ATR enforcement of the S/G2 checkpoint prevents premature S phase shutdown and genome instability

The ATR-enforced S/G2 checkpoint activates during DNA replication to restrain CDK1-dependent phosphorylation of FOXM1 and subsequent transactivation of the G2/M gene network until the end of S phase. However, the extent to which this checkpoint ensures the completion of DNA replication and whether it safeguards genomic integrity has remained unknown. Here, we induce S/G2 checkpoint failure throughout S phase in non-malignant human epithelial cells using multiple ATR pathway inhibitors. Consequently, the mitotic kinase complex cyclin B1-CDK1 prematurely shuts-down the DNA replication program, preventing the completion of genome duplication. In turn, this leads to the retention of inactive replisomes on chromatin and unfired origins into the G2 phase, which induce subsequent accumulation of pan-nuclear ᵧH2AX and mitotic failure. Collectively, these findings indicate the S/G2 checkpoint ensures replication completion and genome stability.