Mechanism and Timing of Mcm2–7 Ring Closure During DNA Replication Origin Licensing

Opening and closing of two ring-shaped Mcm2–7 DNA helicases is necessary to license eukaryotic origins of replication although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process, establishing a topological link between each Mcm2–7 and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule FRET (Förster resonance energy transfer), we monitored S. cerevisiae Mcm2–7 ring opening and closing during origin licensing. The two Mcm2–7 rings are open during initial DNA association and close sequentially, concomitant with release of their associated Cdt1. ATP hydrolysis by Mcm2–7 is coupled to ring closure and Cdt1 release, and failure to load the first Mcm2–7 prevents recruitment of the second Mcm2–7. Our findings identify key mechanisms controlling the Mcm2–7 DNA-entry gate during origin licensing and reveal that the two Mcm2–7 complexes are loaded by a coordinated series of events with implications for bidirectional replication initiation and quality control.