Cdc18 enforces long-term maintenance of the S phase checkpoint by anchoring the Rad3-Rad26 complex to chromatin.

DNA replication is initiated by recruitment of Cdc18 to origins. During S phase, CDK-dependent destruction of Cdc18 occurs. We show that when DNA replication stalls, Cdc18 persists in a chromatin-bound complex including the checkpoint kinases Rad3 and Rad26. Rad26 directly binds Cdc18 and is required for Rad3 recruitment to chromatin. Depletion of Cdc18 when DNA replication is stalled leads to release of Rad3 and Rad26 from chromatin and entry into an aberrant mitosis even though replication intermediates can still be detected. These findings indicate that Cdc18 plays a pivotal role in checkpoint maintenance by anchoring the Rad3-Rad26 complex to chromatin. Cdc18 persistence during DNA-replication arrest requires the S phase checkpoint that inhibits the S phase CDK. We propose that S phase arrest activates the S phase checkpoint blocking mitosis onset and inhibiting Cdc18 degradation, and that the stabilized Cdc18, in turn, anchors Rad3 to chromatin to ensure long-term checkpoint maintenance.