The relationships among three PCNA (proliferating cell nuclear antigen) Subunits in the hyperthermophilic archaeon Sulfolobus tokodaii (StoPCNAs) were analyzed and the effects of two PCNA complexes on the activities of the DNA helicase Hjm, DNA Ligase 1, and Holliday junction specific endonuclease Hjc were tested. There was no strong self-interaction of each StoPCNA. StoPCNA1 and StoPCNA3 interacted with each other, so did StoPCNA2 and StoPCNA3, but no interaction between StoPCNA1 and StoPCNA2 was observed. Two trimeric complexes (designed StoPCNA123 and StoPCNA323) were formed in vitro and it was determined that StoPCNA323 was composed of one StoPCNA2 and two StoPCNA3 subunits, with StoPCNA2 bridging the two StoPCNA3 subunits. Both complexes inhibited the unwinding activity of Hjm and the ligation activity of DNA Ligase I. In contrast, both stimulated the Holliday junction cleavage activity of Hjc. Our results provide further evidence that in crenarchaea, the PCNAs exhibit diversity in subunit interaction and complex formation. (C) 2008 Elsevier Inc. All rights reserved.