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1.  Three Proliferating Cell Nuclear Antigen-Like Proteins Found in the Hyperthermophilic Archaeon Aeropyrum pernix: Interactions with the Two DNA Polymerases 
Journal of Bacteriology  2002;184(3):687-694.
Proliferating cell nuclear antigen (PCNA) is an essential component in the eukaryotic DNA replication machinery, in which it works for tethering DNA polymerases on the DNA template to accomplish processive DNA synthesis. The PCNA also interacts with many other proteins in important cellular processes, including cell cycle control, DNA repair, and an apoptotic pathway in the domain Eucarya. We identified three genes encoding PCNA-like sequences in the genome of Aeropyrum pernix, a crenarchaeal archaeon. We cloned and expressed these genes in Escherichia coli and analyzed the gene products. All three PCNA homologs stimulated the primer extension activities of the two DNA polymerases, polymerase I (Pol I) and Pol II, identified in A. pernix to various extents, among which A. pernix PCNA 3 (ApePCNA3) provided a most remarkable effect on both Pol I and Pol II. The three proteins were confirmed to exist in the A. pernix cells. These results suggest that the three PCNAs work as the processivity factor of DNA polymerases in A. pernix cells under different conditions. In Eucarya, three checkpoint proteins, Hus1, Rad1, and Rad9, have been proposed to form a PCNA-like ring structure and may work as a sliding clamp for the translesion DNA polymerases. Therefore, it is very interesting that three active PCNAs were found in one archaeal cell. Further analyses are necessary to determine whether each PCNA has specific roles, and moreover, how they reveal different functions in the cells.
PMCID: PMC139509  PMID: 11790738
2.  Identification and functional analysis of PCNA1 and PCNA-like1 genes of Phaseolus coccineus 
Journal of Experimental Botany  2009;61(3):873-888.
Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication and in many other processes in eukaryotic cells. Genetic analysis of Phaseolus coccineus showed the presence of at least two PCNA-like genes in the runner bean genome. Two PCNA genes have previously been found in a few plant species including Arabidopsis, tobacco, and maize. In these species, genes were nearly identical. Two cDNAs of P. coccineus PCNA (PcPCNA1 and PcPCNA-like1) have been identified that differ distinctly from each other. Interestingly, both the genetic organization of PcPCNA1 and PcPCNA-like1 genes and their expression patterns were similar, but these were the only similarities between these genes and their products. The identity between PcPCNA1 and PcPCNA-like1 at the amino acid level was only 54%, with PcPCNA-like1 lacking motifs that are crucial for the activity typical of PCNA. Consequently, these two proteins showed different properties. PcPCNA1 behaved like a typical PCNA protein: it formed a homotrimer and stimulated the activity of human DNA polymerase delta. In addition, PcPCNA1 interacted with a p21 peptide and was recognized by an anti-human PCNA monoclonal antibody PC10. By contrast, PcPCNA-like1 was detected as a monomer and was unable to stimulate the DNA polymerase delta activity. PcPCNA-like1 also could not interact with p21 and was not recognized by the PC10 antibody. Our results suggest that PcPCNA-like1 either is unable to function alone and therefore might be a component of the heterotrimeric PCNA ring or may have other, yet unknown functions. Alternatively, the PcPCNA-like1 gene may represent a pseudogene.
PMCID: PMC2814116  PMID: 20007687
DNA polymerase delta; PCNA; Phaseolus coccineus; PRINS; RACE
3.  Functional Interactions of a Homolog of Proliferating Cell Nuclear Antigen with DNA Polymerases in Archaea 
Journal of Bacteriology  1999;181(21):6591-6599.
Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. We cloned the gene encoding a PCNA homolog (PfuPCNA) from an euryarchaeote, Pyrococcus furiosus, expressed it in Escherichia coli, and characterized the biochemical properties of the gene product. The protein PfuPCNA stimulated the in vitro primer extension abilities of polymerase (Pol) I and Pol II, which are the two DNA polymerases identified in this organism to date. An immunological experiment showed that PfuPCNA interacts with both Pol I and Pol II. Pol I is a single polypeptide with a sequence similar to that of family B (α-like) DNA polymerases, while Pol II is a heterodimer. PfuPCNA interacted with DP2, the catalytic subunit of the heterodimeric complex. These results strongly support the idea that the PCNA homolog works as a sliding clamp of DNA polymerases in P. furiosus, and the basic mechanism for the processive DNA synthesis is conserved in the domains Bacteria, Eucarya, and Archaea. The stimulatory effect of PfuPCNA on the DNA synthesis was observed by using a circular DNA template without the clamp loader (replication factor C [RFC]) in both Pol I and Pol II reactions in contrast to the case of eukaryotic organisms, which are known to require the RFC to open the ring structure of PCNA prior to loading onto a circular DNA. Because RFC homologs have been found in the archaeal genomes, they may permit more efficient stimulation of DNA synthesis by archaeal DNA polymerases in the presence of PCNA. This is the first stage in elucidating the archaeal DNA replication mechanism.
PMCID: PMC94121  PMID: 10542158
4.  Nuclear Dynamics of PCNA in DNA Replication and Repair 
Molecular and Cellular Biology  2005;25(21):9350-9359.
The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a “sliding clamp” that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair.
PMCID: PMC1265825  PMID: 16227586
5.  Characterization of proliferating cell nuclear antigen (PCNA) from pathogenic yeast Candida albicans and its functional analyses in S. Cerevisiae 
BMC Microbiology  2015;15:257.
Proliferating cell nuclear antigen (PCNA/POL30) an essential protein forms a homotrimeric ring encircling dsDNA and serves as a molecular scaffold to recruit various factors during DNA replication, repair and recombination. According to Candida Genome Database (CGD), orf19.4616 sequence is predicted to encode C. albicans PCNA (CaPCNA) that has not been characterized yet.
Molecular modeling studies of orf19.4616 using S. cerevisiae PCNA sequence (ScPCNA) as a template, and its subsequent biochemical characterizations suggest that like other eukaryotic PCNAs, orf19.4616 encodes for a conventional homotrimeric sliding clamp. Further we showed by surface plasmon resonance that CaPCNA physically interacted with yeast DNA polymerase eta. Plasmid segregation in genomic knock out yeast strains showed that CaPCNA but not its G178S mutant complemented for cell survival. Unexpectedly, heterologous expression of CaPCNA in S. cerevisiae exhibited slow growth phenotypes, sensitivity to cold and elevated temperatures; and showed enhanced sensitivity to hydroxyurea and various DNA damaging agents in comparison to strain bearing ScPCNA. Interestingly, wild type strains of C. albicans showed remarkable tolerance to DNA damaging agents when compared with similarly treated yeast cells.
Despite structural and physiochemical similarities; we have demonstrated that there are distinct functional differences between ScPCNA and CaPCNA, and probably the ways both the strains maintain their genomic stability. We propose that the growth of pathogenic C. albicans which is evolved to tolerate DNA damages could be controlled effectively by targeting this unique fungal PCNA.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0582-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4634812  PMID: 26537947
Sliding clamp; Native PAGE; Complementation; Hydroxyurea; DNA damage tolerance; Surface plasmon resonance
6.  Reversal of PCNA Ubiquitylation by Ubp10 in Saccharomyces cerevisiae 
PLoS Genetics  2012;8(7):e1002826.
Regulation of PCNA ubiquitylation plays a key role in the tolerance to DNA damage in eukaryotes. Although the evolutionary conserved mechanism of PCNA ubiquitylation is well understood, the deubiquitylation of ubPCNA remains poorly characterized. Here, we show that the histone H2BK123 ubiquitin protease Ubp10 also deubiquitylates ubPCNA in Saccharomyces cerevisiae. Our results sustain that Ubp10-dependent deubiquitylation of the sliding clamp PCNA normally takes place during S phase, likely in response to the simple presence of ubPCNA. In agreement with this, we show that Ubp10 forms a complex with PCNA in vivo. Interestingly, we also show that deletion of UBP10 alters in different ways the interaction of PCNA with DNA polymerase ζ–associated protein Rev1 and with accessory subunit Rev7. While deletion of UBP10 enhances PCNA–Rev1 interaction, it decreases significantly Rev7 binding to the sliding clamp. Finally, we report that Ubp10 counteracts Rad18 E3-ubiquitin ligase activity on PCNA at lysine 164 in such a manner that deregulation of Ubp10 expression causes tolerance impairment and MMS hypersensitivity.
Author Summary
DNA damage is a major source of genome instability and cancer. A universal mechanism of DNA damage tolerance is based on translesion synthesis (TLS) by specialized low-fidelity DNA polymerases capable of replicating over DNA lesions during replication. Translesion synthesis requires the switch between replicative and TLS DNA polymerases, and this switching is controlled through the ubiquitylation of the proliferating-cell nuclear antigen (PCNA), a processivity factor for DNA synthesis. It is thought that DNA polymerase switching is a reversible process that has a favorable outcome for cells in the prevention of irreversible DNA replication forks collapse. However, the low-fidelity nature of TLS polymerases has unfavorable consequences like the increased risk of mutations opposite to DNA lesions. Here we identify Ubp10 as an enzyme controlling PCNA deubiquitylation in the model yeast S. cerevisiae. The identification of Ubp10 is a first step that will allow us to understand its biological significance and its potential role as part of a safeguard mechanism limiting the residence time of TLS DNA polymerases on replicating chromatin in eukaryotes.
PMCID: PMC3400564  PMID: 22829782
7.  The Elongator Complex Interacts with PCNA and Modulates Transcriptional Silencing and Sensitivity to DNA Damage Agents 
PLoS Genetics  2009;5(10):e1000684.
Histone chaperones CAF-1 and Asf1 function to deposit newly synthesized histones onto replicating DNA to promote nucleosome formation in a proliferating cell nuclear antigen (PCNA) dependent process. The DNA replication- or DNA repair-coupled nucleosome assembly pathways are important for maintenance of transcriptional gene silencing and genome stability. However, how these pathways are regulated is not well understood. Here we report an interaction between the Elongator histone acetyltransferase and the proliferating cell nuclear antigen. Cells lacking Elp3 (K-acetyltransferase Kat9), the catalytic subunit of the six-subunit Elongator complex, partially lose silencing of reporter genes at the chromosome VIIL telomere and at the HMR locus, and are sensitive to the DNA replication inhibitor hydroxyurea (HU) and the damaging agent methyl methanesulfonate (MMS). Like deletion of the ELP3, mutation of each of the four other subunits of the Elongator complex as well as mutations in Elp3 that compromise the formation of the Elongator complex also result in loss of silencing and increased HU sensitivity. Moreover, Elp3 is required for S-phase progression in the presence of HU. Epistasis analysis indicates that the elp3Δ mutant, which itself is sensitive to MMS, exacerbates the MMS sensitivity of cells lacking histone chaperones Asf1, CAF-1 and the H3 lysine 56 acetyltransferase Rtt109. The elp3Δ mutant has allele specific genetic interactions with mutations in POL30 that encodes PCNA and PCNA binds to the Elongator complex both in vivo and in vitro. Together, these results uncover a novel role for the intact Elongator complex in transcriptional silencing and maintenance of genome stability, and it does so in a pathway linked to the DNA replication and DNA repair protein PCNA.
Author Summary
During S phase of the cell cycle, not only must DNA sequences be faithfully duplicated, chromatin structures must also be inherited into daughter cells to maintain gene expression states and cell identity. While significant progress has been made in understanding the regulation of DNA replication, how chromatin structures are maintained from one cell division cycle to the next (so-called epigenetic inheritance) is only partially understood. It is believed that the DNA replication-coupled nucleosome assembly process plays an important role in such inheritance as well as maintenance of genome stability. In this process, histone chaperones such as chromatin assembly factor 1 (CAF-1) deposit newly synthesized histones H3–H4, which are acetylated at specific lysine residues, onto replicating DNA in a PCNA dependent reaction. PCNA is a clamp for DNA polymerases and other proteins that are involved in DNA replication and DNA repair. Genetic interactions between lysine acetyltransferase Elp3 and factors involved in DNA replication-coupled nucleosome assembly are described. Elp3 is required for transcriptional silencing and for maintenance of genome stability and binds directly to PCNA. A role for the Elongator complex in response to DNA damage and in maintenance of gene silencing is discussed.
PMCID: PMC2757915  PMID: 19834596
8.  Arabidopsis PCNAs form complexes with selected D-type cyclins 
Proliferating Cell Nuclear Antigen (PCNA) is a key nuclear protein of eukaryotic cells. It has been shown to form complexes with cyclin dependent kinases, cyclin dependent kinase inhibitors and the D-type cyclins which are involved in the cell cycle control. In Arabidopsis two genes coding for PCNA1 and PCNA2 proteins have been identified. In this study by analyzing Arabidopsis PCNA/CycD complexes we tested the possible functional differentiation of PCNA1/2 proteins in cell cycle control. Most out of the 10 cyclins investigated showed only nuclear localization except CycD2;1, CycD4;1, and CycD4;2 which were observed both in the nucleus and cytoplasm. Using the Y2H, BiFC and FLIM-FRET techniques we identified D-type cyclins which formed complexes with either PCNA1 or PCNA2. Among the candidates tested only CycD1;1, CycD3;1, and CycD3;3 were not detected in a complex with the PCNA proteins. Moreover, our results indicate that the formation of CycD3;2/PCNA and CycD4;1/PCNA complexes can be regulated by other as yet unidentified factor(s). Additionally, FLIM-FRET analyses suggested that in planta the distance between PCNA1/CycD4;1, PCNA1/CycD6;1, PCNA1/CycD7;1, and PCNA2/CycD4;2 proteins was shorter than that between PCNA2/CycD4;1, PCNA2/CycD6;1, PCNA2/CycD7;1, and PCNA1/CycD4;2 pairs. These data indicate that the nine amino acid differences between PCNA1 and PCNA2 have an impact on the architecture of Arabidopsis CycD/PCNA complexes.
PMCID: PMC4550699  PMID: 26379676
Arabidopsis; PCNA; D-type cyclins; DNA replication; DNA repair; cell cycle
9.  An in vivo analysis of the localisation and interactions of human p66 DNA polymerase δ subunit 
DNA polymerase δ is essential for eukaryotic DNA replication and also plays a role in DNA repair. The processivity of this polymerase complex is dependent upon its interaction with the sliding clamp PCNA and the polymerase-PCNA interaction is largely mediated through the p66 polymerase subunit. We have analysed the interactions of the human p66 DNA polymerase δ subunit with PCNA and with components of the DNA polymerase δ complex in vivo.
Using the two-hybrid system, we have mapped the interaction domains for binding to the p50 polymerase δ subunit and with PCNA to the N-terminus and the C-terminus of p66, respectively. Co-immunoprecipitation experiments confirm that these interaction domains are functional in vivo. Expression of EGFP-p66 shows that it is a nuclear protein which co-localises with PCNA throughout the cell cycle. p66 is localised to sites of DNA replication during S phase and to repair foci following DNA damage. We have identified a functional nuclear localisation sequence and shown that localisation to replication foci is not dependent upon active nuclear import. Sub-domains of p66 act as dominant negative suppressors of colony formation, suggesting that p66 forms an essential structural link between the p50 subunit and PCNA. Analysis of the C-terminal PCNA binding motif shows that deletion of the QVSITGFF core motif results in a reduced affinity for PCNA, while deletion of a further 20 amino acids completely abolishes the interaction. A reduced affinity for PCNA correlates with reduced targeting to replication foci. We have confirmed the p66-PCNA interaction in vivo using fluorescence resonance energy transfer (FRET) techniques.
We have defined the regions of p66 required for its interaction with PCNA and the p50 polymerase subunit. We demonstrate a functional link between PCNA interaction and localisation to replication foci and show that there is a direct interaction between p66 and PCNA in living cells during DNA replication. The dominant negative effect upon growth resulting from expression of p66 sub-domains confirms that the p66-PCNA interaction is essential in vivo.
PMCID: PMC1187890  PMID: 16000169
10.  Analysis of the role of PCNA-DNA contacts during clamp loading 
Sliding clamps, such as Proliferating Cell Nuclear Antigen (PCNA) in eukaryotes, are ring-shaped protein complexes that encircle DNA and enable highly processive DNA replication by serving as docking sites for DNA polymerases. In an ATP-dependent reaction, clamp loader complexes, such as the Replication Factor-C (RFC) complex in eukaryotes, open the clamp and load it around primer-template DNA.
We built a model of RFC bound to PCNA and DNA based on existing crystal structures of clamp loaders. This model suggests that DNA would enter the clamp at an angle during clamp loading, thereby interacting with positively charged residues in the center of PCNA. We show that simultaneous mutation of Lys 20, Lys 77, Arg 80, and Arg 149, which interact with DNA in the RFC-PCNA-DNA model, compromises the ability of yeast PCNA to stimulate the DNA-dependent ATPase activity of RFC when the DNA is long enough to extend through the clamp. Fluorescence anisotropy binding experiments show that the inability of the mutant clamp proteins to stimulate RFC ATPase activity is likely caused by reduction in the affinity of the RFC-PCNA complex for DNA. We obtained several crystal forms of yeast PCNA-DNA complexes, measuring X-ray diffraction data to 3.0 Å resolution for one such complex. The resulting electron density maps show that DNA is bound in a tilted orientation relative to PCNA, but makes different contacts than those implicated in clamp loading. Because of apparent partial disorder in the DNA, we restricted refinement of the DNA to a rigid body model. This result contrasts with previous analysis of a bacterial clamp bound to DNA, where the DNA was well resolved.
Mutational analysis of PCNA suggests that positively charged residues in the center of the clamp create a binding surface that makes contact with DNA. Disruption of this positive surface, which had not previously been implicated in clamp loading function, reduces RFC ATPase activity in the presence of DNA, most likely by reducing the affinity of RFC and PCNA for DNA. The interaction of DNA is not, however, restricted to one orientation, as indicated by analysis of the PCNA-DNA co-crystals.
PMCID: PMC2824762  PMID: 20113510
11.  PCNA Ubiquitination Is Important, But Not Essential for Translesion DNA Synthesis in Mammalian Cells 
PLoS Genetics  2011;7(9):e1002262.
Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in PcnaK164R/K164R cells, which are resistant to PCNA ubiquitination, compared to Pcna+/+ cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in PcnaK164R/K164R mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.
Author Summary
DNA damage can block replication and lead to mutations, genomic instability, and cancer. In cases when the removal of DNA damage and restoration of the original sequence prior to replication is impossible, cells utilize DNA damage tolerance mechanisms, which help replication to bypass the lesions. A major universal tolerance mechanism is translesion DNA synthesis (TLS), in which specialized low-fidelity DNA polymerases elongate the DNA across the lesion. This is a double-edged sword because the price of completing replication is an increased risk of point mutations opposite the lesion. Thus, TLS regulation is critical for preventing an escalation in mutation rates. A key element in TLS regulation is the attachment of a small protein called ubiquitin to the PCNA protein, a sliding DNA clamp that tethers the DNA polymerases to DNA, which functions to recruit the TLS DNA polymerase to the damaged site in DNA. While in yeast this modification of PCNA is crucial for TLS, there is a debate about its importance in mammals. Here we show that in mammalian cells the modification of PCNA by ubiquitin is important, but there exist secondary yet significant TLS mechanisms that operate in its absence and have an altered mutational outcome.
PMCID: PMC3169526  PMID: 21931560
12.  Molecular modeling and expression of the Litopenaeus vannamei proliferating cell nuclear antigen (PCNA) after white spot syndrome virus shrimp infection 
Results in Immunology  2011;1(1):24-30.
Proliferating cell nuclear antigen (PCNA) is the eukaryotic sliding clamp that tethers DNA polymerase to DNA during replication. The full-length cDNA of the Pacific white shrimp Litopenaeus vannamei PCNA (LvPCNA) was cloned and encoded a protein of 260 amino acids that is highly similar to other Crustacean PCNAs. The theoretical shrimp PCNA structure has all the domains that are necessary for its interaction with template DNA and DNA polymerase. RT-PCR analysis showed that LvPCNA is expressed mainly in muscle and hemocytes and much less in hepatopancreas and gills. LvPCNA mRNA levels are not statistically different in muscle from healthy and challenged shrimp with the white spot syndrome virus (WSSV). In contrast, the mRNA levels of the viral DNA polymerase show a biphasic pattern with expression at 6 h post-infection and later at 24 and 48 h. These results suggest that in shrimp muscle LvPCNA levels are steadily kept to allow viral replication and that WSSV DNA polymerase (WSSV-DNApol) is more responsive towards later stages of infection. More knowledge of the DNA replication machinery would result in a better understanding of the mechanism and components of viral replication, since the WSSV genome does not have all the components required for assembly of a fully functional replisome.
► White Pacific shrimp Litopenaeus vannamei PCNA is very conserved. ► The molecular model is predicted to bind and clamp DNA. ► PCNA mRNA levels do not statistically change upon WSSV infection. ► PCNA mRNA is expressed at much higher levels compared to WSSV DNA polymerase.
PMCID: PMC3787728  PMID: 24371549
Shrimp; PCNA; Proliferating cell nuclear antigen; DNA polymerase; White spot syndrome virus; WSSV
13.  Crystal structure of SUMO-modified proliferating cell nuclear antigen 
Journal of molecular biology  2010;406(1):9-17.
Eukaryotic proliferating cell nuclear antigen (PCNA) is a replication accessory protein that functions in DNA replication, repair, and recombination. The various functions of PCNA are regulated by post-translational modifications including mono-ubiquitylation, which promotes translesion synthesis, and sumoylation, which inhibits recombination. To understand how the SUMO modification regulates PCNA, we generated a split SUMO-modified PCNA protein and showed that it supports cell viability and stimulates DNA polymerase δ activity. We then determined its X-ray crystal structure and found that SUMO occupies a position on the back face of the PCNA ring, which is distinct from the position occupied by ubiquitin in the structure of ubiquitin-modified PCNA. We propose that the back of PCNA has evolved to be a site of regulation that can be easily modified without disrupting ongoing reactions on the front of PCNA, such as normal DNA replication. Moreover, these modifications likely allow PCNA to function as a tool belt, whereby proteins can be recruited to the replication machinery via the back of PCNA and be held in reserve until needed.
PMCID: PMC3193175  PMID: 21167178
DNA replication; DNA recombination; DNA repair; protein-DNA interactions; translesion synthesis
14.  Oxidative damage-induced PCNA complex formation is efficient in xeroderma pigmentosum group A but reduced in Cockayne syndrome group B cells. 
Nucleic Acids Research  1999;27(22):4476-4482.
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.
PMCID: PMC148732  PMID: 10536158
15.  Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon. 
Molecular and Cellular Biology  1997;17(11):6367-6378.
The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.
PMCID: PMC232488  PMID: 9343398
16.  The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation 
The high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (HvPCNA) to a resolution of 2.0 Å.
The overall architecture of HvPCNA is very similar to other known PCNAs, which are highly structurally conserved. Three commonly observed adaptations in halophilic proteins are higher surface acidity, bound ions and increased numbers of intermolecular ion pairs (in oligomeric proteins). HvPCNA possesses the former two adaptations but not the latter, despite functioning as a homotrimer. Strikingly, the positive surface charge considered key to PCNA's role as a sliding clamp is dramatically reduced in the halophilic protein. Instead, bound cations within the solvation shell of HvPCNA may permit sliding along negatively charged DNA by reducing electrostatic repulsion effects.
The extent to which individual proteins adapt to halophilic conditions varies, presumably due to their diverse characteristics and roles within the cell. The number of ion pairs observed in the HvPCNA monomer-monomer interface was unexpectedly low. This may reflect the fact that the trimer is intrinsically stable over a wide range of salt concentrations and therefore additional modifications for trimer maintenance in high salt conditions are not required. Halophilic proteins frequently bind anions and cations and in HvPCNA cation binding may compensate for the remarkable reduction in positive charge in the pore region, to facilitate functional interactions with DNA. In this way, HvPCNA may harness its environment as opposed to simply surviving in extreme halophilic conditions.
PMCID: PMC2737543  PMID: 19698123
17.  Arabidopsis thaliana 
Plant Signaling & Behavior  2013;8(7):e24837.
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.
PMCID: PMC3909058  PMID: 23656863
Arabidopsis thaliana; PCNA; DNA replication; DNA repair; cell cycle
18.  Thermococcus kodakarensis has two functional PCNA homologs but only one is required for viability 
Proliferating cell nuclear antigen (PCNA) monomers assemble to form a ring-shaped clamp complex that encircles duplex DNA. PCNA binding to other proteins tethers them to the DNA providing contacts and interactions for many other enzymes essential for DNA metabolic processes. Most eukarya and euryarchaea have only one PCNA homolog but Thermococcus kodakarensis uniquely has two, designated PCNA1 and PCNA2, encoded by TK0535 and TK0582, respectively. Here, we establish that both PCNA1 and PCNA2 form homotrimers that stimulate DNA synthesis by archaeal DNA polymerases B and D and ATP hydrolysis by the replication factor C complex. In exponentially growing cells, PCNA1 is abundant and present at an ~100-fold higher concentration than PCNA2 monomers. Deletion of TK0582 (PCNA2) had no detectable effects on viability or growth whereas repeated attempts to construct a T. kodakarensis strain with TK0535 (PCNA1) deleted were unsuccessful. The implications of these observations for PCNA1 function and the origin of the two PCNA-encoding genes in T. kodakarensis are discussed.
PMCID: PMC3743106  PMID: 23525944
Archaea; DNA replication; Genetics; PCNA; Structure; Thermococcus kodakarensis
19.  CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis 
Nucleic Acids Research  2014;42(13):8433-8448.
The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.
PMCID: PMC4117764  PMID: 24939902
20.  Vaccinia Virus G8R Protein: A Structural Ortholog of Proliferating Cell Nuclear Antigen (PCNA) 
PLoS ONE  2009;4(5):e5479.
Eukaryotic DNA replication involves the synthesis of both a DNA leading and lagging strand, the latter requiring several additional proteins including flap endonuclease (FEN-1) and proliferating cell nuclear antigen (PCNA) in order to remove RNA primers used in the synthesis of Okazaki fragments. Poxviruses are complex viruses (dsDNA genomes) that infect eukaryotes, but surprisingly little is known about the process of DNA replication. Given our previous results that the vaccinia virus (VACV) G5R protein may be structurally similar to a FEN-1-like protein and a recent finding that poxviruses encode a primase function, we undertook a series of in silico analyses to identify whether VACV also encodes a PCNA-like protein.
An InterProScan of all VACV proteins using the JIPS software package was used to identify any PCNA-like proteins. The VACV G8R protein was identified as the only vaccinia protein that contained a PCNA-like sliding clamp motif. The VACV G8R protein plays a role in poxvirus late transcription and is known to interact with several other poxvirus proteins including itself. The secondary and tertiary structure of the VACV G8R protein was predicted and compared to the secondary and tertiary structure of both human and yeast PCNA proteins, and a high degree of similarity between all three proteins was noted.
The structure of the VACV G8R protein is predicted to closely resemble the eukaryotic PCNA protein; it possesses several other features including a conserved ubiquitylation and SUMOylation site that suggest that, like its counterpart in T4 bacteriophage (gp45), it may function as a sliding clamp ushering transcription factors to RNA polymerase during late transcription.
PMCID: PMC2674943  PMID: 19421403
21.  Chromatin-bound PCNA complex formation triggered by DNA damage occurs independent of the ATM gene product in human cells 
Nucleic Acids Research  2001;29(6):1341-1351.
Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases δ and ɛ, is involved in DNA replication as well as in diverse DNA repair pathways. In quiescent cells, UV light-induced bulky DNA damage triggers the transition of PCNA from a soluble to an insoluble chromatin-bound form, which is intimately associated with the repair synthesis by polymerases δ and ɛ. In this study, we investigated the efficiency of PCNA complex formation in response to ionizing radiation-induced DNA strand breaks in normal and radiation-sensitive Ataxia telangiectasia (AT) cells by immunofluorescence and western blot techniques. Exposure of normal cells to γ-rays rapidly triggered the formation of PCNA foci in a dose-dependent manner in the nuclei and the PCNA foci (40–45%) co-localized with sites of repair synthesis detected by bromodeoxyuridine labeling. The chromatin-bound PCNA gradually declined with increasing post-irradiation times and almost reached the level of unirradiated cells by 6 h. The PCNA foci formed after γ-irradiation was resistant to high salt extraction and the chromatin association of PCNA was lost after DNase I digestion. Interestingly, two radiosensitive primary fibroblast cell lines, derived from AT patients harboring homozygous mutations in the ATM gene, displayed an efficient PCNA redistribution after γ-irradiation. We also analyzed the PCNA complex induced by a radiomimetic agent, Bleomycin (BLM), which produces predominantly single- and double-strand DNA breaks. The efficiency and the time course of PCNA complex induced by BLM were identical in both normal and AT cells. Our study demonstrates for the first time that the ATM gene product is not required for PCNA complex assembly in response to DNA strand breaks. Additionally, we observed an increased interaction of PCNA with the Ku70 and Ku80 heterodimer after DNA damage, suggestive of a role for PCNA in the non-homologous end-joining repair pathway of DNA strand breaks.
PMCID: PMC29758  PMID: 11239001
22.  Readers of PCNA modifications 
Chromosoma  2013;122(4):259-274.
The eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), acts as a central coordinator of DNA transactions by providing a multivalent interaction surface for factors involved in DNA replication, repair, chromatin dynamics and cell cycle regulation. Posttranslational modifications (PTMs), such as mono- and polyubiquitylation, sumoylation, phosphorylation and acetylation, further expand the repertoire of PCNA’s binding partners. These modifications affect PCNA’s activity in the bypass of lesions during DNA replication, the regulation of alternative damage processing pathways such as homologous recombination and DNA interstrand cross-link repair, or impact on the stability of PCNA itself. In this review, we summarise our current knowledge about how the PTMs are “read” by downstream effector proteins that mediate the appropriate action. Given the variety of interaction partners responding to PCNA’s modified forms, the ensemble of PCNA modifications serves as an instructive model for the study of biological signalling through PTMs in general.
Electronic supplementary material
The online version of this article (doi:10.1007/s00412-013-0410-4) contains supplementary material, which is available to authorized users.
PMCID: PMC3714560  PMID: 23580141
PCNA; Ubiquitin; SUMO; Phosphorylation; Acetylation; Posttranslational modifications; DNA replication; Genome stability
23.  HAT3-mediated acetylation of PCNA precedes PCNA monoubiquitination following exposure to UV radiation in Leishmania donovani 
Nucleic Acids Research  2015;43(11):5423-5441.
Histone modifications impact various processes. In examining histone acetyltranferase HAT3 of Leishmania donovani, we find elimination of HAT3 causes decreased cell viability due to defects in histone deposition, and aberrant cell cycle progression pattern. HAT3 associates with proliferating cell nuclear antigen (PCNA), helping load PCNA onto chromatin in proliferating cells. HAT3-nulls show heightened sensitivity to UV radiation. Following UV exposure, PCNA cycles off/on chromatin only in cells expressing HAT3. Inhibition of the ubiquitin-proteasome pathway prior to UV exposure allows accumulation of chromatin-bound PCNA, and reveals that HAT3-nulls are deficient in PCNA monoubiquitination as well as polyubiquitination. While poor monoubiquitination of PCNA may adversely affect translesion DNA synthesis-based repair processes, polyubiquitination deficiencies may result in continued retention of chromatin-bound PCNA, leading to genomic instability. On suppressing the proteasome pathway we also find that HAT3 mediates PCNA acetylation in response to UV. HAT3-mediated PCNA acetylation may serve as a flag for PCNA ubiquitination, thus aiding DNA repair. While PCNA acetylation has previously been linked to its degradation following UV exposure, this is the first report linking a HAT-mediated PCNA acetylation to PCNA monoubiquitination. These findings add a new dimension to our knowledge of the mechanisms regulating PCNA ubiquitination post-UV exposure in eukaryotes.
PMCID: PMC4477661  PMID: 25948582
24.  Target validation and structure–activity analysis of a series of novel PCNA inhibitors 
Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair. Tumor cells express high levels of PCNA, identifying it as a potentially ideal target for cancer therapy. Previously, we identified nine compounds termed PCNA inhibitors (PCNA-Is) that bind directly to PCNA, stabilize PCNA trimer structure, reduce chromatin-associated PCNA, and selectively inhibit tumor cell growth. Of these compounds, PCNA-I1 was most potent. The purpose of this study is to further establish targeting of PCNA by PCNA-I1 and to identify PCNA-I1 analogs with superior potencies. We found that PCNA-I1 does not affect the level of chromatin-associated PCNA harboring point mutations at the predicted binding site of PCNA-I1. Forty-six PCNA-I1 analogs with structures of 1-hydrazonomethyl-2-hydroxy (scaffold A), 2-hydrazonomethyl-1-hydroxy (scaffold B), 2-hydrazonomethyl-3-hydroxy (scaffold C), and 4-pyridyl hydrazine (scaffold D) were analyzed for their effects on cell growth in four tumor cell lines and PCNA trimer stabilization. Compounds in scaffold group A and group B showed the highest trimer stabilization and the most potent cell growth inhibitory activities with a significant potency advantage observed in the Z isomers of scaffold A. The absence of trimer stabilization and growth inhibitory effects in compounds of scaffold group D confirms the essentiality of the hydroxynaphthyl substructure. Compounds structure–activity relationship (SAR)-6 and SAR-24 were analyzed for their effects on and found to reduce chromatin-associated PCNA in tumor cells. This study led to the identification of SAR-24, a compound with superior potencies and potentially improved solubility, which will be used for future development of PCNA-targeting cancer therapies.
PMCID: PMC4324689  PMID: 25729582
Chromatin association; inhibition of tumor cell growth; PCNA; small molecule inhibitors; target validation
25.  Structural insights into the adaptation of proliferating cell nuclear antigen (PCNA) from Haloferax volcanii to a high-salt environment 
The crystal structure of PCNA from the halophilic archaeon H. volcanii reveals specific features of the charge distribution on the protein surface that reflect adaptation to a high-salt environment and suggests a different type of interaction with DNA in halophilic PCNAs.
The sliding clamp proliferating cell nuclear antigen (PCNA) plays vital roles in many aspects of DNA replication and repair in eukaryotic cells and in archaea. Realising the full potential of archaea as a model for PCNA function requires a combination of biochemical and genetic approaches. In order to provide a platform for subsequent reverse genetic analysis, PCNA from the halophilic archaeon Haloferax volcanii was subjected to crystallographic analysis. The gene was cloned and expressed in Escherichia coli and the protein was purified by affinity chromatography and crystallized by the vapour-diffusion technique. The structure was determined by molecular replacement and refined at 3.5 Å resolution to a final R factor of 23.7% (R free = 25%). PCNA from H. volcanii was found to be homotrimeric and to resemble other homotrimeric PCNA clamps but with several differences that appear to be associated with adaptation of the protein to the high intracellular salt concentrations found in H. volcanii cells.
PMCID: PMC2756170  PMID: 19770505
PCNA–DNA interactions; sliding clamps; halophilic environment

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