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At least 19 recordsLinked to original sources

PubMed2001

Reactivity of anti-proliferating cell nuclear antigen (PCNA) murine monoclonal antibodies and human autoantibodies to the PCNA multiprotein complexes involved in cell proliferation.

Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the "protein machinery" for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients.

Animals↗

PubMed2001

Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta.

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.

Amino Acid Motifs↗

PubMed2006

Anti-PCNA autoantibodies preferentially recognize C-terminal of PCNA in patients with chronic hepatitis B virus infection.

We previously reported anti-PCNA autoantibodies in sera from patients with chronic HBV and HCV infection. To analyse the antigenic regions on proliferating cell nuclear antigen (PCNA) that confer autoantibody binding in patients with chronic hepatitis B (HBV) and C (HCV) infection, eight constructs including one wild type PCNA, one mutant type Y114A_PCNA and six C- or N-terminal PCNA truncations were generated. Sera from 185 patients with systemic lupus erythematosus (SLE), 178 with chronic HBV and 163 with chronic HCV infection, and 68 healthy individuals were examined for the presentation of anti-PCNA antibodies by enzyme linked immunosorbent assay (ELISA). By ELISA, anti-PCNA positive sera from patients with SLE, chronic HBV and HCV infection preferentially recognized the wild type PCNA more than the mutant type Y114A_PCNA (P < 0.05). The inhibition of binding by purified full-length rPCNA proteins with anti-PCNA positive sera was shown to exceed 70%. The inhibition of binding by purified truncated rPCNA proteins with sera from patients with chronic HBV and HCV infection and SLE was shown to confer dominant binding in T(L2) and T(L3). Moreover, the higher frequency of inhibition by using T(L3) was found in patients with chronic HBV infection. These data indicate that anti-PCNA autoantibodies preferentially recognize C-terminal of PCNA in patients with chronic HBV infection and may also provide advanced understanding between viral infection and autoimmunity for further study.

Absorption↗

PubMed2000

A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation.

Proliferating cell nuclear antigen is best known as a DNA polymerase accessory protein but has more recently also been shown to have different functions in important cellular processes such as DNA replication, DNA repair, and cell cycle control. PCNA has been found in quaternary complexes with the cyclin kinase inhibitor p21 and several pairs of cyclin-dependent protein kinases and their regulatory partner, the cyclins. Here we show a direct interaction between PCNA and Cdk2. This interaction involves the regions of the PCNA trimer close to the C termini. We found that PCNA and Cdk2 form a complex together with cyclin A. This ternary PCNA-Cdk2-cyclin A complex was able to phosphorylate the PCNA binding region of the large subunit of replication factor C as well as DNA ligase I. Furthermore, PCNA appears to be a connector between Cdk2 and DNA ligase I and to stimulate phosphorylation of DNA ligase I. Based on our results, we propose the model that PCNA brings Cdk2 to proteins involved in DNA replication and possibly might act as an "adaptor" for Cdk2-cyclin A to PCNA-binding DNA replication proteins.

CDC2-CDC28 Kinases↗

PubMed1997

Gene sequence and expression of an analog of proliferating cell nuclear antigen (PCNA) in the alga Tetraselmis chui and detection of the encoded protein with anti-rat PCNA monoclonal antibody.

To identify a phytoplankton cell cycle marker detected by a monoclonal antibody against mammalian proliferating cell nuclear antigen (PCNA) (S. Lin, J. Chang, and E. J. Carpenter, J. Phycol. 30:449-456, 1994), a PCNA gene fragment was isolated by reverse transcription-PCR from the marine unicellular alga Tetraselmis chui Butcher (Prasinophyceae). The gene fragment was 616 bp in length and contained an open reading frame of 205 amino acids. The deduced amino acid sequence showed 80 and 88% similarity to human and rice PCNA, respectively. Southern hybridization indicated that the isolated gene fragment was part of the T. chui genome, with up to three copies in each haploid nucleus. Northern hybridization was used to detect a PCNA mRNA with a size of 1.2 kb from an exponentially growing algal culture. The T. chui gene fragment has been cloned into an expression vector, and a fusion protein was subsequently generated. Anti-rat PCNA simultaneously recognized the PCNA fusion protein and a single 33-kDa band in T.chui total protein extract. Our results indicate that T. chui PCNA is highly similar to its mammalian counterpart and that anti-rat PCNA is a good tool for detecting phytoplankton PCNA in general.

Amino Acid Sequence↗

PubMed1996

p21waf1/cip1 protein associates with the detergent-insoluble form of PCNA concomitantly with disassembly of PCNA at nucleotide excision repair sites.

Evidence is presented that after exposure of normal human fibroblasts to UV-C light, nuclear binding of the proliferating cell nuclear antigen (PCNA) required for nucleotide excision repair, was rapidly triggered in the G1 and G2 phases of the cell cycle. Association to repair sites of the detergent-insoluble form of PCNA reached a peak 15-30 min after irradiation, and then decreased to basal levels within 24-48 h. In contrast, the nuclear association of p21 protein showed a slower kinetics, reaching maximal levels between 24 and 48 h but, similarly to PCNA, occurring 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 resulted 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 showed that p21 was 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.

Cell Cycle↗

PubMed1997

The DNA repair endonuclease XPG binds to proliferating cell nuclear antigen (PCNA) and shares sequence elements with the PCNA-binding regions of FEN-1 and cyclin-dependent kinase inhibitor p21.

Proliferating cell nuclear antigen (PCNA) is a DNA polymerase accessory factor that is required for DNA replication during S phase of the cell cycle and for resynthesis during nucleotide excision repair of damaged DNA. PCNA binds to flap endonuclease 1 (FEN-1), a structure-specific endonuclease involved in DNA replication. Here we report the direct physical interaction of PCNA with xeroderma pigmentosum (XP) G, a structure-specific repair endonuclease that is homologous to FEN-1. We have identified a 28-amino acid region of human FEN-1 (residues 328-355) and a 29-amino acid region of human XPG (residues 981-1009) that contains the PCNA binding activity. These regions share key hydrophobic residues with the PCNA-binding domain of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), and all three competed with one another for binding to PCNA. A conserved arginine in FEN-1 (Arg339) and XPG (Arg992) was found to be crucial for PCNA binding activity. R992A and R992E mutant forms of XPG failed to fully reconstitute nucleotide excision repair in an in vivo complementation assay. These results raise the possibility of a mechanistic linkage between excision and repair synthesis that is mediated by PCNA.

Amino Acid Sequence↗

PubMed2001

Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell nuclear antigen (PCNA) is independent of its in vitro interaction via a consensus PCNA binding region.

Interaction between human flap endonuclease-1 (hFEN-1) and proliferating cell nuclear antigen (PCNA) represents a good model for interactions between multiple functional proteins involved in DNA metabolic pathways. A region of 9 conserved amino acid residues (residues Gln-337 through Lys-345) in the C terminus of human FEN-1 (hFEN-1) was shown to be responsible for the interaction with PCNA. Our current study indicates that 4 amino acid residues in hFEN-1 (Leu-340, Asp-341, Phe-343, and Phe-344) are critical for human PCNA (hPCNA) interaction. A conserved PCNA interaction motif in various proteins from assorted species has been defined as Q(1)X(2)X(3)(L/I)(4)X(5)X(6)F(7)(F/Y)(8), although our results fail to implicate Q(1) (Gln-337 in hFEN-1) as a crucial residue. Surprisingly, all hFEN-1 mutants, including L340A, D341A, F343A, and F344A, retained hPCNA-mediated stimulation of both exo- and flap endonuclease activities. Furthermore, our in vitro assay showed that hPCNA failed to bind to the scRad27 (yeast homolog of FEN-1) nuclease. However, its nuclease activities were significantly enhanced in the presence of hPCNA. Four additional Saccharomyces cerevisiae scRad27 mutants, including multiple alanine mutants and a deletion mutant of the entire PCNA binding region, were constructed to confirm this result. All of these mutants retained PCNA-driven nuclease activity stimulation. We therefore conclude that stimulation of eukaryotic hFEN-1 nuclease activities by PCNA is independent of its in vitro interaction via the PCNA binding region.

Amino Acid Motifs↗

PubMed1998

PCNA binding proteins in Drosophila melanogaster : the analysis of a conserved PCNA binding domain.

The eukaryotic polymerase processivity factor, PCNA, interacts with cell cycle regulatory proteins such as p21(WAF1/Cip1) and Gadd45, as well as with proteins involved in the mechanics of DNA repair and replication. A conserved PCNA-binding motif is found in a subset of PCNA-interacting proteins, including p21, suggesting that the regulation of these interactions is important for the co-ordination of DNA replication and repair. We have identified several classes of protein which bind to Drosophila PCNA. Two of these proteins contain the consensus PCNA-binding domain: one is the Dacapo protein, a Drosophila homologue of p21(WAF1/Cip1), and the second is the transposase encoded by the Pogo DNA transposon . A conserved PCNA-binding domain is also present in a human relative of Pogo , named Tigger , suggesting that this domain has a functional role in this class of transposable element. This raises interesting possibilities for a novel method of transposition in which the transposase might be targeted to replicating DNA. Finally, we have investigated the use of this conserved PCNA-binding domain as a predictor of PCNA-binding capacity.

Amino Acid Sequence↗

PubMed1995

Direct interaction of Gadd45 with PCNA and evidence for competitive interaction of Gadd45 and p21Waf1/Cip1 with PCNA.

We have previously shown (Smith et al., 1994) that antibodies raised against the growth arrest and DNA damage inducible protein Gadd45 co-precipitate proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and repair. Here we demonstrate that Gadd45 can directly bind to PCNA using a Far-western blotting approach. In this assay, a Gadd45 bacterial expression vector was modified to allow synthesis of purified 32P-labeled Gadd45 fusion protein. This protein was used to detect filter bound PCNA protein, while filter bound Gadd45 protein could also be detected by free PCNA molecules. Using recombinant proteins in conjunction with immunoprecipitation and immunoblotting, we show that Gadd45 competes with p21 for binding to PCNA and conversely, p21 blocks the ability of Gadd45 to bind PCNA. In addition, p21 appears to disrupt PCNA trimers whereas Gadd45 has a lesser effect. PCNA trimer disruption was also observed in UV-irradiated cells but not in repair-defective xeroderma pigmentosum group A (XP-A) cells.

Base Sequence↗

PubMed2000

Characterization of MyD118, Gadd45, and proliferating cell nuclear antigen (PCNA) interacting domains. PCNA impedes MyD118 AND Gadd45-mediated negative growth control.

MyD118 and Gadd45 are related genes encoding for proteins that play important roles in negative growth control, including growth suppression and apoptosis. MyD118 and Gadd45 are related proteins that previously were shown to interact with proliferating cell nuclear antigen (PCNA), implicated in DNA replication, DNA repair, and cell cycle progression. To establish the role of MyD118 and Gadd45 interactions with PCNA, in this work we sought to identify the interacting domains and analyze the significance of this interaction in negative growth control. Using complementary in vivo and in vitro interaction assays the N-terminal (1-46) and middle (100-127) regions of PCNA were identified as harboring MyD118- and Gadd45 interacting domains, whereas PCNA interacting domains within MyD118 and Gadd45 were localized to the C termini of these proteins (amino acids 114-156 and 137-165, respectively). These findings provide first evidence that similar domains within MyD118 and Gadd45 mediate interactions with PCNA. Importantly, ectopic expression of MyD118 or Gadd45 N-terminal peptides, lacking the PCNA interacting domain, was found to suppress colony formation or induce apoptosis more efficiently than the full-length proteins. These findings suggest that interaction of MyD118 or Gadd45 with PCNA, in essence, serves to impede negative growth control.

Antigens, Differentiation↗

PubMed2006

Stoichiometric complex formation by proliferating cell nuclear antigen (PCNA) and its interacting protein: purification and crystallization of the DNA polymerase and PCNA monomer mutant complex from Pyrococcus furiosus.

Replicative DNA polymerase interacts with processivity factors, the beta-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 A resolution using synchrotron radiation. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 A.

Crystallization↗

PubMed2005

Proliferating-cell nuclear antigen (PCNA) as an independent prognostic marker in patients after prostatectomy: a comparison of PCNA and Ki-67.

OBJECTIVE: To investigate the prognostic value of prostatic tumour cell proliferation, as measured by Ki-67 and proliferating cell nuclear antigen (PCNA), and to compare these measures in men at low and high risk for progression of tumour. PATIENTS AND METHODS: Two groups of patients with prostate cancer, i.e. 'metastatic' (M, 22) who had pT3b-4aN0M0 and pTanyN1M0, and 'nonmetastatic' (NM, 18), who had < or =pT3aN0M0 disease, were selected from a well-examined and mapped group of 114 treated by radical prostatectomy. Patients in the NM group were selected by the criteria of having a Gleason score of < or = 7. To assess proliferation, 1000 cells were counted at x 400 magnification by two observers and the percentage of tumour cells positively stained with Ki-67 and PCNA defined as the Ki-67 and PCNA labelling index (LI), respectively. The two LI were compared in the NM and M groups, and the correlation of the LIs with pathological stage, progression and prostate-specific antigen (PSA)-free survival evaluated. Prognostic values of the LI were analysed using multivariate analysis. RESULTS: The mean (range) follow-up was 33 (4-78) months. The mean LIs were higher in the M than the NM group for both PCNA and Ki-67 (P = 0.02 and 0.019, respectively). Both LIs were markedly different between the groups when stratified by progression, with both significantly higher in men with progression in the NM group. Both LIs had a significant association with Gleason score, pathological stage, progression and PSA-free survival. In multivariate analysis the PCNA LI, surgical margin status and pathological stage were independent factors for progression. CONCLUSION: Tumour cell proliferation as assessed by Ki-67 or PCNA correlate significantly with progression. The PCNA LI was an independent predictor of progression, especially in patients with a low risk of progression according to predefined criteria.

Aged↗

PubMed1998

Replication factor C disengages from proliferating cell nuclear antigen (PCNA) upon sliding clamp formation, and PCNA itself tethers DNA polymerase delta to DNA.

Replication factor C (RF-C) and proliferating cell nuclear antigen (PCNA) assemble a complex, called sliding clamp, onto DNA. The clamp in turn loads DNA polymerases (pol) delta and epsilon to form the corresponding holoenzymes, which play an essential role in replication of eukaryotic chromosomal DNA and in several DNA repair pathways. To determine the fate of RF-C after loading of PCNA onto DNA, we tagged the RF-C subunit p37 with a protein kinase A recognition motif, so that the recombinant five-subunit RF-C complex could be 32P-labeled and quantitatively detected in femtomolar amounts. Nonspecific binding of RF-C to DNA was minimized by replacing the p140 subunit with an N-terminally truncated p140 subunit lacking the previously identified nonspecific DNA binding domain. Neither of these modifications impaired the clamp loading activity of the recombinant RF-C. Using gel filtration techniques, we demonstrated that RF-C dissociated from the DNA after clamp loading or pol delta holoenzyme assembly, while PCNA or PCNA.pol delta complex remained bound to DNA. PCNA catalytically loaded onto the template-primer was sufficient by itself to tether pol delta and stimulate DNA replication. The readdition of RF-C to the isolated PCNA.DNA complex did not further stimulate pol delta DNA synthesis. We conclude that pol delta holoenzyme consists of PCNA and pol delta core and that RF-C serves only to load PCNA clamp.

Animals↗

PubMed2003

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.

Animals↗

PubMed1985

Purification and N-terminal amino acid sequence of proliferating cell nuclear antigen (PCNA)/cyclin and development of ELISA for anti-PCNA antibodies.

Proliferating cell nuclear antigen (PCNA), also called cyclin, was purified from PBS extract of rabbit thymus by using a combination of ammonium sulfate fractionation, DEAE-Sephacel, HPLC ion exchange, and HPLC gel filtration column chromatography. PCNA was purified more than 600 times and was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. SDS-PAGE showed that a 36 kD protein was selectively isolated in this purification process, and this protein was identified as PCNA by immunoblotting. Other previously identified nuclear antigens, Sm, nRNP, SS-A/Ro, SS-B/La, histone, and DNA, were not detected in this preparation by counterimmunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA). Purified PCNA was used as an antigen to develop ELISA for rapid and specific detection of anti-PCNA in human sera. For further purification, the 36 kD band was electrophoretically eluted from SDS gel slices. The amino acid composition and the first 25 residues from the N-terminus of the protein were determined by using electroeluted PCNA. This amino acid sequence was found to be unique and showed little sequence homology with existent proteins in the protein identification resources databank.

Amino Acid Sequence↗

PubMed2002

A proteomics approach to identify proliferating cell nuclear antigen (PCNA)-binding proteins in human cell lysates. Identification of the human CHL12/RFCs2-5 complex as a novel PCNA-binding protein.

Proliferating cell nuclear antigen (PCNA), a eukaryotic DNA replication factor, functions not only as a processivity factor for DNA polymerase delta but also as a binding partner for multiple other factors. To understand its broad significance, we have carried out systematic studies of PCNA-binding proteins by a combination of affinity chromatography and mass spectrometric analyses. We detected more than 20 specific protein bands of various intensities in fractions bound to PCNA-fixed resin from human cell lysates and determined their peptide sequences by liquid chromatography and tandem mass spectrometry. A search with human protein data bases identified 12 reported PCNA-binding proteins from both cytoplasmic (S100 lysate) and nuclear extracts with substantial significance and four more solely from the nuclear preparation. CHL12, a factor involved in checkpoint response and chromosome cohesion, was a novel example found in both lysates. Further studies with recombinant proteins demonstrated that CHL12 and small subunits of replication factor C form a complex that interacts with PCNA.

Base Sequence↗

PubMed1987

PCNA (cyclin) autoantibodies and monoclonal antibodies reveal similar patterns of cyclin (PCNA) antigen staining in human cultured cells.

Double immunofluorescence and [3H]thymidine autoradiographic analysis of the same field of transformed human amnion cells (AMA) reacted with proliferating cell nuclear antigen (PCNA) autoantibodies and a monoclonal antibody (mAb 19F4) specific for cyclin (PCNA) revealed similar patterns and sequence of cyclin (PCNA) antigen staining during S-phase. These results suggest that immunofluorescence patterns obtained with PCNA autoantibodies reflect in fact patterns of cyclin (PCNA) antigen staining.

Antibodies, Monoclonal↗