FANCJ and RTEL1 facilitate pre-replication complex disassembly following replisome collision.

At the onset of eukaryotic DNA replication, double hexamers of MCM2-7 are assembled into inactive pre-replication complexes (pre-RCs) at replication origins. An excess of pre-RCs is loaded onto DNA, but only a fraction becomes activated to form CMG helicases when origins fire. The replisome, built around CMG, must navigate past dormant origins harboring inactive pre-RCs, but the mechanism by which this is achieved is unclear. Here, we used single-molecule imaging to visualize collisions between replisomes and pre-RCs during DNA replication in Xenopus laevis egg extracts. We show that pre-RCs are frequently removed from DNA upon collision and that efficient pre-RC removal requires the accessory helicases FANCJ and RTEL1. Furthermore, simultaneous depletion of both FANCJ and RTEL1 in human cells leads to pre-RC accumulation on chromatin and DNA damage. Together, our findings reveal a role for FANCJ and RTEL1 in pre-RC removal, ensuring efficient replication fork progression and maintaining genome stability.