Abstract
In DNA replication studies, the mechanism for regulation of the various steps from initiation to elongation is a crucial subject to understand cell cycle control. The eukaryotic minichromosome maintenance (MCM) protein complex is recruited to the replication origin by Cdc6 and Cdt1 to form the pre-replication complex, and participates in forming the CMG complex formation with Cdc45 and GINS to work as the active helicase. Intriguingly, Thermoplasma acidophilum, as well as many other archaea, has only one Gins protein homolog, contrary to the heterotetramer of the eukaryotic GINS made of four different proteins. The Gins51 protein reportedly forms a homotetramer (TaGINS) and physically interacts with TaMCM. In addition, TaCdc6-2, one of the two Cdc6/Orc1 homologs in T. acidophilum reportedly stimulates the ATPase and helicase activities of TaMCM in vitro. Here, we found a reaction condition, in which TaGINS stimulated the ATPase and helicase activities of TaMCM in a concentration dependent manner. Furthermore, the stimulation of the TaMCM helicase activity by TaGINS was enhanced by the addition of TaCdc6-2. A gel retardation assay revealed that TaMCM, TaGINS, and TaCdc6-2 form a complex on ssDNA. However, glutaraldehyde-crosslinking was necessary to detect the shifted band, indicating that the ternary complex of TaMCM-TaGINS-TaCdc6-2 is not stable in vitro. Immunoprecipitation experiment supported a weak interaction of these three proteins in vivo. Activation of the replicative helicase by a mechanism including a Cdc6-like protein suggests the divergent evolution after the division into Archaea and Eukarya.