Cells respond to DNA double-strand breaks (DSBs) by recruiting factors such as the DNAdamage mediator protein MDC1, p53-binding protein 1 (53BP1) and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 via phospho-dependent interactions between the RNF8 forkhead-associated (FHA) domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ATM. We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage suggesting that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.DNA DSBs are highly cytotoxic lesions; and to ensure that they are repaired with minimal impact on genome stability, cells mount a complex DNA-damage response (DDR) that includes the spatial reorganization of DSB repair and signaling proteins into subnuclear structures -ionizing radiation-induced foci (IRIF) -that surround DSB sites (1, 2). Most IRIF formation depends on phosphorylation of the histone variant H2AX (to form γH2AX) by the DNA-PK and ATM protein kinases (3-6). The γH2AX epitope is bound by MDC1 (7-10) that then promotes IRIF formation by other proteins, including 53BP1, Nijmegenbreakage-syndrome protein NBS1 and BRCA1 (11,12). BRCA1 recruitment to IRIF requires its interaction with the ubiquitin-binding protein RAP80 (13)(14)(15)(16) Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts identify RNF8 as the prime ubiquitin ligase for poly-ubiquitination at DSB sites, define its functional importance in the DDR, and establish how RNF8 is recruited to sites of DNA damage via interactions with MDC1.MDC1 is phosphorylated in an ATM-dependent manner in response to ionizing radiation (IR) (11,12). Potential ATM target sites (consensus S/T-Q) cluster in the MDC1 Nterminus, the most notable being four adjacent motifs conforming to the consensus TQXF (Figs. 1A and S1). Significantly, antibodies raised against a peptide encoding phospho-T719 ( Fig. S2A) indicated that it is targeted by ATM in vitro (Fig. 1B), and in vivo ( Fig. 1C and S2B). However, in vitro assays with bacterially-expressed MDC1 fragments revealed that T719 was not the only site of ATM modification. Phosphorylation was only abolished when an "AQXF" mutant protein bearing threonine-to-alanine substitutions in all four TQXF motifs was used as substrate (Fig. 1D). These data and the recent identification of another TQXF site (T752) as an ATM target (17) therefore imply that MDC1 TQXF motifs are likely all modified by ATM and may function redundantly with one another.To address the function of the MDC1 TQXF motifs, we used siRNA to deplete endogenous MDC1 i...