RTF2 and CLASPIN localize CHK1 to the replication fork to control S-phase progression.

The essential kinase ataxia telangiectasia and Rad3-related (ATR) monitors the replicative (S) phase of the cell cycle to ensure faithful propagation of genetic material. While much is known about the mechanisms governing ATR activity at replication forks, how its effector checkpoint kinase 1 (CHK1) is regulated to create a localized hub of CHK1 activity at sites of replication remains poorly understood. Here, we report that replication termination factor 2 (RTF2), an essential replisome-associated protein, is necessary for maintaining CHK1 signaling at replication forks. RTF2 interacts with CHK1 and functions alongside the core replicative helicase complex-interacting protein CLASPIN to tether CHK1 to sites of DNA replication, controlling replication rates and preventing premature mitotic entry. These findings uncover how RTF2 and CLASPIN poise CHK1 at replication forks to facilitate its activation by ATR, creating fork-localized CHK1 activity that fuels unperturbed S-phase progression and promotes genome stability by maintaining the intrinsic S-G2 checkpoint.