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PubMed1993

[Preservation of PCNA in different fixatives and demonstration of PCNA as well as membrane antigen by double labelling technique].

Four fixatives were used with two different fixation times in order to determine the influence of different fixatives on PCNA preservation. The result indicated that the aldehyde-base fixatives preserved PCNA excellently and the appropriate fixative time was 24 hours. The result of alcohol-base fixative on PCNA preservation seemed unsatisfactory and 10% buffer formalin was the best one among the aldehyde-base fixatives for this preservation. Additionally, double-labelling technique for demonstrating PCNA as well as the membrane antigen in the same slide was established. Selection of antigen chromogens suitable to double-labelling is described.

Adenocarcinoma↗
PubMed2000

Essential interaction between the fission yeast DNA polymerase delta subunit Cdc27 and Pcn1 (PCNA) mediated through a C-terminal p21(Cip1)-like PCNA binding motif.

Direct interaction between DNA polymerase delta and its processivity factor proliferating cell nuclear antigen (PCNA) is essential for effective replication of the eukaryotic genome, yet the precise manner by which this occurs is unclear. We show that the 54 kDa subunit of DNA polymerase delta from Schizosaccharomyces pombe interacts directly with Pcn1 (PCNA) both in vivo and in vitro. Binding is effected via a short sequence at the C-terminus of Cdc27 with significant similarity to the canonical PCNA binding motif first identified in the mammalian p21(Cip1) protein. This motif is both necessary and sufficient for binding of Pcn1 by Cdc27 in vitro and is essential for Cdc27 function in vivo. We also show that the Pcn1 binding motif in Cdc27 is distinct from its binding site for Cdc1, the 55 kDa B-subunit of polymerase delta, and present evidence that Cdc27 can bind to Pcn1 and Cdc1 simultaneously. Finally, we show that Cdc27 performs at least two distinct essential functions, one of which is independent of Pcn1 binding.

Amino Acid Sequence↗
PubMed1991

Upstream sequences of rice proliferating cell nuclear antigen (PCNA) gene mediate expression of PCNA-GUS chimeric gene in meristems of transgenic tobacco plants.

The transgenic tobacco plants have been generated that express the E. coli beta-glucuronidase (GUS) gene under control of the promoter from the rice proliferating cell nuclear antigen (PCNA, DNA polymerase auxiliary protein) gene. GUS expression detected in situ by staining with the chromogenic substrate, 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Gluc), was restricted to meristems in the organs of the transgenic tobacco plants. This expression responded to the phytohormones which promote callus formation. Furthermore, in situ thymidine uptake showed that the GUS expression pattern corresponded well to the active sites of DNA synthesis. Deletion analysis of the 5' upstream sequence confined the GUS expression pattern to a fragment extending 263 bp upstream of the transcription start site of the rice PCNA gene. Thus, we have identified this fragment as a main regulatory element of the rice PCNA gene promoter.

Blotting, Northern↗
PubMed1994

Assessment of proliferating cell nuclear antigen (PCNA) in breast cancer using anti-PCNA PC10 and 19A2: correlation with 5-bromo-2'-deoxyuridine or tritiated thymidine labeling and flow cytometric analysis.

We compared immunohistochemical staining by two monoclonal antibodies, PC10 and 19A2, to standard methods for cell proliferation, 3H-TdR or BrdU labeling index (S-LI) and S-phase fraction by flow cytometry (S-flow). One hundred thirty-two breast carcinomas were studied retrospectively using formalin fixed, paraffin embedded archival tissues on which S-LI and S-flow had been obtained originally. Percentages of tumor cells positive with PC10 and 19A2 correlated well (r = 0.736, p < 0.0001), although the mean marking index for 19A2 was lower and closer to reference measurements than the mean PC10 index. Correlations between PC10 or 19A2 vs. S-LI, S-flow or DNA ploidy (DNAI) were significant in a group of 64 tumors obtained between 1988 and June 1992, and poor in another group of 68 tumors obtained between 1985 and 1988, suggesting deterioration of stainability with prolonged storage. Discrimination of faint staining from negative nuclei was difficult on PCNA stained sections. Carnoy fixation did not improve results over those fixed with formalin. S-flow and S-LI predicted relapse-free survival, but PCNA indices did not. We conclude that PC10 and 19A2 immunostaining of formalin or Carnoy fixed archival breast cancer tissue correlated with reference measures of proliferation only in cases of short storage periods. Although statistically significant, levels of correlation were too low to use PCNA indices for prognosis.

Antibodies, Monoclonal↗
PubMed1986

Nuclear patterns of cyclin (PCNA) antigen distribution subdivide S-phase in cultured cells--some applications of PCNA antibodies.

Immunofluorescence studies using PCNA autoantibodies specific for the proliferation-sensitive protein cyclin have revealed dramatic changes in the nuclear distribution of this protein during the S-phase of normal and transformed cells. Patterns of cyclin antigen distribution subdivide S-phase and have provided new cell cycle landmarks. Some of these (nucleolar exclusion or staining), mimic topographical patterns of DNA synthesis thus arguing for a role of this protein in some specific aspect of DNA replication. Cells outside S-phase (G0 included) stain only weakly with PCNA antibodies, stressing the usefulness of this reagent for identifying proliferating cells (S-phase cells) of both normal and malignant origins.

Animals↗
PubMed2003

[Inhibition of lens epithelial cell cycle and PCNA mRNA expression by an antisense oligonucleotides complementary to the messenger RNA of proliferating cell nuclear antigen].

OBJECTIVE: To determine whether antisense oligonucleotides complementary to the messenger RNA of proliferating cell nuclear antigen (PCNA ASODN) inhibit the proliferation of bovine lens epithelial cell (BLEC) by changing the cell cycle and down-regulating the expression of PCNA mRNA and PCNA protein. METHODS: BLECs were cultured in vitro, and the second passage cells were used in this experiment. PCNA ASODN (30 micro mol/L), PCNA SODN (sense oligonucleotides, 30 micro mol/L), basic fibroblast growth factor (bFGF, 10 micro g/L), bFGF (10 ng/ml) + ASODN (30 micro mol/L), bFGF (10 micro g/L) + SODN (30 micro mol/L) were introduced respectively into the medium, and the same amount of PBS was added into the medium as a control. After 24 hours, the cell cycle and the PCNA expression were counted by flow cytometry, and the expression of PCNA mRNA was indicated by Northern ELISA hybridization. RESULTS: PCNA ASODN could decrease the rate of the cells of S phase and down-regulate the expression of PCNA mRNA and PCNA protein. Comparing with the control group, after 24 hours, the rate of cells of S phase was decreased from 15.67% to 7.96%, the expression of PCNA mRNA from 0.266 to 0.176 and the expression of PCNA protein from 55.27% to 12.32%. PCNA ASODN could also inhibit the proliferation of BLEC induced by bFGF, comparing with the bFGF group, the rate of cells of S phase was decreased from 23.4% to 19.9%, the expression of PCNA mRNA from 0.576 to 0.357 and the expression of PCNA protein from 76.4% to 35.48%. CONCLUSIONS: Our results demonstrate that the PCNA ASODN decreases expression of PCNA mRNA and PCNA protein, and stops the cell to enter and progress through S phase. These results provide an important impetus to initiate in vivo studies to determine the feasibility of antisense strategies in the prevention of posterior capsular opacification.

Animals↗
PubMed1987

Proliferating cell nuclear antigen (PCNA)/cyclin in activated human T lymphocytes.

Proliferating cell nuclear antigen (PCNA), also called cyclin, is an intranuclear polypeptide whose synthesis reaches its maximum during the S-phase of the cell cycle. PCNA is expressed in several kinds of proliferating cells, one of which is the mitogen-stimulated human peripheral blood lymphocyte. PCNA expression increases from the late G1 phase through the S-phase of the cell cycle. This study is focused on the regulation of PCNA expression during the G1 phase of human T lymphocytes and on the relationship between PCNA expression and the rate of T cell proliferation. Special use is made of human autoantibodies to PCNA and the development of flow cytometry to label this intranuclear polypeptide. Unstimulated purified human peripheral blood T cells were PCNA negative. T cells treated with monoclonal antibody (64.1) to the CD3 complex expressed receptors for IL 2 but did not express PCNA until exogenous IL 2 was added to the culture. PCNA expression as well as the entry of the cells into S-phase could be inhibited by IL 2 receptor antibodies. Transferrin receptors were expressed only in T cells that were stimulated with both 64.1 and exogenous IL 2. Transferrin receptors were detectable before the cells expressed PCNA. Thus, the onset of PCNA expression is a phase in cell proliferation that follows transferrin receptor expression but preceded DNA synthesis. Drugs like dexamethasone and cyclosporin, which affect the early part of G1, inhibited PCNA expression; whereas cytarabine (ara-C) and hydroxyurea, which affect the S-phase and prevent DNA synthesis, did not block PCNA expression. There was a close correlation between the number of PCNA-positive cells and the number of S-phase cells in unperturbed T cell cultures. The highest level of PCNA expression was seen in cells that were in the first cycle after stimulation. The results show that PCNA expression is regulated by a signal after IL 2 binding and that PCNA labeling is a precise indicator for human T cells that are committed to DNA synthesis. Comparing these results with previous observations on the expression of some other activation-associated antigens, we suggest that the onset of PCNA expression represents a discrete step in T cell activation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Proliferating cell nuclear antigen (PCNA/cyclin) in plant proliferating cells: immunohistochemical and quantitative analysis using autoantibody and murine monoclonal antibodies to PCNA.

Proliferating-cell nuclear antigen (PCNA), also known as cyclin, is synthesized in proliferative cells and recently was identified as DNA polymerase-delta auxiliary protein. In this paper, the association of PCNA to the proliferative cells of plants was analysed using both autoantibodies to PCNA obtained from a patient with systemic lupus erythematosus (SLE) and murine monoclonal antibodies. By immunohistochemical analysis, nuclei of cells around the growing point in soybean root tips reacted strongly with autoantibodies to PCNA in the serum from a patient with SLE. The plant PCNA in root tip cells was purified by ammonium sulfate fractionation, DEAE chromatography, and affinity chromatography. The partially purified plant PCNA was tested by immunoblotting and a 34 kD polypeptide reacted with both the human anti-PCNA autoantibody and a mouse monoclonal antibody against human PCNA (TOB 7). In addition, the purified plant PCNA reacted with both antibodies in enzyme-linked immunosorbent assay (ELISA). The binding of anti-PCNA serum to the animal PCNA was blocked by the plant PCNA in this ELISA. The association of PCNA with growing cells in plants was further confirmed by quantitative sandwich type ELISA using two murine monoclonal antibodies to PCNA, TOB7 and TO17. Those results suggested that PCNA in both plant and animal cells had the same immunological and biochemical characteristics and the plant PCNA might play an important role in cell growth, existing as it does in proliferating plant cells. The concentration of PCNA in soybean germ extract before germination was less than 5 ng ml-1 (protein concentration, 6.8 mg ml-1), but that of the root tip stem including the growing point increased to 887 ng ml-1 (protein concentration 3.8 mg ml-1) in the second day after germination.

Animals↗
PubMed1998

X-ray induced changes in immunostaining of proliferating cell nuclear antigen (PCNA) in V79 hamster fibroblasts.

BACKGROUND: Proliferating cell nuclear antigen (PCNA) ist a 36 kD protein that is involved in DNA-replication and -repair. For V79 hamster cells, a mutated p53 and a so-called "adaptive response", an improved radiation tolerance after pre-irradiation with low X-ray doses hours before definitive irradiation with higher doses have been reported. To better understand the role of PCNA after photon irradiation in vivo, using flow cytometry, we studied the immunochemical PCNA-staining in V79 cells after irradiation with 6-MeV photons with and without serum depletion and with and without low-dose pre-irradiation under different growth conditions. MATERIAL AND METHODS: Using V79 hamster cells, BrdUrd incorporation, total and DNA-bound PCNA were measured for exponential cells and for confluent cells at different times (up to 14 days) after reaching confluence. Cells were either grown with medium containing 10% fetal calf serum (FCS) or 0.5% FCS. Six days after reaching confluence, cells were irradiated with 1 Gy (and 8 Gy for non-serum-depleted cells) (6-MV photons, 2 Gy/min). Then, immunochemical PCNA-staining was measured by flow cytometry at 0, 30, 60 and 120 min after irradiation. For studying the adaptive response, exponentially growing cells and cells that were 6 days in confluence were pretreated with 0.01 Gy, reincubated for 5 h and then definitively treated with 1 Gy and harvested and processed as described above. RESULTS: Four days after reaching confluence, DNA-bound PCNA and BrdUrd content were reduced to a minimum of < 15% positive cells while total PCNA remained essentially unchanged. After irradiation with 1 Gy 6 days after reaching confluence, cells grown with 10% FCS showed a moderate but distinct transient increase in DNA-bound PCNA at 30 min after irradiation. After irradiation with 8 Gy, there was no clear increase at 30 min but a more distinct decrease at 60 min, implying that the increase might occur earlier in the time course at higher doses. Total cellular PCNA and BrdUrd uptake were constant during the first 2 hours after irradiation. In cells that were kept with serum depleted medium for 6 days after reaching confluence, total PCNA was reduced and no changes in either DNA-bound PCNA or BrdUrd-uptake were observed after irradiation. When cells were primed with a dose of 0.01 Gy 5 h before subsequent treatment with 1 Gy, neither for exponentially growing cells nor for those in confluence a significant difference in the detected amount of PCNA (total and DNA-bound) or BrdUrd was observed when compared to cells treated without a priming dose. CONCLUSIONS: The moderate X-ray induced DNA association of PCNA is indicative for ongoing DNA repair but appears to require serum stimuli. However, this p53-independent pathway involving PCNA does not seem to be the most relevant for survival in these rodent cells that tolerate much residual damage. Furthermore, no adaptive response for DNA-association of PCNA could be detected in V79 cells.

Animals↗
PubMed2006

Downregulation of PCNA potentiates p21-mediated growth inhibition in response to hyperoxia.

Prolonged exposure to hyperoxia inhibits cell proliferation in G1 via increased expression of p21. While p21 inhibits proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis, it can also directly lower PCNA abundance; however, it is unclear whether loss of PCNA contributes to growth arrest. Here, we investigate how PCNA loss affects ability of p21 to exert G1 growth arrest of lung epithelial cells exposed to hyperoxia. In A549 cells that express p21 and growth arrest in G1 during hyperoxia, small interfering RNA (siRNA) knockdown of p21 led to G1 checkpoint bypass, increased cell death, and restoration of PCNA expression. Conditional overexpression of the PCNA binding domain of p21 in H1299 cells that do not normally express p21, or exposure to hyperoxia, caused a time-dependent loss of PCNA. Titrating PCNA levels using siRNA to approximate the low amount observed in cells expressing p21 resulted in S phase arrest. While lowering PCNA by itself caused S phase arrest, the combination of hyperoxia and siRNA against PCNA dramatically reduced PCNA abundance resulting in G1 arrest. G1 growth arrest was markedly enhanced upon the addition of p21 to these cells. Our findings suggest a model in which reducing expression of the abundant protein PCNA allows the less abundant protein p21 to be more effective at suppressing the processivity functions of remaining PCNA, thereby fully exerting the G1 checkpoint. Given that high p21 expression is often associated with lower PCNA abundance, our findings are suggestive of a global growth inhibitory mechanism involving p21-mediated PCNA suppression.

Adenocarcinoma↗
PubMed2000

Immunohistochemical localization and correlation of p53 and PCNA expression in breast carcinoma.

The object of the present study is to detect the p53 tumour suppressor gene and proliferation cell nuclear antigen (PCNA) expression in breast carcinoma by immunohistochemistry and correlate them with the prognostic parameters. Total 35 cases of primary breast carcinoma were studied and classified histologically. Paraffin sections were stained by using monoclonal antibody D07 for p53 protein and PC-10 for PCNA. Out of 35 cases, 16 (45.7%) were p53 positive and 25 (71.4%) were PCNA positive. The mean PCNA labelling index (PCNA LI +/- SD) was 58.97 +/- 22.72 in tumors positive for both p53+ and PCNA+ while cases negative for p53- and positive for PCNA+ has higher PCNA LI +/- SD (59.24 +/- 18.97). The difference in the two groups was not significant. Most cases were positive for both p53+ and PCNA+ in the age group < 30 with higher mean PCNA LI +/- SD (62.20 +/- 27.13) than in the group > 30 (57.88 +/- 18.47). In the pre-menopausal group 57.1% cases were positive for p53+ with higher PCNA LI +/- SD (59.94 +/- 24.22). Maximum p53 and PCNA positivity was observed in grade III tumors (63.2% and 84.2%). The mean PCNA LI +/- SD was also highest in grade III carcinomas (66.83 +/- 13.97). No significant correlation was found between p53 and PCNA status with morphological type and tumour size except that logistic regression showed a positive correlation with tumour grade. Therefore the present study suggests that both p53 expression and PCNA are markers of poor differentiation in breast cancer.

Adult↗
PubMed1996

Autoantibodies recognizing proteins copurified with PCNA in patients with connective tissue diseases.

OBJECTIVE: Proliferating cell nuclear antigen (PCNA), one of the target antigen recognized by lupus sera, has been reported to be present as a subnuclear multi-peptide complex. But autoantibodies reacting with components of PCNA complex are poorly understood. To study the specificity of those autoantibodies, immunoreactivities of autoimmune sera against purified PCNA antigen were studied. METHODS: PCNA antigens were purified from rabbit thymus extract by affinity column using murine monoclonal antibodies (mAbs) to PCNA, TOB7, TO17 and TO30. Immunoreactivities of autoimmune sera against purified PCNA were analyzed by WB. RESULTS: PCNA antigen purified by serum AK predominantly showed a 34 kD band specific for PCNA in SDS-PAGE. When antigens were purified by anti-PCNA mAb TOB7 and TO30 which are known to be targeting different epitopes on PCNA antigen, SDS-PAGE analysis showed various mol. wt of proteins in addition to the 34 kD PCNA while both AK and mAbs reacted only with 34 kD PCNA in WB. In WB using PCNA purified by TOB7, various immunoreactivities were observed at 150, 66, 58, 48, 45, 37, 32 and 16 kDa in sera from patients with connective tissue diseases. CONCLUSIONS: These results suggested that many of the proteins copurified with PCNA were also targets of autoimmune responses and these autoantibody expression may be induced through antigen-driven mechanisms.

Animals↗
PubMed2000

Phylogenetic analysis of archaeal PCNA homologues.

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27kDa) and 248 aa (27kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages.

Amino Acid Sequence↗
PubMed1988

Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry.

Proliferating cell nuclear antigen (PCNA)/cyclin is an intranuclear polypeptide antigen that is found in both normal and transformed proliferating cells. We have recently described two mouse monoclonal antibodies reacting with PCNA. In this report we describe the application of these antibodies to the study of proliferating human cells by indirect immunofluorescence microscopy and by flow cytometry. A fixation/permeation procedure was developed in order to obtain satisfactory binding of monoclonal PCNA-specific antibodies to proliferating cells. This method involved fixation with 1% paraformaldehyde followed by methanol treatment. For the staining of cells in suspension with the IgM type monoclonal antibodies lysolecithin was added to the paraformaldehyde solution to achieve a better permeation by the antibody molecules. This procedure gave a good ratio of specific staining relative to the background staining. It also preserved the shape and normal architecture of the cells as judged by visual microscopic observation and by light scatter measurements using a flow cytometer. Furthermore, this fixation technique permits simultaneous labeling of DNA by propidium iodide and PCNA by monoclonal antibodies. PCNA was detected in various types of normal and transformed proliferating cells by indirect immunofluorescence. Quiescent peripheral blood mononuclear cells were PCNA-negative whereas a fraction of lectin-stimulated lymphocytes became PCNA-positive. Similarly, early passages of fetal skin fibroblasts were PCNA-positive but non-proliferating senescent fibroblasts of later passages were PCNA-negative. The association of PCNA-staining by monoclonal antibodies with cell proliferation was confirmed by flow cytometry. Simultaneous labeling of PCNA and DNA showed that the PCNA signal increased during the G1 phase of the cell cycle, reached its maximum in the S-phase, and declined during the G2/M phase. Using cell sorting we demonstrated that mitotic cells had a very low PCNA signal. Thus, monoclonal PCNA-specific antibodies offer a convenient tool for the detection of human cell proliferation by immunofluorescence microscopy and by flow cytometry.

Animals↗
PubMed1996

[Significance of PCNA proliferating fraction for prognosis of ovarian carcinoma].

The prognostic value of the PCNA-proliferative fraction as compared to conventional clinical and histomorphological factors (FIGO-stage, tumour type, histological grading, lymph node status, size of residual tumour) was investigated in 81 ovarian cancer patients. Categorisation of PCNA-expression into tumours with low and high proliferative activity (< 20%/ > or = 20% according to laboratory standards) had the highest prognostic value. Categorisation on the basis of the median value (< or = 34%/> 34%) or classification of PCNA as a continuous variable did not prove advantageous. PCNA-proliferative fraction was significantly directly correlated with histological grading (p = 0.006). Tumours with a high PCNA expression had a greater frequency of macroscopically detectable residual tumours. In univariate survival analyses patients with highly proliferating tumours had a worse outcome than patients with tumour of low proliferation (PCNA </> or = 20%/20%, p = 0.012; PCNA < or = 34%/ > 34%, p = 0.08). The result was consistent in subgroup of FIGO III-tumours (p = 0.031, PCNA < 20%/> or = 20%), of FIGO l-tumours (p = 0.036, PCNA < 20%/> or = 20%), of carcinomas without post-operative residual tumour (p = 0.03 PCNA < 20%/> or = 20%) and also of FIGO III-tumour without residual tumours (p = 0.041 PCNA < 20%/> or = 20%). Multivariate survival analysis comprising all the patients revealed PCNA expression (< 20%/> or = 20%) as an independent prognostic factor second to the size of the residual tumour. In patients with FIGO III-tumours PCNA proves significant as an independent factor after the size of the residual tumour was removed from the model. Thus, PCNA provides additional information which may prove beneficial in determining prognostic estimates for ovarian cancer.

Adult↗
PubMed2000

Two modes of FEN1 binding to PCNA regulated by DNA.

The FEN1 nuclease functions during Okazaki fragment maturation in the eukaryotic cell. Like many other proliferating cell nuclear antigen (PCNA)-binding proteins, FEN1 interacts with the interdomain connector loop (IDCL) of PCNA, and PCNA greatly stimulates FEN1 activity. A yeast IDCL mutant pcna-79 (IL126,128AA) failed to interact with FEN-1, but, surprisingly, pcna-79 was still very active in stimulating FEN1 activity. In contrast, a C-terminal mutant pcna-90 (PK252,253AA) showed wild-type binding to FEN1 in solution, but poorly stimulated FEN1 activity. When PCNA was loaded onto a DNA substrate coupled to magnetic beads, it stabilized retention of FEN1 on the DNA. In this DNA-dependent binding assay, pcna-79 also stabilized retention of FEN1, but pcna-90 was inactive. Therefore, in the absence of DNA, FEN1 interacts with PCNA mainly through the IDCL. However, when PCNA encircles the DNA, the C-terminal domain of PCNA rather than its IDCL is important for binding FEN1. An FF-->GA mutation in the PCNA-interaction domain of FEN1 severely decreased both modes of interaction with PCNA and resulted in replication and repair defects in vivo.

Amino Acid Motifs↗
PubMed1999

Oxidative damage-induced PCNA complex formation is efficient in xeroderma pigmentosum group A but reduced in Cockayne syndrome group B cells.

Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases delta and epsilon, is essential for both DNA replication and repair. PCNA is required in the resynthesis step of nucleotide excision repair (NER). After UV irradiation, PCNA translocates into an insoluble protein complex, most likely associated with the nuclear matrix. It has not previously been investigated in vivo whether PCNA complex formation also takes place after oxidative stress. In this study, we have examined the involvement of PCNA in the repair of oxidative DNA damage. PCNA complex formation was studied in normal human cells after treatment with hydrogen peroxide, which generates a variety of oxidative DNA lesions. PCNA was detected by two assays, immunofluorescence and western blot analyses. We observed that PCNA redistributes from a soluble to a DNA-bound form during the repair of oxidative DNA damage. PCNA complex formation was analyzed in two human natural mutant cell lines defective in DNA repair: xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). XP-A cells are defective in overall genome NER while CS-B cells are defective only in the preferential repair of active genes. Immunofluorescent detection of PCNA complex formation was similar in normal and XP-A cells, but was reduced in CS-B cells. Consistent with this observation, western blot analysis in CS-B cells showed a reduction in the ratio of PCNA relocated as compared to normal and XP-A cells. The efficient PCNA complex formation observed in XP-A cells following oxidative damage suggests that formation of PCNA-dependent repair foci may not require the XPA gene product. The reduced PCNA complex formation observed in CS-B cells suggests that these cells are defective in the processing of oxidative DNA damage.

Blotting, Western↗