Palm, G.; Butryn, A.; Canal, B.; Pühringer, T.; Gross, M. H.; Diffley, J. F. X.; Costa, A.

Eukaryotic DNA replication initiation requires controlled, temporally separated steps to preserve genome stability. The MCM replicative helicase is loaded on duplex DNA as an inactive double hexamer, which nucleates the assembly of dimeric Cdc45-MCM-GINS-Pol epsilon (dCMGE) replisomes. Mcm10 splits dCMGE into two, generating divergent replication forks, but the mechanism is unknown. Using ATPase-defective yeast MCM variants that slow origin melting, we captured five reaction intermediates that explain the structural mechanism. Two Mcm10 molecules bridge across the CMGE dimer, bracing two converging helicases. The restrained MCM motors pull apart the two DNA filaments, such that each lagging strand becomes ejected through the Mcm2-5 gate. Our reconstituted structures resemble the double CMG stabilized by metazoan DONSON, pointing to an origin DNA melting mechanism conserved across evolution.