The Rap80-BRCC36 de-ubiquitinating enzyme complex antagonizes RNF8-Ubc13-dependent ubiquitination events at DNA double strand breaks

Abstract

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...

The Lys63-deubiquitylating Enzyme BRCC36 Limits DNA Break Processing and Repair

“…Depletion of BRCC36 compromised the G 2 /M cell cycle checkpoint, impaired BRCA1 recruitment onto DSBs, resulted in hyperactive homologous recombination (HR)-based gene conversion events and hypersensitized the cell to genotoxic stress (7)(8)(9)(10)(11)(12). Consistent with the idea that dysregulated DNA repair promotes tumorigenesis, aberrant expression of BRCC36 has been associated with breast carcinomas (19) and nasopharyngeal carcinomas (20) and represents a promising prognostic marker and a potential target in the therapies for these cancer types.…”

“…Indeed, BRCC36 resides within the BRCA1-A macromolecular protein complex, which comprises the ubiquitin-binding protein RAP80, BRCC45/BRE, Abraxas/CCDC98, Merit40/NBA1, and BRCA1. The BRCA1-A complex is targeted to chromatin domains surrounding DNA double strand breaks (DSBs) by the ubiquitin-binding protein RAP80, which preferentially binds to K63-Ub adducts generated by the ubiquitin-conjugating enzyme UBC13 (3,(7)(8)(9)(10)(11)(12). Abraxas plays a major scaffolding role to support the integrity of the BRCA1-A assembly and directly anchors the tumor suppressor BRCA1 via a phosphorylation-dependent interaction (13)(14)(15).…”

NBA1/MERIT40 and BRE Interaction Is Required for the Integrity of Two Distinct Deubiquitinating Enzyme BRCC36-containing Complexes

“…NBA1 deficiency leads to decreased levels of Abraxas, BRE, and BRCC36 (16,17,20). In this study, we found that NBA1 and BRE interaction plays an important role in maintaining the integrity of both of the BRCC36-containing complexes.…”

Deubiquitylases From Genes to Organism

“…The activity of POH1 is enigmatic. Based on in vitro enzymatic and structural studies of AMSH, BRCC3, and POH1, it has been proposed that the JAMM family proteins may collectively possess a stringent specificity for Lys63 ubiquitin chain linkages (44,45,123,171,225,235). However, elegant preceding work suggested that POH1 activity on proteasomal substrates 1) indirectly requires ATPase activity presumably for unfolding of the substrate, 2) is coupled to proteasomal degradation, and 3) completely removes ubiquitin by cleavage at the base of the ubiquitin chain (266,284).…”