Arabidopsis thaliana proliferating cell nuclear antigen has several potential sumoylation sites

Abstract

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.

Imidazole-free purification of His 3 -tagged recombinant proteins using ssDNA aptamer-based affinity chromatography

“…Immobilized metal ion affinity chromatography (IMAC) is a simple and a fast technique which has been successfully used for the purification of many recombinant proteins, produced in bacteria [1][2][3], yeast [4,5], mammalian cells [6,7] and insect cells [8,9], under either native or denatured conditions. This method is based on the reversible interaction between the histidine tag (His-tag), which is attached either at the N-or C-terminus of the purified protein, and divalent metal ions, e.g.…”

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

“…Selected combinations of PCNA1, PCNA2 and CycA1 (BiFC control) fused with full length, N-terminal or C-terminal GFP fragments were transiently overexpressed in Nicotiana benthamiana epidermal cells. 21,26 The results of this…”

“…[19][20][21][22] One of the broadly studied PCNA post-translational modifications is ubiquitination. 20,22 The stalled replication forks induce PCNA ubiquitination at lysine 164 and activate postreplication repair.…”

Similarities and differences between Arabidopsis PCNA1 and PCNA2 in complementing the yeast DNA damage tolerance defect

“…It is also of great interest to notice that in a reported in vitro assay, AtPCNA can be ubiquitinated in the presence of Ubc2 (Rad6) but absence of any E3 [11]. Furthermore, the same group also reported that the AtPCNA-K164R mutant proteins can still be ubiquitinated [11] or sumoylated [10], raising a possibility that AtPCNA1-K200 and AtPCNA2-K262 may serve as alternative residues for PCNA covalent modifications in vivo.…”

“…Although the molecular mechanisms of DDT are thought to be conserved in plants [6][7][8][9], and AtPCNA can be sumoylated [10] and ubiquitinated [11] in vitro, the involvement of plant PCNA in DDT has not yet been demonstrated in vivo. The Arabidopsis genome encodes two PCNAs differing in only nine amino acid residues (Fig.…”