CHK1 is an integral regulator of DNA replication in human cells.

CHK1, a key serine/threonine kinase, is essential for cell cycle progression and genome maintenance in response to DNA damage and/or replication stress. However, its functions during normal DNA replication remain to be defined. Here, we employed the dTAG system to achieve rapid and selective CHK1 depletion in cells and examined the consequences of its acute loss. CHK1 degradation led to rapid cell death, with significant loss of viability within 16 h and complete lethality by 48 h, indicating critical roles of CHK1 during normal DNA replication. Rescue experiments demonstrated that only full-length, catalytically active CHK1 could restore cell survival, emphasizing the essential role of its kinase function and ATR-dependent phosphorylation. CHK1 depletion triggered extensive DNA damage, as evidenced by increased γH2AX and RPA2 phosphorylation, and caused S-phase arrest, replication fork collapse, and failure to enter mitosis. Interestingly, cells arrested at the G1/S boundary, which do not undergo DNA replication, were still sensitive to CHK1 depletion. These data reveal a critical role of CHK1 in suppressing replication fork progression even in the absence of DNA replication. Thus, our results highlight CHK1's indispensable role in the management of replication fork stability and cell cycle progression, providing a refined mechanistic understanding of its function during normal cell proliferation.