RSF1-Dependent PAR Turnover Promotes 53BP1 Liquid Condensate Formation at DNA Damage Sites.

Liquid-liquid phase separation (LLPS) is essential for proper DNA damage signaling. The sequential formation of PAR- and 53BP1-based condensates must be tightly controlled in space and time, however the molecular regulators of this process remain insufficiently defined. Here, we show that RSF1 contains a substantial proportion of intrinsically disordered regions (IDRs) and participates in the sequential assembly of liquid condensates at DSB sites. RSF1 depletion severely abolishes 53BP1 optoDroplet formation without affecting FUS condensates, but causes persistent PAR chains due to defective PARG recruitment. Domain mapping reveals that the C-terminal region of the IDR in RSF1 is sufficient to support 53BP1 condensate formation. Mechanistically, RSF1 controls the timely extinction of PAR chains by promoting the recruitment of PARG, but not PARP1, to DNA damage sites. This regulation enables the transition from PAR-based scaffolds to 53BP1 condensates. Importantly, defects in RSF1-mediated condensate formation correlate with impaired transcription of p53 target genes, including p21CIP1. Together, these findings establish RSF1 as a molecular coordinator that links PAR turnover to 53BP1 condensate assembly, thereby coupling the spatial and temporal regulation of LLPS with p53-dependent transcriptional responses at DNA lesions.