Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM

In preparation for bidirectional replication, the origin recognition complex (ORC) loads two MCM helicases forming a head-to-head double hexamer (DH) around DNA(1,2). How DH formation occurs is debated. Single-molecule experiments suggest a sequential mechanism whereby ORC-dependent loading of the first hexamer drives second hexamer recruitment(3). In contrast, biochemical data show that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites(4,5). We visualized MCM loading using time-resolved EM, to identify DH formation intermediates. We confirm that both hexamers are recruited via the same interaction between the MCM and ORC C-terminal domains, and identify the mechanism for coupled MCM loading. A first loaded hexamer locked around DNA is recognized by ORC, which unexpectedly engages the N-terminal homo-dimerization interface of MCM. In this configuration, ORC is poised to direct second hexamer recruitment in an inverted orientation, suitable for DH formation. Our data reconcile two apparently contrasting models.