Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase

The eukaryotic replicative helicase CMG is a closed ring around double-stranded (ds)DNA at origins yet must transition to single-stranded (ss)DNA for helicase action. CMG must also handle repair intermediates, such as reversed forks that lack ssDNA. Here, using correlative single-molecule fluorescence and force microscopy, we show that CMG harbors a ssDNA gate that enables transitions between ss and dsDNA. When coupled to DNA polymerase, CMG remains on ssDNA, but when uncoupled, CMG employs this gate to traverse forked junctions onto dsDNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA to nucleate a functional replisome. The gate—distinct from that between Mcm2/5 used for origin loading—is intrinsic to CMG; however, Mcm10 promotes strand passage by enhancing the affinity of CMG to DNA. This gating process may explain the dsDNA-to-ssDNA transition of CMG at origins and help preserve CMG on dsDNA during fork repair. [Display omitted] •Eukaryotic CMG helicase employs a gate in its ring to switch between ss and dsDNA•Gating enables CMG to vacate a replication fork when uncoupled from DNA polymerase•CMG diffuses on dsDNA and uses this gate to enter a fork and restart replication•Mcm10, an essential replisome factor, tethers CMG to DNA during the gating process A “gate” in the eukaryotic CMG helicase allows it to switch between single- and double-stranded DNA, providing an explanation for how replication forks can continue past DNA lesions and restart after stalling