To counteract the threat of genomic instability introduced by DNA double-strand breaks (DSBs), cells have evolved DNA-damage response pathways that rapidly deploy repair proteins when and where they are needed. Mobilization is mediated by post-translational modifications. In mammals, early events in the DNA-damage response include recruitment of the E3 ubiquitin ligases RNF8 and RNF168 to break sites, where they add K63-linked ubiquitin chains to the surrounding chromatin. Ubiquitination creates a binding target for RAP80, a protein that contains tandem ubiquitin-interacting motifs (UIMs); in turn, RAP80 recruits BRCA1 to the DNA break site. Now, Guzzo et al. show that RAP80 also contains a SUMO-interacting motif (SIM) and that BRCA1 is recruited through RAP80 interactions with hybrid SUMO-ubiquitin chains. The authors identified a consensus SIM in RAP80 that confers binding to SUMO-2 polymers in vitro and that is required in conjunction with the UIMs to recruit RAP80 to DSBs induced by ionizing radiation in U2OS cells. Because the RAP80 SIM is proximal to the UIMs, a dual SUMO-ubiquitin recognition mechanism was queried by assaying binding to chains of diubiquitin conjugated to SUMO-2. Indeed, RAP80-binding affinity for the hybrid chain was 80-fold greater than that observed for either SUMO or ubiquitin alone. Ubiquitin and SUMO signals are integrated in vivo by RNF4, a SUMO-targeted E3 ligase that generates hybrid chains. Endogenous RNF4 localizes to DNA-repair foci upon ionizing-radiation treatment, and recruitment of both RAP80 and BRCA1 is reduced when RNF4 is depleted by siRNA. Transfection of an RNF4 wild-type construct, but not a RING-domain mutant, restores RAP80 and BRCA1 recruitment to DSBs in depleted cells, which indicates that RNF4 ubiquitination activity is required to generate RAP80 binding signals. These results establish roles for both ubiquitin and SUMO in regulating BRCA1 localization to DNA breaks and provide a foundation to investigate the potential roles of SUMO-ubiquitin hybrid chains in signaling cell stress. (Sci. Signal. 5, ra88, 2012)