SPARK-ID: Dynamic DSB-sensor interactomes reveal modular nuclear repair networks coordinated by connector proteins

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 {gamma}H2AX 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. Graphical AbstractThree DSB sensors (MRE11, Ku80, SIRT6) orchestrate DNA repair through dynamic protein-protein interaction networks that expand upon DSB induction. Proximity labeling reveals distinct sensor interactomes that share a functional core of nuclear processes while incorporating sensor-specific interactors for pathway specialization. These networks contain connector nodes that coordinate multiple parallel nuclear functions, enabling functional diversification and conferring robustness to the DNA damage response. The sensors reshape their interactome structure and composition to integrate and constrain cellular responses through organized macromolecular complexes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/740882v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1bb112dorg.highwire.dtl.DTLVardef@64cc4dorg.highwire.dtl.DTLVardef@1a0e2d1org.highwire.dtl.DTLVardef@18b3e8b_HPS_FORMAT_FIGEXP M_FIG C_FIG