An ORC/Cdc6/MCM2-7 Complex Is Formed in a Multistep Reaction to Serve as a Platform for MCM Double-Hexamer Assembly

• Published in: Molecular cell Vol. 50; no. 4; pp. 577 - 588
• Main Authors: Fernández-Cid, Alejandra, Riera, Alberto, Tognetti, Silvia, Herrera, M. Carmen, Samel, Stefan, Evrin, Cecile, Winkler, Christian, Gardenal, Emanuela, Uhle, Stefan, Speck, Christian
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23-05-2013
• Subjects: Adenosine Triphosphate - metabolism; adenosinetriphosphatase; ATP; Binding Sites - genetics; Blotting, Western; cell cycle; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Chromosomal Proteins, Non-Histone - genetics; Chromosomal Proteins, Non-Histone - metabolism; Cyclin-Dependent Kinases - metabolism; DNA; DNA, Fungal - genetics; DNA, Fungal - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; eukaryotic cells; Hydrolysis; loading; Models, Biological; Multiprotein Complexes - genetics; Multiprotein Complexes - metabolism; Mutation; Origin Recognition Complex - genetics; Origin Recognition Complex - metabolism; Phosphorylation; Protein Binding; proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2013.03.026

In Saccharomyces cerevisiae and higher eukaryotes, the loading of the replicative helicase MCM2-7 onto DNA requires the combined activities of ORC, Cdc6, and Cdt1. These proteins load MCM2-7 in an unknown way into a double hexamer around DNA. Here we show that MCM2-7 recruitment by ORC/Cdc6 is blocked by an autoinhibitory domain in the C terminus of Mcm6. Interestingly, Cdt1 can overcome this inhibitory activity, and consequently the Cdt1-MCM2-7 complex activates ORC/Cdc6 ATP-hydrolysis to promote helicase loading. While Cdc6 ATPase activity is known to facilitate Cdt1 release and MCM2-7 loading, we discovered that Orc1 ATP-hydrolysis is equally important in this process. Moreover, we found that Orc1/Cdc6 ATP-hydrolysis promotes the formation of the ORC/Cdc6/MCM2-7 (OCM) complex, which functions in MCM2-7 double-hexamer assembly. Importantly, CDK-dependent phosphorylation of ORC inhibits OCM establishment to ensure once per cell cycle replication. In summary, this work reveals multiple critical mechanisms that redefine our understanding of DNA licensing.
► A Cdt1 interaction with MCM2-7 alleviates an autoinhibitory activity in Mcm6 ► OCM establishment occurs fast, while MCM2-7 double-hexamer formation is slow ► Both Orc1 and Cdc6 ATP-hydrolysis promote Cdt1 release and OCM formation ► The OCM represents a platform for double-hexamer assembly