Sustained poly(ADP-ribosyl)ation limits RPA loading and attenuates ATR-CHK1 signaling upon replication fork collapse

Abstract Poly(ADP-ribosyl)ation (PARylation) is a transient post-translational modification catalyzed by PARP enzymes and reversed by the glycohydrolase PARG. Inhibition of PARG causes sustained PARylation, a toxic state that could be exploited for cancer therapy. However, the mechanisms by which excessive PARylation drives toxicity and cell death remain incompletely understood. Here, we examined how persistent PARylation influences cellular responses to replication stress and DNA damage. We found that during replication stress, the decline of ATR checkpoint activity marks the transition from fork stalling to fork collapse, a context in which stabilized poly(ADP-ribose) reduces chromatin-bound RPA. This loss of RPA, a key activator of the checkpoint, further reduces ATR signaling, revealing a feedback loop between unrestrained PARylation and checkpoint control. Our results identify a checkpoint-dependent consequence of unregulated PARP activity during fork collapse and highlight the PARylation-RPA axis as a critical determinant of PARG inhibitor cytotoxicity.