Abstract
<jats:p>DNA double-strand breaks (DSBs) activate repair pathways that must be coordinated with other cellular functions. Although DSB sensors SIRT6, Ku80, and MRE11 initiate repair, how they organize these nuclear processes remains unknown. SPARK-ID is a proximity-labeling strategy mapping DDR interactome dynamics. Using these sensors as baits, we resolved chromatin-associated interactomes from damage formation to recovery. The sensors shared an enriched repair core while capturing distinct interactors, allowing temporal specialization: SIRT6ID was biased toward RNA and chromatin regulation, Ku80ID toward telomere-associated and translational programs, and MRE11ID toward recombination and DNA synthesis. Modularity analysis showed these functions are organized into modules linked by "connectors". Among them, Nucleolin linked DNA repair, RNA-metabolism, and nucleolar modules. Nucleolin depletion rewired DSB-sensor interactomes, altered repair-associated complex composition, expanded γH2AX domains, and reduced BRCA1, 53BP1, and phospho-ATM foci. Altogether, SPARK-ID reveals modular DSB-sensor interactomes whose robustness depends on connectors that integrate and constrain the DNA damage response.</jats:p>