RPA-independent activation of the ATR/CHK1 pathway

The ATR/CHK1 pathway governs a crucial intra-S-phase checkpoint that safeguards genome stability under replication stress by stabilizing stalled replication forks and ensuring high-fidelity DNA replication. Traditionally, activation of this pathway is thought to rely on replication protein A (RPA)-coated single-stranded DNA, which recruits the ATR-ATRIP complex to sites of stalling fork, positioning RPA as essential for ATR signaling. In this study, we report a surprising and previously unrecognized phenomenon: acute depletion of RPA2 triggers robust ATR/CHK1 activation through an RPA-independent mechanism. Using 293A and RPE-1 cells engineered with an inducible RPA2-dTAG degron system, we observed increased phosphorylation of CHK1 at Ser296 and Ser345 in the absence of RPA. Notably, this elevated CHK1 phosphorylation was abolished by ATR inhibition, confirming its dependence on ATR kinase activity. Mechanistic analyses further revealed that this RPA-independent activation requires the checkpoint mediators RAD9 and TOPBP1. These findings uncover dual mechanisms, both RPA-dependent and -independent, of ATR/CHK1 pathway activation, highlighting a robust and flexible replication stress response network that preserves genome integrity even when canonical signaling is disrupted.