the resting cells of the phloem tissues. The biochemical role of PCNA in rolling circle replication (RCR) of geminivirus DNA has not been explored in detail. The initiation of RCR of the bipartite genome of a geminivirus, Indian mung bean yellow mosaic virus (IMYMV), is mainly controlled by viral protein Rep (or AL1 or AC1). The role of host PCNA in RCR of IMYMV was revealed by studying the physical and functional interactions between recombinant PCNA and recombinant IMYMV Rep. Pea nuclear PCNA as well as recombinant pea PCNA showed binding to recombinant Rep in experiments involving both affinity chromatography and yeast two-hybrid approaches. The contacting amino acid residues of PCNA seemed to be present throughout a wide region of the trimeric protein, while those of Rep appeared to be localized only in the middle part of the protein. The site-specific nicking-closing activity and the ATPase function of IMYMV Rep were impaired by PCNA. These observations lead to interesting speculations about the control of viral RCR and dynamic profiles of protein-protein interactions at the RCR origin of the geminiviruses.
Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication and in many other processes in eukaryotic cells. Genetic analysis of Phaseolus coccineus showed the presence of at least two PCNA-like genes in the runner bean genome. Two PCNA genes have previously been found in a few plant species including Arabidopsis, tobacco, and maize. In these species, genes were nearly identical. Two cDNAs of P. coccineus PCNA (PcPCNA1 and PcPCNA-like1) have been identified that differ distinctly from each other. Interestingly, both the genetic organization of PcPCNA1 and PcPCNA-like1 genes and their expression patterns were similar, but these were the only similarities between these genes and their products. The identity between PcPCNA1 and PcPCNA-like1 at the amino acid level was only 54%, with PcPCNA-like1 lacking motifs that are crucial for the activity typical of PCNA. Consequently, these two proteins showed different properties. PcPCNA1 behaved like a typical PCNA protein: it formed a homotrimer and stimulated the activity of human DNA polymerase delta. In addition, PcPCNA1 interacted with a p21 peptide and was recognized by an anti-human PCNA monoclonal antibody PC10. By contrast, PcPCNA-like1 was detected as a monomer and was unable to stimulate the DNA polymerase delta activity. PcPCNA-like1 also could not interact with p21 and was not recognized by the PC10 antibody. Our results suggest that PcPCNA-like1 either is unable to function alone and therefore might be a component of the heterotrimeric PCNA ring or may have other, yet unknown functions. Alternatively, the PcPCNA-like1 gene may represent a pseudogene.
DNA polymerase delta; PCNA; Phaseolus coccineus; PRINS; RACE
The proliferating cell nuclear antigen (PCNA) is an essential component of DNA replication, cell cycle regulation, and epigenetic inheritance. High expression of PCNA is associated with poor prognosis in patients with breast cancer. The 5′-region of the PCNA gene contains two computationally-detected estrogen response element (ERE) sequences, one of which is evolutionarily conserved. Both of these sequences are of undocumented cis-regulatory function. We recently demonstrated that estradiol (E2) enhances PCNA mRNA expression in MCF7 breast cancer cells. MCF7 cells proliferate in response to E2.
Here, we demonstrate that E2 rapidly enhanced PCNA mRNA and protein expression in a process that requires ERα as well as de novo protein synthesis. One of the two upstream ERE sequences was specifically bound by ERα-containing protein complexes, in vitro, in gel shift analysis. Yet, each ERE sequence, when cloned as a single copy, or when engineered as two tandem copies of the ERE-containing sequence, was not capable of activating a luciferase reporter construct in response to E2. In MCF7 cells, neither ERE-containing genomic region demonstrated E2-dependent recruitment of ERα by sensitive ChIP-PCR assays.
We conclude that E2 enhances PCNA gene expression by an indirect process and that computational detection of EREs, even when evolutionarily conserved and when near E2-responsive genes, requires biochemical validation.
The cell cycle regulator p21 interacts with and inhibits the DNA replication and repair factor proliferating cell nuclear antigen (PCNA). We have defined a 39 amino acid fragment of p21 which is sufficient to bind PCNA with high affinity (Kd 10-20 nM). This peptide can inhibit DNA replication in vitro and microinjection of a GST fusion protein containing this domain inhibited S phase in vivo. Despite its high affinity for PCNA, the free 39 amino acid peptide does not have a well-defined structure, as judged 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 was 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 was defined as both necessary and sufficient for binding to p21.
In addition to its role as a processivity factor in DNA replication, proliferating cell nuclear antigen (PCNA) may function in the regulation of cell cycle progression. We present genetic evidence that PCNA interacts with the gene product of CDC44, an essential nucleotide-binding protein that encodes the large subunit of yeast replication factor C (K. Fien and B. Stillman, personal communication). Mutations in POL30 (PCNA) suppress cold-sensitive alleles of cdc44 that contain mutations in or near nucleotide-binding consensus domains, but they do not suppress a null allele. Thus, it appears that PCNA interacts with Cdc44p but cannot substitute for its function. pol30 mutations suppress additional phenotypes of cdc44 mutations, including the cold sensitivity that they were selected to suppress. This observation suggests an intimate association between PCNA and Cdc44p. Each of five independent pol30 mutants contains a unique single mutation that maps to a localized region on one face of the predicted three-dimensional structure of PCNA. This face identifies a region likely to be important for functional interaction between the CDC44 and POL30 gene products.