Launching a ubiquitination cascade at DNA breaks.

T he BRCA1 gene was cloned in 1994 as the first tumor suppressor of hereditary breast cancer, and it has been heavily studied ever since. The Brca1 protein is multifunctional and critical for the maintenance of genomic stability. Among its many roles, Brca1 is part of an E3 ubiquitin ligase important for homologous recombination (HR) and signaling of double-strand DNA breaks (DSBs). In response to DSBs, the checkpoint kinases ATM and ATR phosphorylate the histone variant H2AX adjacent to the breaks, which then recruits the mediator protein Mdc1 and other DNA repair and damage signaling proteins, including Brca1. Once recruited to DSBs, Brca1 colocalizes with these repair and signaling proteins in discrete nuclear foci. Both H2AX and Mdc1 are required for the formation of Brca1 foci, but how they contribute to Brca1 recruitment has not been clear. Part of this ‘‘missing link’’ was recently revealed. Rap80, a Brca1associating protein, targets Brca1 to DSBs through its UIM domains, which recognize polyubiquitin chains (1–3). In this issue of PNAS, Wang and Elledge (4) present the discovery of Rnf8 as an E3 ubiquitin ligase that recognizes phosphorylated proteins at DSBs and generates the polyubiquitin chains recognized by Rap80, thereby connecting the phosphorylationand ubiquitination-regulated steps during the recruitment of Brca1. One important clue to the E3 ligase required for Brca1 recruitment is the specific binding of the UIM domains of Rap80 to Lys-63 (K63)-linked polyubiquitin chains (4). To generate polyubiquitin chains on substrates, an E3 ubiquitin ligase must work with an E2 ubiquitinconjugating enzyme. Thus far, Ubc13 is the only known E2 conjugating enzyme that supports the formation of K63-linked polyubiquitin chains. In fact, Ubc13 was shown to be required for the formation of Brca1 foci (5). Ubc13 is known to interact with several E3 ligases (6). By screening these E3 ligases with siRNAs, Wang and Elledge (4) discovered that Rnf8 is the only E3 required for the formation of Brca1 foci. Independently of Wang and Elledge (4), three other groups (7–9) also found that Rnf8 is required for Brca1 recruitment by using different approaches. From an RNAi screen, Kolas et al. (7) identified Rnf8 as a protein required for the formation of 53BP1 foci at DSBs. Huen et al. (8) and Mailand et al. (9), on the other hand, noticed that, in addition to a RING-finger domain characteristic for certain E3 ligases, Rnf8 possesses an FHA domain that can function as phosphopeptide-binding modules in some DNA repair and damage signaling proteins. Both Wang and Elledge (4) and Huen et al. (8) showed that a region of Rnf8 containing the FHA domain but not the RING finger is sufficient for foci formation at DSBs. The formation of Rnf8 foci requires H2AX and Mdc1 but not Brca1, Nbs1, and 53BP1 (8). A recent study by Elledge and colleagues (10) identified nine damage-induced phosphorylation sites (SQ/TQ) in Mdc1. Through a phosphopeptide library screen, Huen et al. (8) identified pTXXY/F as the optimal