Involvement Of Proliferating Cell Nuclear Antigen Research Articles

Cytoplasmic PCNA is located in the actin belt and involved in osteoclast differentiation.

Osteoporosis (OP) is an age-related osteolytic disease and characterized by low bone mass and more prone to fracture due to active osteoclasts. Proliferating cell nuclear antigen (PCNA) has been long identified as a nuclear protein playing critical roles in the regulation of DNA replication and repair. Recently, a few studies have demonstrated the cytoplasmic localization of PCNA and its function associated with apoptosis in neutrophil and neuroblastoma cells. However, the involvement of PCNA, including the cytoplasmic PCNA, in the osteoclast differentiation remains unclear. In the present study, we show that PCNA is translocated from nucleus to cytoplasm during the RANKL-induced osteoclast differentiation, and localized in the actin belt of mature osteoclast. Knockdown of PCNA significantly affected the integrity of actin belt, the formation of multinucleated osteoclasts, the expression of osteoclast-specific genes, and the in vitro bone resorption. Interactomic study has revealed β-actin as the major interacting partner of the cytoplasmic PCNA, suggesting that cytoplasmic PCNA might play a critical role in the differentiation of osteoclast through regulation of actin-cytoskeleton remodeling. Taken together, our results demonstrate the critical role of cytoplasmic PCNA during the process of osteoclast differentiation, and provided a potential therapeutic target for treatment of osteoclast-related bone diseases.

Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation.

The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) mediates Okazaki fragment maturation through tight coordination of the activities of DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (Lig1). Little is known regarding the mechanism of partner switching on PCNA and the involvement of PCNA's three binding sites in coordinating such processes. To shed new light on PCNA-mediated Okazaki fragment maturation, we developed a novel approach for the generation of PCNA heterotrimers containing one or two mutant monomers that are unable to bind and stimulate partners. These heterotrimers maintain the native oligomeric structure of PCNA and exhibit high stability under various conditions. Unexpectedly, we found that PCNA heterotrimers containing only one functional binding site enable Okazaki fragment maturation by efficiently coordinating the activities of Pol δ, FEN1, and Lig1. The efficiency of switching between partners on PCNA was not significantly impaired by limiting the number of available binding sites on the PCNA ring. Our results provide the first direct evidence, to our knowledge, that simultaneous binding of multiple partners to PCNA is unnecessary, and if it occurs, does not provide significant functional advantages for PCNA-mediated Okazaki fragment maturation in vitro. In contrast to the "toolbelt" model, which was demonstrated for bacterial and archaeal sliding clamps, our results suggest a mechanism of sequential switching of partners on the eukaryotic PCNA trimer during DNA replication and repair.

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

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. 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. 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.

Effect of deprival of LH on Leydig cell proliferation: involvement of PCNA, cyclin D3 and IGF-1

The levels of proliferating cell nuclear antigen (PCNA) and cyclin D3 which are known markers of cellular proliferation were monitored by immunoblotting in progenitor Leydig cells (PLC), immature Leydig cells (ILC) and adult Leydig cells (ALC) isolated from 21, 35 and 90 day old rats, respectively which represent the Leydig cells at different stages of development. The levels of PCNA and cyclin D3 were highest in PLC, intermediate in ILC and lowest in ALC. Following administration of an antiserum to LH to deprive endogenous LH in 21 day old rats, a significant decrease in the levels of PCNA and Cyclin D3 were observed suggesting the involvement of Lutenizing hormone (LH) in PLC proliferation. In support of this observation, Bromodeoxyuridine (BrdU) incorporation was highest in PLC when compared with ILC and ALC, and administration of LH antiserum to 21 day old rats led to a total absence of BrdU incorporation by the isolated PLC. Also, there was a decrease in the level of IGF-1 and IGF-1 receptor mRNA levels by 55 and 35%, respectively as assessed by semi-quantitative RT-PCR. In addition, the PLC isolated from rats deprived of endogenous LH incorporated much less BrdU following addition of IGF-1. These results, which are obtained using an in vivo model system establish that LH has a very important role in Leydig cell proliferation in immature rats.

Detection of chromatin-bound PCNA in mammalian cells and its use to study DNA excision repair.

Compelling evidence indicates that proliferating cell nuclear antigen (PCNA) is an indispensable factor not only in DNA replication but in nucleotide excision repair (NER), alternative pathway of base excision repair (BER), and mismatch repair. The common function of PCNA in each of these is to assist in the initiation of DNA synthesis by providing a scaffolding clamp as a trimer catalyzed by RF-C at the 3'-OH terminus of a nascent DNA strand, to which DNA polymerase delta or epsilon can bind. Interestingly, DNA synthesis is reported to be ingeniously inhibited in replication, but not in NER owing to the interaction with CDKN1A (formerly known as p21/WAF1/CIP1). Furthermore, several proteins, XPG, FEN1, and DNA ligase I, recently were shown to competitively bind to the same region of PCNA, the interdomain connector loop, to which DNA polymerase delta or epsilon also binds. PCNA therefore seems to have a regulatory role in these DNA transactions. The in vitro reconstituted experimental system has been a powerful tool to obtain these lines of evidence, but another approach, immunofluorescence studies, also has been a contributor. In fact, the involvement of PCNA in DNA replication, NER, and BER has for the first time been indicated by a unique method that makes visible only in vivo chromatin-bound PCNA. The usefulness of this method and the importance of cooperative studies done with in vitro and in vivo experimental systems is discussed in terms of DNA excision repair.