Tight CDK1 inhibition elicits domain-specific pre-RC setting in S-phase, suppressing CFS instability

Genome integrity requires replication to be completed before chromosome segregation. This coordination essentially relies on replication-dependent activation of a dedicated checkpoint that inhibits CDK1, delaying mitotic onset. Under-replication of Common Fragile Sites (CFSs), however, escapes surveillance, which generates mitotic chromosome breaks. Using human cells, we asked whether this leakage results from insufficient CDK1 inhibition under modest stresses used to destabilize CFSs. We found that tight CDK1 inhibition does suppress CFS instability in so-stressed cells. Molecular combing and Repli-Seq analyses consistently showed a burst of replication initiations in mid S-phase across large origin-poor domains shaped by transcription, including CFSs. CFS rescue and extra-initiations strikingly require availability of essential pre-Replication Complex (pre-RC) proteins during the S-phase, showing that CDK1 inhibition promotes targeted and mistimed pre-RC reloading. In addition to delay mitotic onset, tight checkpoint activation therefore advances replication completion of origin-poor domains at risk of under-replication, two complementary roles preserving genome stability. Repli-Seq experiments of human lymphoblastoid cells grown in the following conditions: Normal media (1 sample), 600 nM aphidicolin for 16h (1 sample), 600 nM aphidicolin and 10μM CDK1 inhibitor for 16h (two samples), 150μM HU for 16h (1 sample) and 150μM HU and 100mM Atr inhibitor for 16h.