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 double-stranded (ds)DNA at origins of replication yet must transition to single-stranded (ss)DNA for helicase action. CMG must also handle DNA repair intermediates, such as reversed forks that lack ssDNA. The mechanism by which CMG switches between dsDNA- and ssDNA-binding remains enigmatic. In collaboration with the O’Donnell Lab, we employ smCFFM to visualize purified yeast CMG and other replisome proteins on various DNA substrates. This approach enables us to, for the first time, observe that CMG loads onto ssDNA directly from solution and that CMG originally residing at a forked junction, when decoupled from the DNA polymerase, departs the fork and undergoes diffusion on duplex DNA. We further show 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 CMG that potentially helps its preservation on dsDNA during fork repair and allows its dsDNA-to-ssDNA transition at origins.