RESUMEN
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.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN , Complejo de Reconocimiento del Origen/metabolismo , Pyrococcus furiosus/metabolismo , Origen de Réplica , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , ADN Helicasas/genética , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Complejo de Reconocimiento del Origen/química , Complejo de Reconocimiento del Origen/genética , Conformación Proteica , Pyrococcus furiosus/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The initiation step is a key process to regulate the frequency of DNA replication. Although recent studies in Archaea defined the origin of DNA replication (oriC) and the Cdc6/Orc1 homolog as an origin recognition protein, the location and mechanism of duplex opening have remained unclear. We have found that Cdc6/Orc1 binds to oriC and unwinds duplex DNA in the hyperthermophilic archaeon Pyrococcus furiosus, by means of a P1 endonuclease assay. A primer extension analysis further revealed that this localized unwinding occurs in the oriC region at a specific site, which is 12-bp long and rich in adenine and thymine. This site is different from the predicted duplex unwinding element (DUE) that we reported previously. We also discovered that Cdc6/Orc1 induces topological changes in supercoiled oriC DNA, and that this process is dependent on the AAA+ domain. These results indicate that topological alterations of oriC DNA by Cdc6/Orc1 introduce a single-stranded region at the 12-mer site, that could possibly serve as an entry point for Mcm helicase.