PARG inhibition sequesters nuclear PAR-binding proteins, including XRCC1 and its partners, into nuclear condensates to elicit cytotoxicity.

DNA breaks activate PARP1/2 to synthesize poly(ADP-ribose) (PAR), which relaxes chromatin and recruits DNA repair factors. Normally, PAR is short-lived, rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG). While PARP1/2 inhibitors are established therapies for homologous recombination (HR)-deficient cancers, predictive biomarkers for PARG inhibition (PARGi) remain undefined. Using parallel genome-wide CRISPR screens with PARP and PARG inhibitors, we show that PARGi is synthetically lethal with loss of several PAR-binding factors, including XRCC1-LIG3, POLB, ALC1/CHD1L, ARH3, and PARG itself, but notably not with HR deficiency. Conversely, loss of PARP1, NMNAT1 (required for nuclear NAD⁺ synthesis), or UNG (upstream of APE1 cleavage and PARP1 activation), confers PARGi resistance. Mechanistically, PARGi induces time- and dose-dependent formation of PARP1-and PAR-dependent nuclear condensates containing XRCC1 and associated repair factors in otherwise undamaged cells. These condensates do not harbor active DNA breaks but instead sequester PAR-binding repair proteins, depleting their available nuclear pool and impairing their recruitment to genuine DNA breaks. While our analysis focused on XRCC1, PARG inhibition likely sequesters additional PAR- and PARP1-binding proteins. Thus, we propose that PARGi sequesters PAR-binding proteins to elicit toxicity, explaining the essentiality of PARG (but not PARP1) and identifying the loss of PAR-binding factors as candidate predictive biomarkers for PARG-targeted therapy.