Cryo-EM structures of G4-stalled CMG reveal inchworm mechanism of DNA translocation

DNA G-quadruplexes (G4s) are non-B-form DNA secondary structures that threaten genome stability by impeding DNA replication. To elucidate how G4s induce replication fork arrest, we have characterized fork collisions with preformed G4s in the parental DNA using fully reconstituted yeast and human replisomes. We demonstrate that a single G4 in the leading strand template is sufficient to stall replisomes by blocking the CMG helicase. An ensemble of high-resolution cryo-EM structures of stalled yeast and human CMG complexes reveals that the G4 is fully folded and lodged inside the CMG central channel. The stalled CMG is conformationally constrained and arrests in the transition between translocation states. Unexpectedly, our analysis suggests that CMG employs an unprecedented inchworm mechanism to translocate on DNA. These findings illuminate the eukaryotic replication fork mechanism under both normal and perturbed conditions.