DePARylation prevents DNA replication-driven PARP1 condensation

PARG, the primary enzyme responsible for the reversal of PARP1-mediated poly(ADP-ribosyl)ation, has attracted considerable attention as a therapeutic target in cancer. Yet, the mechanisms underlying PARG inhibitor (PARGi) efficacy remain elusive. Herein, we demonstrate that PARGi prolongs PARP1 residence at damaged chromatin in a manner mechanistically distinct from PARP-inhibitor-induced trapping. That is, PARGi triggers the formation of PAR-driven, FUS-enriched PARP1 nuclear condensates upon DNA damage. Importantly, unrestrained S-phase PARylation during Okazaki fragment maturation also elicited PARP1 condensation in a manner directly reflecting intrinsic PARGi sensitivity, with FEN1 co-inhibition enhancing both PARP1 condensate formation and cytotoxicity. Finally, we discover that dePARylation prevents the rapid nuclear extrusion of PARP1 upon S-phase entry, a phenomenon that is reversible and could undermine PARP1-dependent nuclear processes. Together, our findings reveal that dePARylation precludes the replication-driven condensation of PARP1 and identify Okazaki fragment maturation as a targetable vulnerability that exacerbates condensation and PARGi cytotoxicity.