The Interaction Between Pcna And Serine/Threonine Protein Kinase In The Hyperthermophilic Archaeon

Archaea, the third domain of life, are different from eubacteria and eukaryotes in evolution. In recent years, it has become evident that the archaeal replication machinery is most likely a core version of that found in eukaryotes. The extreme thermophilic archaea have become model organisms for studying the DNA replication regulation mechanisms of higher eukaryotes. In this study, we found for the first time that the proliferating cell nuclear antigen (PCNA) proteins interacted with serine/threonine protein kinase in the S. tokodaii and these interactions were found to have an effect on the regulation of PCNA function. The results are as follows:(1) Using bacterial two-hybrid technique and Pull-down/Western blotting assays, we showed that three PCNA protein subunits had physical interaction with ST0686.(2) Three PCNA subunits, PCNA1, PCNA2, and PCNA3 monomers could be phosphorylated by ST0686 in vitro. Three PCNA subunits inhibited their respective phosphorylations with each other. PCNA trimer was most weakly phosphorylated if compared with their monomers. (3) All three PCNA subunits were found to stimulate StPolB1 polymerase activity. The stimulation activity was observed to be significantly enhanced when either of PCNA monomers was phosphorylated by ST0686 kinase. Strikingly, PCNA trimer stimulated the StPolB1 polymerization activity with a strong effect. However, the stimulation was obviously inhibited if the PCNA trimer was phosphorylated. (4) The forms of three phosphorylated PCNA proteins were detected by Co-Immunoprecipitation from the archaeal cells.In this study, we presented the first evidence on the physical interaction and functional linkage between PCNA and protein kinase ST0686 in an archaeon. This could offer a powerful tool for addressing as yet unidentified issues in eukaryotes due to a similarity between archaeal and eukaryotic DNA replication.