One ORC with Many Faces

Abstract

The eukaryotic chromosomal Origin Recognition Complex (ORC) is a six-protein ATPase complex that not only binds to origin DNA, but together with Cdc6, also loads the Mcm2-7 helicase onto DNA. We have visualized by cryo-EM how the yeast ORC alters its structure as it interacts with and responds to its partners. The yeast ORC is a bi-lobed structure with Orc1, 4, and 5 in one lobe and Orc2, 3, and 6 in the other. ORC alone is flexible and binding to DNA rigidifies the structure. The replication initiator Cdc6 profoundly changes the ORC structure. Cdc6 completes a ring by bridging the bi-lobed structure from the side, and in doing so, nearly doubles the DNase I footprint. Cdc6 also causes Orc1 to rotate against Orc4. These changes lie at the heart of the molecular switch that transforms the ORC from a passive DNA binder to an active helicase loader. Using cryo-EM, we have caught ORC in the act of loading the first MCM2-7 hexamer. More precisely, we have captured a 1.1-MDa 14-protein complex comprised of ORC-Cdc6-Cdt1-Mcm2-7 in the presence of ATP-γS. ORC is transformed into a spiral and has partially loaded the Mcm2-7 hexamer onto dsDNA, as DNA density is visible going through the ORC-Cdc6 spiral and into the Mcm2-7 central chamber. This loading mechanism bears striking similarity to the loading of PCNA ring by the RFC ATPase spiral. ORC will eventually recruit a second MCM2-7 hexamer and deposit a Mcm2-7 double hexamer on dsDNA before letting go of the latter. We have established the architecture of the double hexamer, providing insights into Dbf4-dependent Cdc7 kinase regulation.