Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork

Abstract The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), that have important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3-3.5 Å resolution cryo-EM structures comprising CMG, Ctf4, Csm3/Tof1 and Mrc1 at a replication fork. The structures provide high-resolution views of CMG:DNA interactions, revealing the mechanism of strand separation. Furthermore, they illustrate the topology of Mrc1 in the replisome and show Csm3/Tof1 ‘grips’ duplex DNA ahead of CMG via a network of interactions that are important for efficient replication fork pausing. Our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.