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.     

Arabidopsis thaliana: Proliferating cell nuclear antigen 1 and 2 possibly form homo- and hetero-trimeric complexes in the plant cell

The proliferating cell nuclear antigen (PCNA) is a key component of the eukaryotic DNA replication machinery. It also plays an important role in DNA repair mechanisms. Despite the intense scientific research on yeast and human PCNA, information describing the function of this protein in plants is still very limited. In the previous study Arabidopsis PCNA2 but not PCNA1 was proposed to be functionally important in DNA polymerase η-dependent postreplication repair. In addition to the above study, PCNA2 but not PCNA1 was also shown to be necessary for Arabidopsis DNA polymerase λ-dependent oxidative DNA damage bypass. Taking into account the reported differences between PCNA1 and PCNA2, we tested the idea of a possible cooperation between PCNA1 and PCNA2 in the plant cell. In a bimolecular fluorescence complementation assay an interaction between PCNA1 and PCNA2 was observed in the nucleus, as well as in the cytoplasm. This finding, together with our previous results, indicates that PCNA1 and PCNA2 may cooperate in planta by forming homo- and heterotrimeric rings. The observed interaction might be relevant when distinct functions for PCNA1 and PCNA2 are considered.     

Platelet-activating factor (PAF) receptor binding antagonists from Alpinia officinarum

The bioassay-guided purification of ether extracts of Alpinia officinarum led to the isolation of two new compounds 6-hydroxy-1,7-diphenyl-4-en-3-heptanone (1) and 6-(2-hydroxy-phenyl)-4-methoxy-2-pyrone (4) as well as three known compounds 1,7-diphenyl-4-en-3-heptanone (2), 1,7-diphenyl-5-methoxy-3-heptanone (3), and apigenin (5). Their structures were established on the basis of spectral methods. All three diarylheptanoids 1, 2, and 3 exhibited potent PAF receptor binding inhibitory activities with an IC₅₀ of 1.3, 5.0, and 1.6 μM, respectively. These studies have identified diarylheptanoids as a novel class of potent PAF antagonists.     

A heterochromatin protein 1 (HP1) dimer and a proliferating cell nuclear antigen (PCNA) protein interact in vivo and are parts of a multiprotein complex involved in DNA replication and DNA repair

Heterochromatin protein 1 (HP1) is a small non-histone chromosomal protein known as a dominant suppressor of position-effect variegation and a major component of heterochromatin. Posttranslationally modified HP1, through interaction with protein partners from different groups, can be involved in a number of nuclear processes, including gene activation, chromatin remodeling, replication and DNA repair. Using bimolecular fluorescence complementation assay and live cell imaging, we demonstrate that HP1β and PCNA, a key player in DNA replication, are closely spaced components of a multiprotein complex involved in replication, both in S phase and during DNA repair, and that the functional complex requires formation of an HP1 dimer.     

Characterization of plant proliferating cell nuclear antigen (PCNA) and flap endonuclease-1 (FEN-1), and their distribution in mitotic and meiotic cell cycles

The biochemical and cell cycle-dependent properties of proliferating cell nuclear antigen (OsPCNA) and flap endonuclease-1 (OsFEN-1) were characterized from rice (Oryza sativa). OsPCNA was physically associated with OsFEN-1 and increased the flap-endonuclease activity of OsFEN-1 by 2.5-fold. Northern and Western blotting analysis revealed that OsPCNA and OsFEN-1 were present in meristematic tissues such as cultured cells, shoot apical meristem and root apical meristem. No expression was detected in the mature leaves, although they were exposed to UV. Both of these proteins were localized in the nuclei of the interphase cells including G1, S and G2, and in the nuclear region at telophase. The distribution patterns of plant PCNA and FEN-1 in meiotic cell progression were investigated using microsporocytes of lily (Lilium longiflorum cv. Hinomoto). During the leptotene to pachytene stages, PCNA and FEN-1 were localized in the nuclear region. The florescence gradually disappeared from diplotene to metaphase I. Interestingly, signals for PCNA formed 10-20 intense spots at leptotene. The number of spots decreased to 1-5 at zygotene and finally to 1 at pachytene. The roles of OsPCNA and OsFEN-1 in mitotic and meiotic cell cycles are discussed.     

Platelet-activating factor (PAF) receptor-binding antagonist activity of Malaysian medicinal plants

Forty-nine methanol extracts of 37 species of Malaysian medicinal plants were investigated for their inhibitory effects on platelet-activating factor (PAF) binding to rabbit platelets, using 3H-PAF as a ligand. Among them, the extracts of six Zingiberaceae species (Alpinia galanga Swartz., Boesenbergia pandurata Roxb., Curcuma ochorrhiza Val., C. aeruginosa Roxb., Zingiber officinale Rosc. and Z. zerumbet Koenig.), two Cinnamomum species (C. altissimum Kosterm. and C. pubescens Kochummen.), Goniothalamus malayanus Hook. f. Momordica charantia Linn. and Piper aduncum L. are potential sources of new PAF antagonists, as they showed significant inhibitory effects with IC50 values ranging from 1.2 to 18.4 microg ml(-1).     

Role of human DNA2 (hDNA2) as a potential target for cancer and other diseases: A systematic review

DNA nuclease/helicase 2 (DNA2), a multi-functional protein protecting the high fidelity of genomic transmission, plays critical roles in DNA replication and repair processes. In the maturation of Okazaki fragments, DNA2 acts synergistically with other enzymes to cleave the DNA-RNA primer flaps via different pathways. DNA2 is also involved in the stability of mitochondrial DNA and the maintenance of telomeres. Moreover, DNA2 potentially participates in controlling the cell cycle by repairing the DNA replication faults at main checkpoints. In addition, previous evidences demonstrated that DNA2 also functions in the repair process of DNA damages, such as base excision repair (BER). Currently, large studies revealed the structures and functions of DNA2 in prokaryotes and unicellular eukaryotes, such as bacteria and yeast. However, the studies that highlighted the functions of human DNA2 (hDNA2) and the relationships with other multifunctional proteins are still elusive, and more precise investigations are immensely needed. Therefore, this review mainly encompasses the key functions of DNA2 in human cells with various aspects, especially focusing on the genome integrity, and also generalizes the recent insights to the mechanisms related to the occurrence of cancer and other diseases potentially linked to the mutations in DNA2.     

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.     

Pathophysiological role of growth differentiation factor 15 (GDF15) in obesity,cancer, and cachexia

Growth differentiation factor 15 or macrophage inhibitory cytokine-1 (GDF15/MIC-1) is a divergent member of the transforming growth factor β superfamily and has a diverse pathophysiological roles in cancers, cardiometabolic disorders, and other diseases. GDF15 controls hematopoietic growth, energy homeostasis, adipose tissue metabolism, body growth, bone remodeling, and response to stress signals. The role of GDF15 in cancer development and progression is complicated and depends on the specific cancer type, stage, and tumor microenvironment. Recently, research on GDF15 and GDF15-associated signaling has accelerated due to the identification of the GDF15 receptor: glial cell line-derived neurotrophic factor (GDNF) family receptor α-like (GFRAL). Therapeutic interventions to target GDF15 and/or GFRAL revealed the mechanisms that drive its activity and might improve overall outcomes of patients with metabolic disorders and cancer. This review highlights the structure and functions of GDF15 and its receptor, emphasizing the pleiotropic role of GDF15 in obesity, tumorigenesis, metastasis, immunomodulation, and cachexia.     

The PCNA-associated factor KIAA0101/p15(PAF) binds the potential tumor suppressor product p33ING1b

The KIAA0101/p15(PAF)/OEATC-1 protein was initially isolated in a yeast two-hybrid screen for proliferating cell nuclear antigen (PCNA) binding partners, and was shown to bind PCNA competitively with the cell cycle regulator p21(WAF). PCNA is involved in DNA replication and damage repair. Using polyclonal antisera raised against a p15(PAF) fusion protein, we have shown that in a range of mammalian tumor and non-tumor cell lines the endogenous p15(PAF) protein localises to the nucleus and the mitochondria. Under normal conditions no co-localisation with PCNA could be detected, however following exposure to UV it was possible to co-immunoprecipitate p15(PAF) and PCNA from a number of cell lines, suggesting a UV-enhanced association of the two proteins. Overexpression of p15(PAF) in mammalian cells was also found to protect cells from UV-induced cell death. Based on similarities between the behaviour of p15(PAF) and the potential tumor suppressor product p33ING1b, we have further shown that these two proteins interact in the same complex in cell cultures. This suggests that p15(PAF) forms part of a larger protein complex potentially involved in the regulation of DNA repair, apoptosis and cell cycle progression.     

Poly(ADP-ribose) polymerase: A guardian of the genome that facilitates DNA repair by protecting against DNA recombination

We have studied the clonogenic survival response to X-rays and MNNG of V79 Chinese hamster cells and two derivative cell lines, ADPRT54 and ADPRT351, deficient in poly(ADP-ribose) polymerase (PARP) activity. Under conditions of exponential growth, both PARP-deficient cell lines are hypersensitive to X-rays and MNNG compared to their parental V79 cells. In contrast, under growth-arrested, confluent conditions, V79 and PARP-deficient cells become similarly sensitive to X-rays and MNNG suggesting that PARP may be involved in the repair of X-ray or MNNG-induced DNA damage in logarithmically growing cells but not in growth-arrested confluent cells. This suggestion, however, creates a dilemma as to how PARP can be involved in DNA repair in only selected growth phases while it is functionally active in all growth phases. To explain these paradoxical results and resolve this dilemma we propose a hypothesis based on the consistent observation that inhibition of PARP results in a significant increase in sister chromatid exchange (SCEs). Thus, we propose that PARP is a guardian of the genome that protects against DNA recombination. We have extended this theme to provide an explanation for our results and the studies done by many others.     

Targeting of O6-MeG DNA methyltransferase (MGMT) to mitochondria protects against alkylation induced cell death

Mitochondrial DNA (mtDNA) mutations are implicated in pathogenesis of human diseases including cancer. To prevent mutations cells have developed repair systems to counteract harmful genetic changes caused by DNA damaging agents. One such DNA repair protein is the O(6)-Methylguanine-DNA methyltransferase (MGMT) that prevents certain types of alkylation damage. Yet, the role of MGMT in preventing alkylation induced DNA damage in mtDNA is unclear. We explored the idea of increasing cell survival after alkylation damage by overexpressing MGMT in mitochondria. We show that overexpression of this repair protein in mitochondria increases cell survival after treatment with the DNA damaging agent MNNG.     

Presence of platelet-activating factor in squirrel monkey (Saimiri boliviensis) spermatozoa: seasonal differences

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) (PAF) is a potent signaling phospholipid which has pleiotropic biological properties in addition to platelet activation. PAF has been detected in the spermatozoa in a number of species. The concentration of PAF is inversely related to human spermatozoal quality. There are no reports on the presence of PAF in nonhuman primate spermatozoa. Therefore, the primary objective of this study was to determine if PAF is present in the spermatozoa from the squirrel monkey (which is a seasonal breeder). A second objective was to determine if PAF levels change from the breeding to the nonbreeding season. Endogenous lipids were extracted from mature Bolivian squirrel monkeys (Saimiri boliviensis) spermatozoa and assayed for the presence of PAF by [¹²⁵I] radioimmunoassay. PAF was detected in all samples assayed. PAF levels were significantly higher (P< 0.01) during the breeding season (mean: 3.58 ng/10⁶ spermatozoa) than the nonbreeding season (mean: 0.76 ng/10⁶ spermatozoa). The data demonstrate that PAF is present in squirrel monkey spermatozoa, with higher levels found during the breeding season. Additional studies are warranted to elucidate the role of PAF in spermatozoa function. Am. J. Primatol. 45:301–305, 1998. © 1998 Wiley-Liss, Inc.     

The MCM8/9 complex: A recent recruit to the roster of helicases involved in genome maintenance

There are several DNA helicases involved in seemingly overlapping aspects of homologous and homoeologous recombination. Mutations of many of these helicases are directly implicated in genetic diseases including cancer, rapid aging, and infertility. MCM8/9 are recent additions to the catalog of helicases involved in recombination, and so far, the evidence is sparse, making assignment of function difficult. Mutations in MCM8/9 correlate principally with primary ovarian failure/insufficiency (POF/POI) and infertility indicating a meiotic defect. However, they also act when replication forks collapse/break shuttling products into mitotic recombination and several mutations are found in various somatic cancers. This review puts MCM8/9 in context with other replication and recombination helicases to narrow down its genomic maintenance role. We discuss the known structure/function relationship, the mutational spectrum, and dissect the available cellular and organismal data to better define its role in recombination.     

Growth-phase-dependent response to DNA damage in poly(ADP-ribose) polymerase deficient cell lines: basis for a new hypothesis describing the role of poly(ADP-ribose) polymerase in DNA replication and repair

We have studied the role of poly(ADP-ribose) polymerase in the repair of DNA damage induced by x-ray and N-methyl N-nitro-N-nitrosoguanidine (MNNG) by using V79 chinese hamster cells, and two derivative mutant cell lines, ADPRT54 and ADPRT351, that are deficient in poly(ADP-ribose) polymerase activity. Under exponentially growing conditions these mutant cell lines are hypersensitive to x-irradiation and MNNG compared to their parental V79 cells which could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in the repair of DNA damage. However, the level of DNA strand breaks induced by x-irradiation and MNNG and their rates of repair are similar in all the cell lines, thus suggesting that it may not be the difference in strand break formation or in its rate of repair that is contributing to the enhanced cell killing in exponentially growing poly(ADP-ribose) polymerase deficient cell lines. In contrast, under growth-arrested conditions, all three cell lines become similarly sensitive to both x-irradiation and MNNG, thus suggesting that poly(ADP-ribose) polymerase may not be involved in the repair of DNA damage in growth-arrested cells. These paradoxical results could be interpreted to suggest that poly(ADP-ribose) polymerase is involved in DNA repair in a cell-cycle-dependent fashion, however, it is functionally active throughout the cell cycle. To resolve this dilemma and explain these results and those obtained by many others, we propose that the normal function of poly(ADP-ribose) polymerase is to prevent DNA recombination processes and facilitate DNA ligation.     

Human DNA polymerase lambda possesses terminal deoxyribonucleotidyl transferase activity and can elongate RNA primers: implications for novel functions

DNA polymerase lambda is a novel enzyme of the family X of DNA polymerases. The recent demonstration of an intrinsic 5'-deoxyribose-5'-phosphate lyase activity, a template/primer dependent polymerase activity, a distributive manner of DNA synthesis and sequence similarity to DNA polymerase beta suggested a novel beta-like enzyme. All these properties support a role of DNA polymerase lambda in base excision repair. On the other hand, the biochemical properties of the polymerisation activity of DNA polymerase lambda are still largely unknown. Here we give evidence that human DNA polymerase lambda has an intrinsic terminal deoxyribonucleotidyl transferase activity that preferentially adds pyrimidines onto 3'OH ends of DNA oligonucleotides. Furthermore, human DNA polymerase lambda efficiently elongates an RNA primer hybridized to a DNA template. These two novel properties of human DNA polymerase lambda might suggest additional roles for this enzyme in DNA replication and repair processes.     

Platelet activating factor (PAF) involvement in endotoxin-induced hypotension in rats. Studies with PAF-receptor antagonist kadsurenone

Evidence from three types of experiments indicates that platelet activating factor (PAF)1 is an important mediator of endotoxin-induced hypotension in rats. a) Endotoxin infusion stimulates the time-dependent appearance of PAF in the blood. b) PAF infusion results immediately (less than 30 sec) in hypotension while endotoxin-induced hypotension takes 3-5 min to occur, allowing time for PAF production. c) Infusion of the specific PAF-receptor antagonist kadsurenone (2.2 mumole/kg bolus, 0.9 mumoles/min/kg continuous infusion), which inhibits PAF-induced hypotension by 67%, causes a 67% reversal of endotoxin-elicited hypotension. An additional finding of this study is that rats respond hypotensively to each of a series of low-dose PAF infusions but only to the first low-dose endotoxin infusion. These endotoxin-refractory rats do respond to subsequent PAF infusions.     

Release, purification, and characterization of platelet-activating factor (PAF)

Platelet-activating factor (PAF) is a phospholipid mediator, released by basophils, macrophages and neutrophils under immunological and non immunological stimuli. It aggregates platelets and liberates their vasoactive contents. We studied the "spontaneous" release of PAF from hog blood leukocytes : optimal conditions were 22 degrees C, pH 9.5 in BSA and Ca2+-containing Tyrode's. This release was inhibited by the Ca2+-chelating agent, EDTA, and by the phospholipase A2 inhibitor, bromophenacyl bromide. Disruption of the cells did not yield PAF, indicating that it is not a "preformed" mediator. A preparative procedure for the extraction and purification of bulk quantities of PAF was developed. Purification was performed by silicic acid columns followed by high pressure liquid chromatography. The active fraction was eluted between sphingomyelin and lysophosphatidylcholine. The PAF purest fractions were still contaminated with these phospholipids as shown by thin layer chromatography and chemical ionization mass spectrometry. PAF activity was not affected by treatment with diazomethane, acetylation or hydrogenation. Our results combined with those obtained from our previous studies of the PAF structure using specific phospholipases indicate that PAF is a glycero-phospholipid devoid of ester function at position 1. This allowed us to establish precise criteria to distinguish PAF from other aggregating agents.     

RIF1: A novel regulatory factor for DNA replication and DNA damage response signaling

DNA double strand breaks (DSBs) are highly toxic to the cells and accumulation of DSBs results in several detrimental effects in various cellular processes which can lead to neurological, immunological and developmental disorders. Failure of the repair of DSBs spurs mutagenesis and is a driver of tumorigenesis, thus underscoring the importance of the accurate repair of DSBs. Two major canonical DSB repair pathways are the non-homologous end joining (NHEJ) and homologous recombination (HR) pathways. 53BP1 and BRCA1 are the key mediator proteins which coordinate with other components of the DNA repair machinery in the NHEJ and HR pathways respectively, and their exclusive recruitment to DNA breaks/ends potentially decides the choice of repair by either NHEJ or HR. Recently, Rap1 interacting factor 1 has been identified as an important component of the DNA repair pathway which acts downstream of the ATM/53BP1 to inhibit the 5′–3′ end resection of broken DNA ends, in-turn facilitating NHEJ repair and inhibiting homology directed repair. Rif1 is conserved from yeast to humans but its function has evolved from telomere length regulation in yeast to the maintenance of genome integrity in mammalian cells. Recently its role in the maintenance of genomic integrity has been expanded to include the regulation of chromatin structure, replication timing and intra-S phase checkpoint. We present a summary of these important findings highlighting the various aspects of Rif1 functions and discuss the key implications for genomic integrity.