CMG helicase harbors a single-stranded DNA gate that enables replication fork activation (Wasserman et al., Cell 2019)
Correlative single-molecule fluorescence and force microscopy to study the eukaryotic CMG helicase. (A) Schematic of the experimental setup. One optical trap was moved relative to the other to apply different amounts of force to the DNA tether. (B) An example kymograph showing that CMG (green) switches between non-diffusive (high force) and diffusive (low force) modes. SsDNA regions are generated at high force. (C) Cartoon illustration of a gate in the CMG ring that allows it to transition between ss and dsDNA. Mcm10 tethers CMG to DNA during the gating process.
The eukaryotic replicative helicase CMG is a closed ring around dsDNA at origins, yet must transition to ssDNA for helicase action. The mechanism by which the topologically closed CMG switches from dsDNA to ssDNA binding remains enigmatic. In collaboration with the O'Donnell Lab at The Rockefeller University, Wasserman et al. employed correlative single-molecule fluorescence-force microscopy to visualize purified yeast CMG and other replisome proteins on various DNA substrates. This approach enabled us to, for the first time, observe that CMG loads onto ssDNA directly from solution and make a surprising discovery that CMG originally residing at a forked junction can depart the fork and undergo diffusion on duplex DNA and that a diffusing CMG on dsDNA can re-enter a fork and nucleate replisome assembly for replication restart. These findings unveil a ssDNA gate in the closed-ring-shaped architecture of CMG that allows it to transition between ss and dsDNA, which has important implications in replication initiation and DNA repair.