Replisome preservation by a single-stranded DNA gate in the CMG helicase

The eukaryotic replicative helicase CMG is assembled at replication origins and is thought to remain topologically closed until termination. Upon encountering a lesion, CMG must vacate a stalled fork to allow DNA repair. However, the fate of CMG under these stress conditions remains unclear. Here, using correlative single-molecule fluorescence and force microscopy, we show that when uncoupled from a DNA polymerase, CMG opens a single-stranded (ss) DNA gate to traverse a forked junction and reside on double-stranded (ds) DNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA for continued fork progression. The accessory protein Mcm10 is required for robust ssDNA gating. These results reveal an Mcm10-induced pathway that preserves CMG on DNA and allows it to access a repaired fork for swift replication recovery.