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  • br Acknowledgement AM and SK thank ICMR

    2019-09-10


    Acknowledgement AM and SK thank ICMR, New Delhi, India for the financial assistance BIC/12(32)/2012. SK is the recipient of JRF from the project. RK and GJ thank Bristol-Myer Squibb (Grant # 34003085), USA for financial support.
    Introduction Proliferating Cell Nuclear Antigen (PCNA) is a highly conserved protein from yeast to human and plant cells [[1], [2], [3]]. PCNA acts as a molecular platform for protein-protein interactions for numerous cellular processes like DNA replication, DNA repair, 10537 pathway control, chromatin remodeling, sister chromatid cohesion and others [[3], [4], [5]]. PCNA is structured by three identical monomers that form a ring in which the carboxy end of one monomer interacts with the amino end of the other monomer, i.e., it is a homotrimer. It is to this structure that replicative DNA polymerases bind, conferring the required processivity for DNA replication [6]. It has been suggested that PCNA can form dimers of trimers that may be physiologically active both in animal [7] and in plant cells [8]. In mammals, PCNA function during the cell cycle can be regulated by its association to p21KIP1 protein (KIP, kinase inhibitor protein), an inhibitor of G1 Cyclin-CDK complexes [9]. In vitro assays have shown that p21 interferes with PCNA-DNA polymerase δ interaction affecting DNA replication. Methyl-directed mismatch (MMR) repair seems also to be abrogated [10]. PCNA interacts with cell cycle regulators forming a quaternary complex that includes p21, a cyclin dependent kinase (CDK) and cyclins D (CycD), suggesting complex regulatory models [9,11]. PCNA appears to act as a docking protein to facilitate interaction between different proteins during DNA replication. For example, binding of CycA/CDK2 to PCNA stimulates phosphorylation of DNA ligase I, Replication Factor C (RFC) and Flap endonuclease-1 (FEN1) [11,12]. In plants, the function of PCNA as a docking protein seems conserved. In pea axilary buds, PCNA associates to CycD3 [13]. The two Arabidopsis PCNA isoforms associate to almost all AtCycDs in nuclei, except CycD1;1, CycD3;1 and CycD3;3 [14]. Also, Tandem Affinity Purification assays (TAP) showed AtPCNA to be in replication and repair complexes, together with DNA polymerases POLD1 and POLD2, and in cell cycle complexes with CycA2;3 and E2F Transcription Factors. Such complexes are enriched with proteins whose genes contain E2F motifs in their promoters [15], suggesting that these complexes are formed in a specific moment of the cell cycle, the transition towards the S phase. Arabidopsis PCNA can bind to synthetic peptides derived from p21 protein [16], but there is no p21 protein in plants. There is however a family of KIP-related proteins, or KRPs, that contain a sequence of amino acids present in p21, that are related to its inhibitory activity [17,18], and that inhibit kinase activity in CycD/CDKs complexes in plants [[19], [20], [21]]. PCNA-associated kinase activity on the Retinoblastoma-Related (RBR) protein was inhibited by a peptide containing the carboxyl end of maize KRP4;2 in a doses dependent manner, indicating association of PCNA to active Cyc/CDK heterodimers that are susceptible to KRP inhibition [22]. However, no interaction PCNA-KRP has been demonstrated so far. During maize germination DNA replication starts by 12 h after imbibition [23]. PCNA protein levels increase since the start of imbibition and stabilize by the time replication starts [24]. During this process, the homotrimer of PCNA would seem to form part of protein complexes of molecular mass fluctuating between 160 and 200 kDa [25]. Among these, PCNA binds G1 Cycs/CDKs complexes that contain kinase activity (on histone H1 or RBR as substrates), in which PSTAIRE motif-containing CDKs and Cycs D2;2; D4;2 or D5;3 can be found [[24], [25], [26], [27], [28]]. The association of CycD4;2 and CDKA to chromatin-bound PCNA during maize germination was reported [8], strongly suggesting the function of PCNA as a platform for CycD/CDK complexes, perhaps playing a role in the progression of the cell cycle.