Structure of the Pre-Initiation Complex Explains CMGE Biogenesis

When cells enter S phase, bidirectional DNA replication is initiated through the kinase-regulated recruitment of three activators (Cdc45, GINS and Pol epsilon) to a duplex DNA-loaded double hexamer of MCM ATPases. Together these proteins form two CMGE helicases that establish divergent replication forks as they become separated ¹ . To understand CMGE biogenesis, we reconstituted the pre-Initiation Complex with purified yeast proteins. The cryo-EM structure shows a set of firing factors caught in the act of assembling two symmetric CMGEs. We show how stepwise complex formation reshapes MCM in preparation for DNA opening and we explain how ATP promotes firing-factor ejection and CMGE maturation. While we find that Sld2 promotes GINS recruitment to MCM as expected, it also aids efficient separation of the CMGE dimer, and it is essential for lagging strand ejection from MCM. These findings have direct implications for our understanding of the metazoan Sld2 ortholog, RECQL4, pointing to a replication-fork establishment mechanism conserved across eukaryotes.