Abstract

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, it remains unclear whether a repaired fork can be reactivated by the same CMG that is retained on DNA, or if fork restart relies on a distinct replisome traveling from another origin. 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 ssDNA gating. These results reveal an Mcm10-induced pathway that preserves CMG on DNA and allows it to access an empty fork for swift replication recovery.

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