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

Abstract

Multisubunit protein assemblies offer integrated functionalities for efficient cell signal transduction control. One example of such protein assemblies, the BRCA1-A macromolecular complex, couples ubiquitin recognition and metabolism and promotes cellular responses to DNA damage. Specifically, the BRCA1-A complex not only recognizes Lys 63 -linked ubiquitin (K63-Ub) adducts at the damaged chromatin but is endowed with K63-Ub deubiquitylase (DUB) activity. To explore how the BRCA1-A DUB activity contributes to its function at DNA double strand breaks (DSBs), we used RNAi and genome editing approaches to target BRCC36, the protein subunit that confers the BRCA1-A complex its DUB activity. Intriguingly, we found that the K63-Ub DUB activity, although dispensable for maintaining the integrity of the macromolecular protein assembly, is important in enforcing the accumulation of the BRCA1-A complex onto DSBs. Inactivating BRCC36 DUB attenuated BRCA1-A functions at DSBs and led to unrestrained DSB end resection and hyperactive DNA repair. Together, our findings uncover a pivotal role of BRCC36 DUB in limiting DSB processing and repair and illustrate how cells may physically couple ubiquitin recognition and metabolizing activities for fine tuning of DNA repair processes. Active hydrolysis of ubiquitin polymers by deubiquitylases (DUBs)2 has emerged as important biochemical processes that underlie diverse signal transduction pathways, including cell responses to DNA damage (1). To date, around 94 human DUBs have been identified and are classified into five families: ubiquitin C-terminal hydrolases, ubiquitin-specific proteases, ovarian tumor proteases, Josephins, and JAB1/MPN/MOV4 metalloproteases. Notably, the JAB1/MPN/MOV4 metalloprotease family members are zinc-dependent metalloproteases, and the other families are cysteine proteases (1).BRCC36, originally reported as the BRCA1/BRCA2-containing complex subunit 36 (2), is endowed with DUB activity for lysine 63-linked ubiquitin polymers (K63-Ub). BRCC36 DUB relies on its zinc-dependent JAB1/MPN/MOV4 metalloprotease domain (3) and its interaction with Abraxas/KIAA0157 (3-5). Although BRCC36 forms distinct nuclear and cytoplasmic complexes (5, 6) and plays pleiotropic roles during cell proliferation, the K63-Ub DUB is arguably best known for its strong links with the breast and ovarian susceptible gene product BRCA1 in DNA damage responses (DDRs). 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, 7-12). 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 (13-15). Indeed, BRCA1, via it...

Three-Dimensional Architecture of the Human BRCA1-A Histone Deubiquitinase Core Complex

“…In addition to the heterotrimeric species, partial dissociation was observed, enabling a pairwise interaction map to be generated. These data clearly show that incorporation of BRCC45 and MERIT40 into the BRCA1-A complex is independent of BRCC36, as reported previously (Ng et al., 2016), and are consistent with a linear association, whereby BRCC45 forms a bridge between MERIT40 and the Abraxas scaffold. The VWA (von Willebrand factor type A) domain core of MERIT40 was therefore modeled into the terminal regions of the arm segments using the crystal structure of the homologous domain from Rpn10 (PDB: 2X5N; Figure S3A).…”

Mitochondrial DNA oxidation induces imbalanced activity of NLRP3/NLRP6 inflammasomes by activation of caspase-8 and BRCC36 in dry eye

“…BRCC36 is known to participate in DNA damage response in the BRCA1-A complex recruited by the chromatin at double-strand break sites, as well as in the cytosolic BRISC complex. Recently BRCC36 is also known as a critical regulator of NLRP3 deubiquitination and inflammasome activation [46, 47]. Thus we hypothesize that mtDNA oxidative damage may stimulate BRCC36 activity to priming and activate NLRP3 inflammasome, an innate immune response to environmental stress via ROS-mediated oxidative injury.…”

ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair

“…ZMYM3 accumulates at DNA damage sites, promotes survival to DSB-inducing agents, and regulates DSB repair by HR. Depletion of the BRCA1-A complex members RAP80, ABRA1, and BRCC36 leads to hyperresection, increased HR efficiency (Coleman and Greenberg 2011;Hu et al 2011b;Ng et al 2016), and defective BRCA1 IRIF formation (Chen et al 2006;Kim et al 2007b;Liu et al 2007;Feng et al 2009;Shao et al 2009;Wang et al 2009;Hu et al 2011a). The loss of ZMYM3 also reduces BRCA1 IRIFs but, unlike other BRCA1-A complex members, reduces HR repair.…”