PARG inhibition reduces ssDNA levels and limits RPA loading upon replication fork collapse

Abstract Poly(ADP-ribosyl)ation (PARylation) is a transient post-translational modification catalyzed by PARP enzymes and reversed by PARG. PARG inhibition causes sustained PARylation and is being explored as an anticancer strategy, but its cellular consequences remain incompletely understood. Here, we examine how persistent PARylation influences cellular responses to replication stress and DNA damage. We show that sustained PARylation reduces phosphorylated and chromatin-bound RPA most strongly under fork-stalling conditions that progress toward fork collapse. This effect requires PARP1 activity and is restrained by intact ATR–CHK1 signaling, as checkpoint inhibition renders otherwise resistant cells permissive for PARG inhibitor-associated phosphorylated RPA loss from the chromatin. The reduction of RPA phosphorylation is not dependent on BRCA1 and it is not accompanied by increased RAD51 loading. Instead, reduced chromatin-bound RPA coincides with decreased exposed ssDNA. Our results identify a checkpoint-dependent fork-collapse state in which sustained PARylation limits ssDNA and RPA levels.