Phosphatidylinositol 3-phosphate promotes 53BP1 condensate-like assembly at DNA double-strand breaks

53BP1 nuclear bodies are dynamic structures with properties resembling biomolecular condensates, but the molecular determinants that govern 53BP1 higher-order assembly at DNA double-strand breaks (DSBs) remain to be established. Here, we show that 53BP1 condensation is stimulated by phosphatidylinositol 3-phosphate (PI(3)P) in vitro through a highly specific interaction with its C-terminal tandem BRCT domain (tBRCT). Consequently, mutational inactivation of the 53BP1 tBRCT domain compromised PI(3)P binding and suppressed 53BP1 optodroplet formation in vivo. We further show that rapid 53BP1 clustering following DNA damage precedes its stable assembly on DSB-flanking chromatin, requires its tBRCT, and is suppressed by sequestration of nuclear PI(3)P. Taken together, our findings identify PI(3)P binding as a mechanism that promotes the formation and maturation of 53BP1 condensate-like assemblies on damaged chromatin.