Dynamics of Replication Fork Progression Following Helicase–Polymerase Uncoupling in Eukaryotes

Leading-strand polymerase stalling at DNA damage impairs replication fork progression. Using biochemical approaches, we show this arises due to both slower template unwinding following helicase–polymerase uncoupling and establishment of prolonged stalled fork structures. Fork slowing and stalling occur at structurally distinct lesions, are always associated with continued lagging-strand synthesis, are observed when either Pol ε or Pol δ stalls at leading-strand damage, and do not require specific helicase–polymerase coupling factors. Hence, the key trigger for these replisome-intrinsic responses is cessation of leading-strand polymerization, revealing this as a crucial driver of normal replication fork rates. We propose that this helps balance the need for sufficient uncoupling to activate the DNA replication checkpoint with excessive destabilizing single-stranded DNA exposure in eukaryotes.