Wojciech Strzalka

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.

Crystal structures of the Arabidopsis thaliana proliferating cell nuclear antigen 1 and 2 proteins complexed with the human p21 C‐terminal segment

The proliferating cell nuclear antigen (PCNA) is well recognized as one of the essential cellular components of the DNA replication machinery in all eukaryotic organisms. Despite their prominent importance, very little biochemical and structural information about plant PCNAs is available, in comparison with that obtained from other eukaryotic organisms. We have determined the atomic resolution crystal structures of the two distinct Arabidopsis thaliana PCNAs (AtPCNA), both complexed with the C‐terminal segment of human p21. Both AtPCNAs form homotrimeric ring structures, which are essentially identical to each other, including the major contacts with the p21 peptide. The structure of the amino‐terminal half of the p21 peptide, containing the typical PIP box sequence, is remarkably similar to those observed in the previously reported crystal structures of the human and archaeal PCNA‐PIP box complexes. Meanwhile, the carboxy‐terminal halves of the p21 peptide in the plant PCNA complexes are bound to the protein in a unique manner, most probably because of crystal packing effects. A surface plasmon resonance analysis revealed high affinity between each AtPCNA and the C‐terminal fragment of human p21. This result strongly suggests that the interaction is functionally significant, although no plant homologs of p21 have been identified yet. We also discovered that AtPCNA1 and AtPCNA2 form heterotrimers, implying that hetero‐PCNA rings may play critical roles in cellular signal transduction, particularly in DNA repair.

Identification and functional analysis of PCNA1 and PCNA-like1 genes of Phaseolus coccineus

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.

Photoactive Protochlorophyllide-Enzyme Complexes Reconstituted with PORA, PORB and PORC Proteins of A. thaliana : Fluorescence and Catalytic Properties

Photoactive Pchlide-POR-NADPH complexes were reconstituted using protochlorophyllide (Pchlide) and recombinant light-dependent protochlorophyllide oxidoreductase (POR) proteins, His₆-PORA, His₆-PORB and His₆-PORC, from Arabidopsis thaliana. We did not observe any differences in the kinetics of the protochlorophyllide photoreduction at room temperature among the PORA, PORB and PORC proteins. In contrast, the PORC protein showed lower yield of Chlide formation than PORA and PORB when preincubated in the dark for 30 min and then illuminated for a short time. The most significant observation was that reconstituted Pchlide-POR-NADPH complexes showed fluorescence maxima at 77 K similar to those observed for highly aggregated Pchlide-POR-NADPH complexes in prolamellar bodies (PLBs) in vivo. Homology models of PORA, PORB and PORC of Arabidopsis thaliana were developed to compare predicted structures of POR isoforms. There were only slight structural differences, mainly in the organisation of helices and loops, but not in the shape of whole molecules. This is the first comparative analysis of all POR isoforms functioning at different stages of A. thaliana development.

RAD5a ubiquitin ligase is involved in ubiquitination of Arabidopsis thaliana proliferating cell nuclear antigen

The proliferating cell nuclear antigen (PCNA) is post-translationally modified by ubiquitin in yeast and mammalian cells. It is widely accepted that in yeast mono- and polyubiquitinated PCNA is involved in distinct pathways of DNA postreplication repair. This study showed an interaction between plant ubiquitin and PCNA in the plant cell. Using different approaches, it was demonstrated that Arabidopsis RAD5a ubiquitin ligase is involved in the post-translational modification of plant PCNA. A detailed analysis of the properties of selected Arabidopsis ubiquitin-conjugating enzymes (AtUBC) has shown that a plant homologue of yeast RAD6 (AtUBC2) is sufficient to monoubiquitinate AtPCNA in the absence of ubiquitin ligase. Using different combinations of selected AtUBC proteins together with AtRAD5a, it was demonstrated that plants have potential to use different pathways to ubiquitinate PCNA. The analysis of Arabidopsis PCNA1 and PCNA2 did not demonstrate substantial differences in the ubiquitination pattern between these two proteins. The major ubiquitination target of Arabidopsis PCNA, conserved in eukaryotes, is lysine 164. Taken together, the presented results clearly demonstrate the involvement of Arabidopsis UBC and RAD5a proteins in the ubiquitination of plant PCNA at lysine 164. The data show the complexity of the plant ubiquitination system and open new questions about its regulation in the plant cell.

Arabidopsis thaliana proliferating cell nuclear antigen has several potential sumoylation sites

Proliferating cell nuclear antigen (PCNA) is post-translationally modified in yeast and animal cells. Major studies carried out in the last decade have focused on the role of sumoylated and ubiquitinated PCNA. Using different approaches, an interaction between plant PCNA and SUMO both in vivo and in bacteria has been demonstrated for the first time. In addition, identical sumoylation patterns for both AtPCNA1 and 2 were observed in bacteria. The plant PCNA sumoylation pattern has been shown to differ significantly from that of Saccharomyces cerevisiae. This result contrasts with a common opinion based on previous structural analysis of yeast, human, and plant PCNAs, which treats PCNA as a highly conserved protein even between species. Analyses of AtPCNA post-translational modifications using different SUMO proteins (SUMO1, 2, 3, and 5) revealed similar modification patterns for each tested SUMO protein. Potential target lysine residues that might be sumoylated in vivo were identified on the basis of in bacteria AtPCNA mutational analyses. Taken together, these results clearly show that plant PCNA is post-translationally modified in bacteria and may be sumoylated in a plant cell at various sites. These data open up important new perspectives for further detailed studies on the role of PCNA sumoylation in plant cells.

New transgenic line of Arabidopsis thaliana with partly disabled zeaxanthin epoxidase activity displays changed carotenoid composition, xanthophyll cycle activity and non-photochemical quenching kinetics

Zeaxanthin epoxidase (ZE, E.C. 1.14.13.90), an enzyme belonging to the lipocalin superfamily, catalyses the conversion of zeaxanthin to antheraxanthin and violaxanthin. These reactions are part of the xanthophyll biosynthetic pathway and the xanthophyll cycle. The role of carotenoids in the dissipation of excessive light energy has been widely studied using mutants with a disabled carotenoid biosynthetic pathway. In this paper, the transgenic line MaZEP7 with partially disabled ZE activity is described and compared with wild-type plants and npq2 mutant lacking active ZE. We examined the presence and the abundance of aba1 transcripts, measured pigment composition, xanthophyll cycle functioning and chlorophyll fluorescence in all three lines. The MaZEP7 line contains additional copies of the aba1 gene introduced by agroinfiltration, but no enhanced aba1 transcript level was observed. In addition, ZE activity in MaZEP7 was impaired, resulting in an altered xanthophyll profile. In dark-adapted plants, violaxanthin and neoxanthin levels were lower than in wild-type plants, whereas antheraxanthin and zeaxanthin levels were considerably higher. The presence of lutein epoxide was also observed. Violaxanthin levels changed only minimally during light exposition, whereas antheraxanthin was converted to zeaxanthin and there was no epoxidation during the course of the experiment indicating disturbed xanthophyll cycle functioning. The amounts of carotenoids and chlorophylls on a dry weight basis and chl a/chl b ratio were similar in all lines. The presence of epoxidated pigments in MaZEP7 plants indicates that epoxidation occurs, but it is likely very slow. Chlorophyll fluorescence measurements showed that the dependence of electron transport rates on light intensity for the MaZEP7 line resembled the npq2 mutant. Kinetic measurements showed that the MaZEP7 line exhibited very rapid induction and a high steady-state value of non-photochemical quenching.

Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography

Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of many different His6-tagged recombinant proteins. On the one hand, it is a powerful technique but on the other hand it has its disadvantages. In this report, we present the development of a unique ssDNA aptamer for the purification of His3-tagged recombinant proteins. Our study shows that stability of the His3-tag/H3T aptamer complex can be controlled by the sodium ion concentration. Based on this feature, we demonstrate that H3T aptamer resin was successfully employed for the purification of three out of four tested His3-tagged recombinant proteins from an E. coli total protein extract using imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins is superior when purified with the help of the H3T aptamer in comparison with Ni-NTA resin.

The impact of immobilized metal affinity chromatography (IMAC) resins on DNA aptamer selection

DNA aptamers are single-stranded oligonucleotides which can form various secondary and tertiary structures. They can recognize a broad range of targets ranging from small molecules, such as ions, vitamins, antibiotics, to high molecular weight structures, including enzymes and antibodies. DNA aptamers are extensively studied as a potential source of new pharmaceutical drugs due to their inexpensive synthesis, low immunogenicity, and high specificity. The commonly used aptamer selection procedure is systematic evolution of ligands by exponential enrichment (SELEX) where the target molecule is immobilized on an appropriate chromatography resin. For peptide/protein targets, immobilized metal affinity chromatography (IMAC) resins are frequently used. There is a broad range of commercially available resins which can be used for IMAC. They are characterized by different metal ions, linker types, and bead materials. In this study, we tested the impact of different IMAC resins on the DNA aptamer selection process during eight SELEX cycles. A histidine-tagged 29 amino acid peptide corresponding to the interdomain connecting loop of human proliferating cell nuclear antigen was used as a selection target. Different resin materials containing the same metal ion (Co2+) were tested. Simultaneously, agarose resins containing identical linkers, but different metal ions (Co2+, Cu2+, Ni2+, and Zn2+) were analyzed. The results of this study clearly demonstrated the impact of the metal ion and resin material on the DNA aptamer selection progress. The presented data indicate that for successful IMAC resin-based SELEX, the determination of the optimal resin might be crucial.

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.