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Launching a Ubiquitination Cascade at DNA Breaks

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Launching a Ubiquitination Cascade at DNA Breaks COMMENTARY Launching a ubiquitination cascade at DNA breaks Xiaohong H. Yang* and Lee Zou*†‡ *Massachusetts General Hospital Cancer Center and †Department of Pathology, Harvard Medical School, Charlestown, MA 02129 he BRCA1 gene was cloned in tion to a RING-finger domain charac- transiently expressed H2A and H2AX in 1994 as the first tumor suppres- teristic for certain E3 ligases, Rnf8 cells requires Rnf8 and that purified Rnf8 sor of hereditary breast cancer, possesses an FHA domain that can promotes H2A polyubiquitination in vitro. and it has been heavily studied function as phosphopeptide-binding How is Brca1 recruited to polyubiq- Tever since. The Brca1 protein is multifunc- modules in some DNA repair and dam- uitin chains? Previous studies by three tional and critical for the maintenance of age signaling proteins. Both Wang and groups have identified Abra1, Rap80, genomic stability. Among its many roles, Elledge (4) and Huen et al. (8) showed and Brcc36 as the components of a Brca1 is part of an E3 ubiquitin ligase that a region of Rnf8 containing the Brca1-containing complex (the Brca1 A important for homologous recombination FHA domain but not the RING finger complex) (1–3). Among these proteins, (HR) and signaling of double-strand DNA is sufficient for foci formation at DSBs. Rap80 bears two UIM domains capable breaks (DSBs). In response to DSBs, the The formation of Rnf8 foci requires of recognizing polyubiquitin chains. Al- checkpoint kinases ATM and ATR phos- H2AX and Mdc1 but not Brca1, Nbs1, though these UIM domains alone are phorylate the histone variant H2AX adja- and 53BP1 (8). A recent study by sufficient for foci formation, Wang and cent to the breaks, which then recruits the Elledge and colleagues (10) identified Elledge (4) found that the optimal local- mediator protein Mdc1 and other DNA nine damage-induced phosphorylation ization of Rap80 to DSBs depends on repair and damage signaling proteins, in- sites (SQ/TQ) in Mdc1. Through a phos- both the UIM domains and the interac- cluding Brca1. Once recruited to DSBs, phopeptide library screen, Huen et al. tion between Rap80 and Abra1. Brcc36, Brca1 colocalizes with these repair and (8) identified pTXXY/F as the optimal which associates with Rap80 through signaling proteins in discrete nuclear foci. Abra1, also is required for the forma- Both H2AX and Mdc1 are required for tion of Rap80 foci (4). Thus, with the the formation of Brca1 foci, but how they UIM domains of Rap80 directly recog- contribute to Brca1 recruitment has not The Brca1 protein nizing polyubiquitin chains, the Rap80– been clear. Part of this ‘‘missing link’’ was Abra1–Brcc36 module of the Brca1 A recently revealed. Rap80, a Brca1- is multifunctional complex targets Brca1 to DSBs. associating protein, targets Brca1 to DSBs In addition to revealing the role of through its UIM domains, which recog- and critical for Rnf8 in Brca1 recruitment, Wang and nize polyubiquitin chains (1–3). In this Elledge (4) also present a comprehensive issue of PNAS, Wang and Elledge (4) the maintenance of analysis of the anatomy of the Brca1 A present the discovery of Rnf8 as an E3 complex. They found that Abra1 functions ubiquitin ligase that recognizes phosphor- genomic stability. as a scaffold in this complex. The N and ylated proteins at DSBs and generates the C termini of Abrca1 interact with Brca1 polyubiquitin chains recognized by Rap80, and Rap80, respectively. Abra1 and thereby connecting the phosphorylation- motif recognized by the FHA domain of Brcc36 associate with each other through and ubiquitination-regulated steps during Rnf8. Mdc1 contains four TQXF motifs the coiled-coil domain on each protein. the recruitment of Brca1. that may be phosphorylated by ATM Rap80 uses an internal region to associate One important clue to the E3 ligase and recognized by Rnf8. The interaction with Abra1 and depends on Abra1 to as- required for Brca1 recruitment is the spe- of Rnf8 with Mdc1, as well as the re- sociate with Brca1 and Brcc36. Previous cific binding of the UIM domains of cruitment of Rnf8 to DSBs, is disrupted studies also have suggested that the Brca1 Rap80 to Lys-63 (K63)-linked polyubiq- by mutations in the four TQXF motifs A complex contains additional compo- uitin chains (4). To generate polyubiquitin of Mdc1 or the FHA domain of Rnf8 nents, including Bard1 and Brcc45 (2). chains on substrates, an E3 ubiquitin (7–9). Together, these results strongly Because Abra1, Bach1, and CtIP interact ligase must work with an E2 ubiquitin- suggest that Rnf8 is recruited to DSBs with the BRCT domains of Brca1 in a conjugating enzyme. Thus far, Ubc13 is by phosphorylated Mdc1. mutually exclusive manner (1), the Abra1- the only known E2 conjugating enzyme How does Rnf8 generate polyubiquitin containing Brca1 A complex is distinct that supports the formation of K63-linked chains at DSBs? Rnf8 can function with from the Bach1-containing B and CtIP- polyubiquitin chains. In fact, Ubc13 was multiple E2 ubiquitin-conjugating en- containing C complexes. shown to be required for the formation of zymes to form K63- or K48-linked polyu- The studies by Wang and Elledge (4) Brca1 foci (5). Ubc13 is known to interact biquitin chains. Because Ubc13 is the only and the other three groups (7–9) suggest with several E3 ligases (6). By screening known E2 that generates K63-linked an attractive model for the recruitment these E3 ligases with siRNAs, Wang and chains and because both Ubc13 and Rnf8 of Brca1 A complex to DSBs. In the Elledge (4) discovered that Rnf8 is the are required for the formation of Brca1 presence of DSBs, H2AX is phosphory- only E3 required for the formation of foci, Ubc13 and Rnf8 likely function as an lated by ATM in the vicinity of the Brca1 foci. E2–E3 complex at DSBs to catalyze the breaks, allowing Mdc1 to be recruited Independently of Wang and Elledge formation of K63-linked chains. Both his- through a phosphorylation-directed in- (4), three other groups (7–9) also found tones H2A and H2AX are known to be that Rnf8 is required for Brca1 recruit- ubiquitinated in response to DNA damage ment by using different approaches. and are good candidates for the substrates Author contributions: X.H.Y. and L.Z. wrote the paper. From an RNAi screen, Kolas et al. (7) of Rnf8–Ubc13. H2AX, in particular, was The authors declare no conflict of interest. identified Rnf8 as a protein required for shown to be ubiquitinated in a Ubc13- See companion article on page 20759. the formation of 53BP1 foci at DSBs. dependent manner (5). Consistently, ‡To whom correspondence should be addressed. E-mail: Huen et al. (8) and Mailand et al. (9), Mailand et al. (9) and Huen et al. (8) [email protected]. on the other hand, noticed that, in addi- showed that the polyubiquitination of © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710916105 PNAS ͉ December 26, 2007 ͉ vol. 104 ͉ no. 52 ͉ 20645–20646 Downloaded by guest on September 27, 2021 teraction. The localization of Mdc1 to questions. The foremost question is why Furthermore, conflicting results were re- DSBs enables ATM to phosphorylate and how Rnf8 and Brca1, two distinct ported on whether the phosphorylation of Mdc1, thus presenting binding sites for ubiquitin ligases, function together at H2AX is a prerequisite for its polyubiq- the FHA domain of Rnf8. The recruited DSBs. The Brca1-containing E3 ligase can uitination and whether Brca1 is important Rnf8–Ubc13 then generates polyubiq- interact with different E2s, such as Ubc13 for the ubiquitination of H2AX (5, 8, 15). uitin chains at DSBs, which are subse- and UbcH5, to assemble either K63- or How the Brca1 A complex functions at quently recognized by Rap80 in the K6-linked polyubiquitin chains (12–14). DSBs also is unclear. Despite the clear Brca1 A complex. This model not only The Brca1–UbcH5 interaction and the in requirement of Rnf8 for the recruitment provides a mechanism by which Brca1 is vitro E3 ligase activity of Brca1 are stimu- of the Brca1 A complex, depletion of recruited to DSBs but also connects the lated by DNA damage in a Ubc13- Rnf8 did not affect the phosphorylation initial ATM-mediated phosphorylation dependent manner (5), suggesting that of a number of ATM/ATR substrates (9). events to the subsequent ubiquitination both the activity and specificity of Brca1 Furthermore, although CtIP is ubiquiti- cascade catalyzed by Rnf8 and Brca1. are regulated by DSBs. Interestingly, the nated by Brca1 in response to DSBs (16), It is important to note that Rnf8 is UIM domains of Rap80 recognize not it is unlikely a substrate of the Brca1 A required for the foci formation of both only K63-linked but also K6-linked polyu- complex because of the mutually exclusive Brca1 and 53BP1 (7, 9). Unlike the biquitin chains (2), suggesting a possible binding of CtIP and Abra1 to Brca1. Brca1 A complex, 53BP1 is not known role for Brca1 in propagating itself at It is important to note that Brca1 ex- to directly recognize polyubiquitin DSBs. Yet another layer of regulation ists in multiple complexes in cells (1). chains. Instead, 53BP1 uses its tandem may come from the deubiquitinase Bach1, a unique component of the B Tudor domains to bind to histone H4 Brcc36, a component of the Brca1 A com- complex, is implicated in HR (17). CtIP, dimethylated at K20 (11). Because the plex. Biochemical reconstitution of the a unique component of the C complex, methylation of H4 K20 is not induced by Rnf8–Ubc13 and Brca1 A complexes is required for the G2/M checkpoint in DNA damage, it was proposed that the would help to address how Rnf8, Brca1, response to DSBs (16).
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