Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation

Background

PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome.

Scope

This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution.

Conclusions

Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.     

Proliferating cell nuclear antigen (PCNA) may function as a double homotrimer complex in the mammalian cell

The diverse function of proliferating cell nuclear antigen (PCNA) may be regulated by interactions with different protein partners. Interestingly, the binding sites for all known PCNA-associating proteins are on the outer surface or the C termini ("front") sides of the PCNA trimer. Using cell extracts and purified human PCNA protein, we show here that two PCNA homotrimers form a back-to-back doublet. Mutation analysis suggests that the Arg-5 and Lys-110 residues on the PCNA back side are the contact points of the two homotrimers in the doublet. Furthermore, short synthetic peptides encompassing either Arg-5 or Lys-110 inhibit double trimer formation. We also found that a PCNA double trimer, but not a homotrimer alone, can simultaneously accommodate chromatin assembly factor-1 and polymerase delta. Together, our data supports a model that chromatin remodeling by chromatin assembly factor-1 (and, possibly, many other cellular activities) are tightly coupled with DNA replication (and repair) through a PCNA double trimer complex.     

Proliferating cell nuclear antigen (PCNA) is expressed in activated rat skeletal muscle satellite cells

Skeletal muscle satellite cells from uninjured muscle of adult animals are generally found to be in a quiescent state, and when cultured, they remain quiescent in vitro for a period of time which is directly related to the age of the donor animal. A technique for studying the activation of satellite cells in primary cultures has been developed and employs proliferating cell nuclear antigen (PCNA) as a marker for entrance into the S phase of the cell cycle. PCNA is a protein involved in DNA replication and is maximally expressed in S phase of the cell cycle. We monitored PCNA expression in satellite cells isolated from young (3 week) and adult (9 month) rats, and our results indicate that satellite cells begin to accumulate PCNA prior to changes in cell number in both age groups. Using ELISA techniques, we demonstrated that addition of an extract of crushed muscle (CME) activated satellite cells and significantly reduced the length of the lag phase in cells from both age groups. Addition of bFGF shortened the lag phase of PCNA synthesis in satellite cells from 3-week-old rats but had no effect on the kinetics of PCNA expression in cells from 9-month-old rats. Based on our experiments, PCNA expression can be used as a marker to follow the entry of satellite cells into the cell cycle in primary mass cultures. © 1993 Wiley-Liss, Inc.     

Proliferating cell nuclear antigen (PCNA/cyclin) immunocytochemistry as a labeling index in mouse lung tissues

Summary Proliferating cell nuclear antigen is expressed in cells from late G₁ through the S-phase of the cell cycle. Therefore, antibodies directed against this molecule should provide a probe for labeling immunocytochemically the nuclei of proliferating cells. Herein we demonstrate the feasibility and reliability of this technique by quantifying immunostained pulmonary nuclei. We applied polyclonal and monoclonal antisera to alveolar and bronchiolar pulmonary epithelial cells in various proliferative states in tissue-sections and in vitro. A/J mice had a slightly higher labeling index than C57BL/6J mice, and proliferation in both strains increased dramatically after butylated hydroxytoluene treatment produced compensatory hyperplasia of Type-II pneumocytes. Immunostaining in fetal and neonatal lung samples from mice was higher than in adults. Spontaneous lung adenomas had a higher labeling index than the surrounding normal lung tissue. In addition, new data contained herein demonstrate a strain difference in proliferation of bronchiolar epithelial cells, and quantify the extent to which BHT-induced lung damage increases these proliferative rates. This mammalian nuclear antigen did not cross-react with antiserum to a functionally related bacterial protein, the beta subunit of E. coli DNA polymerase-III holoenzyme.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

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.     

Proliferating cell nuclear antigen (PCNA) and human malignant tumor nucleolar antigens (HMTNA) in nucleoli of human hematological malignancies

Lymphoma (Lymphocytic non-Hodgkin's malignant lymphoma) and leukemic (chronic lymphocytic, acute and chronic myeloid, myelomonocytic leukemia) cells were studied by indirect immunofluorescence to evaluate the presence of proliferating cell nuclear antigen (PCNA) and human malignant tumor nuclear antigen (HMTNA) in their nucleoli. Most cells in lymph node smears of lymphocytic non-Hodgkin's malignant lymphoma (NHML) developed a bright nucleolar fluorescence with HMTNA antibodies. PCNA was detected in nucleoli of a limited number of cells which apparently represent the proliferating cell population in these lymphomas. Similarly, in the bone marrow smears of patients with chronic lymphocytic leukemia most cells possessed a nucleolar fluorescence for HMTNA and PCNA was present in nucleoli of a limited number of cells. In the bone marrow smears of patients with myeloid or myelomonocytic leukemias most blastic or monocytoid cells also developed a bright nucleolar fluorescence with HMTNA antibodies and PCNA was present only in a small percentage of these cells. Leukemic cells with PCNA in their nucleoli like thekhuntigen might represent a proliferating cell population in late G1-early S phase.     

Proliferating cell nuclear antigen (PCNA) regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries

Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer granulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries.     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

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.     

Influence of preoperative dexamethasone therapy on proliferating cell nuclear antigen (PCNA) expression in comparison to other parameters in meningiomas.

We conducted a trial in 42 benign and malignant meningiomas to assess a possible influence of preoperative dexamethasone therapy on mitotic index, labelling indices of proliferating cell nuclear antigen (PCNA), progesterone receptor, epidermal growth factor receptor (EGF-R), c-erbB-2 oncoprotein, cathepsin D, gamma-gamma enolase as well as the mean number of silver-stained nucleolar organizer region-associated proteins (AgNORs). Tumors with preceding dexamethasone therapy for more than 1 day display significantly less immunohistochemical staining for PCNA. A correlation between the labelling index of PCNA and the degree of malignancy could not be identified. There was no significant effect of preoperative dexamethasone therapy on the other parameters. Our data suggest that dexamethasone may selectively inhibit the expression of PCNA in the G1/S-phase of the cell cycle. Thus, we emphasize the necessity to heed factors, e.g. dexamethasone, which may affect the expression of proliferating markers.     

Immunochemical and biochemical analysis of the proliferating cell nuclear antigen (PCNA) in HeLa cells

PCNA, also known a cyclin, a protein of molecular weight (MW) 35 000, accumulates in the nuclei of dividing and transformed cells and reacts with autoantibodies from certain lupus patients. Using an indirect immunofluorescence technique, we show that lupus sera containing anti-PCNA antibodies reveal a heterogeneous nuclear fluorescence pattern upon reaction with asynchronous HeLa cells, whereas with synchronized cells a sequence of distinct patterns is disclosed on progression through the cell cycle. Cell-free translation of HeLa cell mRNA followed by immunoprecipitation with anti-PCNA sera shows a single protein with the same apparent MW as PCNA labelled in vivo, suggesting that PCNA is not derived from a larger precursor protein and not grossly modified by post-translational events. However, a group of at least nine nuclear polypeptides ranging in MW from about 12 000 to 110 000 are recognized by immunoblotting with anti-PCNA sera, indicating either that additional antigenic sites are produced on denaturation of native proteins or that additional autoantibodies are present in these sera. We also show that PCNA and several of these polypeptides are associated with nuclear structures containing chromatin.     

Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, was quantified in the cell lines SP2/0 and MOLT-4 and in mouse splenocytes induced to proliferate in vitro with mitogens. Autoantibody from a patient with systemic lupus erythematosus was used to label PCNA in cell suspensions after the cells had been fixed and permeabilized. In the same cells DNA was stained by propidium iodide. The cells were then analysed by flow cytometry for PCNA and DNA content. The PCNA profiles in proliferating spleen cells and the cell lines were similar. Most G0-G1 cells did not express significant amount of PCNA. A dramatic increase in PCNA immunofluorescence was observed in late G1 cells, and further increases were observed in S-phase cells. G2-M cells showed a reduced level of PCNA immunofluorescence relative to S-phase cells but were still elevated relative to G0-G1 cells. Proliferating cells arrested at the G1-S boundary by exposure to cytosine arabinoside showed an increased PCNA immunofluorescence as compared to unstimulated cells.     

Conversion from mitosis to meiosis: morphology and expression of proliferating cell nuclear antigen (PCNA) and Dmc1 during newt spermatogenesis

The conversion from mitosis to meiosis is a phenomenon specific to the cellular progenitors of gametes; however, the mechanism or mechanisms responsible for this conversion are poorly understood. To this end, some morphological and molecular changes that occur during the initiation of meiosis in newt spermatogenesis are reported in the present paper. In situ morphologic studies revealed that spermatogonial stages comprise two phases: early mitotic generations (G1-G4) and late mitotic generations (G5-G8). Morphologic conversion from secondary spermatogonia to primary spermatocytes occurred during the intermediate stage of premeiotic DNA replication. The expression of proliferating cell nuclear antigen (PCNA), a DNA polymerase-delta auxiliary protein, in spermatogonia was weak in G1, highest during DNA synthesis (S), decreased in G2 and was not detectable in dividing cells. Complementary DNA for newt homologs of DMC1 (disrupted meiotic cDNA), which is an Escherichia coli RecA-like protein specifically active during meiosis, were isolated. The newt Dmc1 mRNA was first expressed significantly during the preleptotene stage and this continued into the spermatid stage. These observations present a basis for investigating the mechanism(s) controlling the conversion of newt spermatogonial cells from mitosis to meiosis.     

Drosophila replication and repair proteins: proliferating cell nuclear antigen (PCNA).

Proliferating cell nuclear antigen (PCNA), a protein intimately involved in both replication and repair, has been identified in eukaryotes at all levels of evolution. Is primary sequence, Drosophila melanogaster PCNA is 73% identical to mammalian PCNA. Moreover, it is able to substitute for mammalian PCNA in at least one intricate cell-free replication assay. Mutations in the gene for Drosophila PCNA, including some that are temperature sensitive, have been reported. Procedures are described for the biochemical purification of wild-type PCNA from a population of 6- to 18-h-old Drosophila embryos. Procedures were also developed for purification of unmodified wild-type Drosophila PCNA after induction of expression in Escherichia coli. An NH(2)-terminally His-tagged but otherwise wild-type Drosophila PCNA, as well as mutant His-tagged PCNA, were also engineered and purified to apparent homogeneity. Finally, an in situ polyacrylamide gel technique allows DNA polymerase assays to be performed on portions of single adults as well as single Drosophila embryos. This assay should tremendously facilitate systematic genetic studies of metazoan replication and repair.     

Proliferating cell nuclear antigen and Ki-67 antigen expression in human haematopoietic cells during growth stimulation and differentiation

Abstract. By flow cytometric dual parameter analysis of proliferating cell nuclear antigen (PCNA) and the Ki-67 antigen a detailed cell cycle analysis can be performed. In this study the co-ordinated expression of these two growth-related antigens was investigated in human haematopoietic cells at entrance into the cell cycle as well as at exit from the cycle. In mitogen-stimulated peripheral blood lymphocytes entering the first cell cycle, the Ki-67 antigen was found to be expressed in S phase cells and not in G₁ cells. Thus, the Ki-67 antigen expression in PCNA-positive S phase cells differed between continuously cycling cells and cells entering the cell cycle. Based on this difference, it was possible to visualize and evaluate the recruitment of cells into the first cell cycle from a resting stage. This new cell cycle parameter can give additional information concerning tumour growth. The Ki-67 antigen was also studied during different stages of G₁ and was found to be expressed at high levels in early G₁ cells compared with other parts of G₁.     

Proliferating cell nuclear antigen: More than a clamp for DNA polymerases

DNA metabolic events such as replication, repair and recombination require the concerted action of several enzymes and cofactors. Nature has provided a set of proteins that support DNA polymerases in performing processive, accurate and rapid DNA synthesis. Two of them, the proliferating cell nuclear antigen and its adapter protein replication factor C, cooperate to form a moving platform that was initially thought of only as an anchor point for DNA polymerases δ and ε. It now appears that proliferating cell nuclear antigen is also a communication point between a variety of important cellular processes including cell cycle control, DNA replication, nucleotide excision repair, post-replication mismatch repair, base excision repair and at least one apoptotic pathway. The dynamic movement of proliferating cell nuclear antigen on and off the DNA renders this protein an ideal communicator for a variety of proteins that are essential for DNA metabolic events in eukaryotic cells.     

Reduced stability and increased dynamics in the human proliferating cell nuclear antigen (PCNA) relative to the yeast homolog

Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes. The amino acid sequence of human PCNA is 35% identical to the yeast homolog, and the two proteins have the same 3D crystal structure. In this report, we give evidence that the budding yeast (sc) and human (h) PCNAs have highly similar structures in solution but differ substantially in their stability and dynamics. hPCNA is less resistant to chemical and thermal denaturation and displays lower cooperativity of unfolding as compared to scPCNA. Solvent exchange rates measurements show that the slowest exchanging backbone amides are at the β-sheet, in the structure core, and not at the helices, which line the central channel. However, all the backbone amides of hPCNA exchange fast, becoming undetectable within hours, while the signals from the core amides of scPCNA persist for longer times. The high dynamics of the α-helices, which face the DNA in the PCNA-loaded form, is likely to have functional implications for the sliding of the PCNA ring on the DNA since a large hole with a flexible wall facilitates the establishment of protein-DNA interactions that are transient and easily broken. The increased dynamics of hPCNA relative to scPCNA may allow it to acquire multiple induced conformations upon binding to its substrates enlarging its binding diversity.