DNA double strand breaks (DSBs) initiate reversible cellular checkpoint and repair activities. Whereas many of the activating events at DSBs have recently been elucidated, the mechanisms used to terminate responses at these sites are largely undefined. Here we report a pathway required to reverse RNF8-Ubc13 dependent ubiquitination events on chromatin flanking DSBs. Inhibition of the Rap80-BRCC36 de-ubiquitinating enzyme complex partially restored DSB-associated ubiquitin levels following RNF8 knockdown or proteasome inhibition. Similarly, BRCC36 knockdown or expression of a BRCC36 de-ubiquitinating enzyme-inactive mutant rescued both 53BP1 recruitment to DSBs and ionizing radiation-induced ␥H2AX ubiquitination following RNF8 depletion, and mitigated ionizing radiation sensitivity resulting from RNF8 deficiency. Thus, concomitant and opposing RNF8-Ubc13 ubiquitin ligase and Rap80-BRCC36 ubiquitin hydrolysis activities are responsible for determining steady-state ubiquitin levels at DNA DSBs. These findings reveal a Rap80-BRCC36 dependent pathway that is required for appropriate DSB recruitment and repair responses.BRCA1 ͉ DNA damage ͉ ubiquitin M ultiple, parallel signaling pathways converge upon DNA double-strand breaks (DSBs) to initiate temporal and spatial coordination of checkpoint and repair responses. These DNA damage response activities are mediated in part by accumulation of DNA repair proteins at both chromatin and non-nucleosomal DNA regions flanking DSBs (1-3). Repair factor targeting occurs within minutes of DSB induction, providing strong evidence that molecular recognition events rapidly develop at sites of DNA damage (1,4,5). Following the resolution of DNA damage, repair proteins dissociate from DSBs, thus alleviating cell cycle checkpoint responses and allowing resumption of cell proliferation. It is unclear, however, if DSB termination and activation pathways occur simultaneously in an equilibrium process during the earliest stages of repair, or if DNA damage response resolution occurs in a step-wise manner subsequent to the completion of DNA repair.Some of the recruitment events at DSBs have recently been elucidated for the breast and ovarian cancer suppressor protein BRCA1 (Breast Cancer 1, early onset) and the checkpoint and repair protein 53BP1 (p53 Binding Protein 1) (6-12). Histone H2AX phosphorylation by the PI3K-like kinase members ATM (Ataxia Telangiectasia Mutated) and DNA-PKcs (DNADependent Protein Kinase catalytic subunit) occurs at chromatin flanking DSBs to initiate a direct interaction between phosphorylated serine 139 of H2AX (␥H2AX) and the MDC1 (Mediator of DNA Damage Checkpoint Protein 1) protein (13-15). PI3K-like kinase phosphorylation of MDC1 (13, 16) creates a binding site for the E3 ubiquitin ligase RNF8 (Ring Finger 8). In conjunction with the E2 enzyme Ubc13 (ubiquitin-conjugating 13), RNF8 ubiquitinates histones H2A and H2AX in a K63-linked manner to create a docking site for DNA repair proteins to accumulate at DSBs (6-8, 17). These DSB chromatin-associated K63-link...

Rap80 targets the breast cancer suppressor protein BRCA1 along with Abraxas and the BRCC36 deubiquitinating enzyme (DUB) to polyubiquitin structures at DNA double-strand breaks (DSBs). These DSB targeting events are essential for BRCA1-dependent DNA damage response-induced checkpoint and repair functions. Here, we identify MERIT40 (Mediator of Rap80 Interactions and Targeting 40 kD)/(C19orf62) as a Rap80-associated protein that is essential for BRCA1-Rap80 complex protein interactions, stability, and DSB targeting. Moreover, MERIT40 is required for Rap80-associated lysine 63 -ubiquitin DUB activity, a critical component of BRCA1-Rap80 G2 checkpoint and viability responses to ionizing radiation. Thus, MERIT40 represents a novel factor that links BRCA1-Rap80 complex integrity, DSB recognition, and ubiquitin chain hydrolytic activities to the DNA damage response. These findings provide new molecular insights into how BRCA1 associates with independently assembled core protein complexes to maintain genome integrity.[Keywords: C19orf62; HSPC142; MERIT40; BRCA1; Rap80; Abraxas; BRCC36] Supplemental material is available at http://www.genesdev.org.

SUMMARY Lysine63-linked ubiquitin (K63-Ub) chains represent a particular ubiquitin topology that mediates proteasome-independent signaling events. The deubiquitinating enzyme (DUB) BRCC36 segregates into distinct nuclear and cytoplasmic complexes that are specific for K63-Ub hydrolysis. RAP80 targets the five-member nuclear BRCC36 complex to K63-Ub chains at DNA double-strand breaks. The alternative four-member BRCC36 containing complex (BRISC) lacks a known targeting moiety. Here we identify Serine Hydroxymethyltransferase (SHMT) as a heretofore-unappreciated component that fulfills this function. SHMT directs BRISC activity at K63-Ub chains conjugated to the type 1 interferon (IFN) receptor chain 1 (IFNAR1). BRISC-SHMT2 complexes localize to and deubiquitinate actively engaged IFNAR1, thus limiting its K63-Ub mediated internalization and lysosomal degradation. BRISC deficient cells and mice exhibit attenuated responses to IFN and are protected from IFN-associated immunopathology. These studies reveal a novel mechanism of DUB regulation, and suggest a therapeutic use of BRISC inhibitors for treating pathophysiologic processes driven by elevated IFN responses.

BRCC36 is a JAMM (JAB1/MPN/Mov34 metalloenzyme) domain, lysine 63-ubiquitin (K63-Ub)-specific deubiquitinating enzyme (DUB) and a member of two protein complexes: the DNA damage-responsive BRCA1-RAP80 complex, and the cytoplasmic BRCC36 isopeptidase complex (BRISC). The presence of several identical constituents in both complexes suggests common regulatory mechanisms and potential competition between K63-Ub-related signaling in cytoplasmic and nuclear compartments. Surprisingly, we discover that BRCC36 DUB activity requires different interactions within the context of each complex. Abraxas and BRCC45 were essential for BRCC36 DUB activity within the RAP80 complex, whereas KIAA0157/Abro was the only interaction required for DUB activity within the BRISC. Poh1 also required protein interactions for activity, suggesting a common regulatory mechanism for JAMM domain DUBs. Finally, BRISC deficiency enhanced formation of the BRCA1-RAP80 complex in vivo, increasing BRCA1 levels at DNA double strand breaks. These findings reveal that JAMM domain DUB activity and K63-Ub levels are regulated by multiple mechanisms within the cell.The mammalian genome is remarkably stable despite an estimated 10 5 mutagenic events/cell cycle (1). This exquisite fidelity can be attributed to the multiple and varied activities of the DNA damage response (DDR). 4 To maintain genome integrity, eukaryotic cells activate the DDR, a complex signaling network that integrates and coordinates DNA damage recognition, cell cycle checkpoints, and DNA repair (2, 3). Recent evidence implicates ubiquitin chain formation, recognition, and breakdown at the site of the genomic lesion as an essential component of the DDR. Ubiquitin is a 76-amino acid protein that can be attached to a target protein via an isopeptide linkage between the ⑀-amino lysine residue of a target protein and the C-terminal glycine residue within ubiquitin. Protein ubiquitination can have quite complex outcomes resulting from the considerable structural information embedded within ubiquitin polymers. Specifically, a single ubiquitin monomer can be extended through the ubiquitination of any one of seven lysines or through the N terminus, creating polyubiquitin chains (4). These different ubiquitin topologies result in the formation of diverse structures resulting in vastly different biological outcomes. The canonical Lys 48 -linked polyubiquitination of proteins signals for proteasomal degradation (5); conversely, Lys 63 -linked polyubiquitin has been implicated in non-degradative signals in response to both cytoplasmic and nuclear cues. Specifically, Lys 63 -linked polyubiquitin is involved in both the recruitment and retention of DNA repair factors at sites of DNA damage (6 -8).BRCA1 is central to the DDR and forms a number of mutually exclusive macromolecular complexes, each with discrete activities (9, 10). Recently, it has been shown that the core RAP80 complex plays an important role in facilitating BRCA1 localization to DNA double strand breaks (DSBs). The RAP80 complex is a five-...