53BP1 condensates function as bioreactors for NHEJ directed DNA repair and insulators to determine pathway choice

Genomic integrity requires efficient resolution of DNA damage. Non-homologous end joining (NHEJ) is the primary mechanism of DNA double strand break (DSB) repair in mammalian cells and is mediated by 53BP1, a tumor suppressor involved in preventing DSB end-resection and homologous recombination. NHEJ repair foci form following DSB formation, however how mesoscale assembly occurs and whether 53BP1 is the driver of this process are unknown. Also, despite knowledge of the identify of key pathway molecules, the specific functions of mesoscale repair condensates in DNA repair and pathway selectivity are unknown. To address these gaps, we determined the minimal domain of 53BP1 sufficient for phase separation in vitro and identified the key residues that governing its condensation. Utilizing a separation-of-function mutant, we demonstrate that 53BP1and its protein condensation is the core driver of NHEJ foci formation. 53BP1 condensates function as bioreactor compartments, increasing the effective concentration of substrates near double strand breaks and are essential for efficient DNA damage resolution. Additionally, we show that 53BP1 condensates function as insulators around DSB sites to prevent end-resection and direct repair pathway selectivity. Collectively our work reveals a specialized compartment for DNA repair through the spatial clustering of 53BP1 molecules into repair foci essential to maintain genome integrity.