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.

Licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential Mcm4 motif that permit loading of two Mcm2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant Mcm2-7 forms initial hexamer-hexamer interactions; however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant Mcm2-7 reveal a limited time window for double-hexamer formation following second Mcm2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, Mcm10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation.

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