Human replisomes direct a dedicated leading-strand primase for fork restart

Abstract Disruptions to leading-strand synthesis impair and stall replication forks because DNA synthesis by the leading-strand polymerase, Pol ε, is essential for optimal template unwinding by the replicative CMG (CDC45–MCM–GINS) helicase 1,2 . Failure to efficiently restart and/or protect stalled replication forks is a major source of genomic instability 3–6 . Stalled forks can be restarted by a variety of mechanisms, including repriming downstream of the impediment 4 . In human cells, the primase–polymerase PRIMPOL promotes fork progression during replication stress 7–10 , but how its activity is coordinated by the replisome is unresolved. Here, by reconstituting replication fork stalling and restart with purified human proteins, we show that the CMG helicase directs PRIMPOL for efficient replication fork restart. Unexpectedly, the interaction between PRIMPOL and replication protein A 9,11 is not critical for fork restart in this system. We show that PRIMPOL binds directly to CMG, and a cryo-electron microscopy structure illustrates how this targets PRIMPOL specifically for leading-strand priming to minimize the size of post-replicative gaps. Ablating the CMG–PRIMPOL interaction abolishes fork restart in vitro and PRIMPOL-dependent DNA damage tolerance in human cells. Our work establishes PRIMPOL as an integrated component of the human replisome dedicated to leading-strand priming.