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. [Display omitted] •Upon RS, Chk1 can reactivate during G2 phase, preventing mitosis onset•Timely Chk1 reactivation in G2 phase relies on both Cdk2/1 and Plk1 activities•A subset of cells divides despite G2 Chk1 activity, revealing checkpoint adaptation•G2 phase Chk1 reactivation translates to G1 arrest in daughter cells Replication stress (RS) causes under-replicated DNA to persist at mitosis, challenging chromosomal integrity. Lebrec et al. use a FRET-based sensor and report that Chk1 reactivates in G2 phase following RS, efficiently preventing mitosis. Chk1 signaling is overridden through checkpoint adaptation, which culminates in a p53-dependent G1 arrest of the daughter cells.