Cdc6/Orc1 from Pyrococcus furiosus may act as the origin recognition protein and Mcm helicase recruiter

Archaea have one or more Cdc6/Orc1 proteins, which share sequence similarities with eukaryotic Cdc6 and Orc1. These proteins are involved in the initiation process of DNA replication, although their specific function has not been elucidated, except for origin recognition and binding. We showed that the Cdc6/Orc1 protein from the hyperthermophilic archaeon Pyrococcus furiosus specifically binds to the oriC region in the whole genome. However, it remains unclear how this initiator protein specifically recognizes the oriC region and how the Mcm helicase is recruited to oriC. In the current study, we characterized the biochemical properties of Cdc6/Orc1 in P. furiosus. The ATPase activity of the Cdc6/Orc1 protein was completely suppressed by binding to DNA containing the origin recognition box (ORB). Limited proteolysis and DNase I-footprint experiments suggested that the Cdc6/Orc1 protein changes its conformation on the ORB sequence in the presence of ATP. This conformational change may have an unknown, important function in the initiation process. Results from an in vitro recruiting assay indicated that Mcm is recruited onto the oriC region in a Cdc6/Orc1-dependent, but not ATP-dependent, manner. However, some other function is required for the functional loading of this helicase to start the unwinding of the replication fork DNA.