Chk1 dynamics in G2 phase upon replication stress predict daughter cell outcome

In human cells, ATR/Chk1 signaling couples S phase exit with the expression of mitotic inducers and prevents premature Mitosis upon replication stress (RS). Nonetheless, under-replicated DNA can persist at Mitosis, prompting chromosomal instability. To decipher how the DNA replication checkpoint (DRC) allows cells to enter Mitosis over time upon RS, we developed a FRET-based Chk1 activity sensor. During unperturbed growth, a basal Chk1 activity level is sustained throughout S phase and relies on replication origin firing. Incremental RS triggers stepwise Chk1 over-activation that delays S-phase, suggesting a rheostat-like role for DRC coupled with the replication machinery. Upon RS, Chk1 is inactivated as DNA replication terminates but surprisingly is reactivated in a subset of G2 cells, which relies on CDK1/2 and PLK1 and prevents mitotic entry. Cells can override active Chk1 signaling and reach Mitosis onset, revealing checkpoint adaptation. Cell division following Chk1 reactivation in G2 results in a p53/p21-dependent G1 arrest, eliminating the daughter cells from proliferation.