Abraxas and RAP80 Form a BRCA1 Protein Complex Required for the DNA Damage Response

Abstract

The BRCT repeats of BRCA1 are essential for tumor suppression. Phospho-peptide affinity proteomic analysis identified a novel protein, Abraxas, that directly binds the BRCA1 BRCT repeats through a phospho-SXXF motif. Abraxas binds BRCA1 mutually exclusively with BACH1 and CTIP, forming a third Brca1 complex. Abraxas recruits the ubiquitin-interacting motif (UIM) containing protein Rap80 to BRCA1. Both Abraxas and Rap80 are required for DNA damage resistance, G2/M checkpoint control and DNA repair. Rap80 is required for a subset of Brca1-foci formation in response to IR and the UIM domains alone are capable of foci formation. The Rap80/Abraxas complex may help recruit Brca1 to DNA damage sites in part through recognition of ubiquitinated proteins.The Brca1 tumor suppressor is associated with hereditary breast and ovarian cancer and has significant roles in DNA repair, cell cycle checkpoint control and maintenance of genomic stability (1, 2). Brca1 contains a N-terminal RING domain, a SQ cluster domain phosphorylated by DNA damage signaling kinases ATM/ATR(3), and two C-terminal BRCT repeats. Brca1 forms a heterodimeric complex with Bard1 (4) that exhibits E3 ubiqutin ligase activity(5-7). Many tumorigenic mutations in BRCA1 disrupt the BRCT repeats which constitute a phospho-peptide recognition domain that binds peptides containing a pSxxF motif (8-11).To identify proteins that bind the BRCT domains of Brca1, we combined peptide affinity purification and stable isotope labeling with amino acids in cell culture (SILAC) (12-14) to identify phospho-peptides that directly bind to the BRCT domain of Brca1 and to quantify their abundance in the presence of DNA damage by mass spectrometry. Proteins from cells grown in heavy medium and cells grown in light medium treated with 10 Gy IR were prepared, mixed (1:1) and digested with Trypsin. Tryptic peptides bound to Gst-Brca1-BRCT were identified by mass spectrometry analysis. We searched for phospho-peptides that contained pSXXF motif and compared the peptides to the list of proteins we recently identified in an ATM/ATR substrate screen (Matsuoka et al., in preparation (Fig. 1B). We named this protein Abraxas for the Greek god and the gene Abra1. SILAC quantification analysis also indicated a doubly phosphorylated peptide, GEGFYS#RS#PTF containing p-S404 and p-S406, was enriched approximately 8-fold after DNA damage (Fig. S1). Both p-S406, and doubly, p-S404 p-S406, phosphorylated Abraxas peptides bound purified GST-Brca1-BRCT, while p-S404 and unphosphorylated peptides did not (Fig. 1C), a result confirmed using BIACORE analysis (Fig. S2A).Abraxas is well conserved in vertebrates (Fig. S3). Bioinformatics analysis also revealed KIAA0157, which is 39% identical to Abra1 in the N-terminal two-thirds of the protein (AA 1-260) we call the "ABR" domain. This protein, now named Abro1 (Abraxas Brother 1) ( Fig. 1D), is also conserved in vertebrates ( Fig. S3) but lacks the pSXXF motif and does not bind to Brca1 (data not shown).Full length Abraxas binds the BRCT-repeats...

Degradation of Human RAP80 is Cell Cycle Regulated by Cdc20 and Cdh1 Ubiquitin Ligases

“…The BRCA1-A complex contains many subunits, including a coiled coil domain-containing protein (CCDC98/ABRAXAS), the BRCA1/BRCA2-containing complex subunit 45/brain and reproductive organ-expressed protein (BRCC45/BRE), the BRCA1/BRCA2-containing complex subunit 36 (BRCC36), mediator of RAP80 interactions and targeting subunit of 40/new component of the BRCA1-A complex (MERIT40/NBA1), and breast cancer 1 (BRCA1; refs. [6][7][8][9][10][11][12][13][14]. This recruitment is required for the activity of BRCA1 at the DNA damage checkpoint and for DNA repair.…”

“…Receptor-associated protein 80 (RAP80) plays an important role in signal transduction in the DNA damage response by recruiting the BRCA1-A complex to DNA damage sites in a lysine 63-mediated polyubiquitin-binding dependent manner (6)(7)(8)(9)(10)(11)(12)(13)(14). The BRCA1-A complex contains many subunits, including a coiled coil domain-containing protein (CCDC98/ABRAXAS), the BRCA1/BRCA2-containing complex subunit 45/brain and reproductive organ-expressed protein (BRCC45/BRE), the BRCA1/BRCA2-containing complex subunit 36 (BRCC36), mediator of RAP80 interactions and targeting subunit of 40/new component of the BRCA1-A complex (MERIT40/NBA1), and breast cancer 1 (BRCA1; refs.…”

A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

“…18,19 53BP1 recognizes dimethylated histone H4 lysine 20 through its Tudor domain and histone H2A K15 monoubiquitinated by RNF168 via a newly identified ubiquitination-dependent recruitment (UDR) motif. 20,21 The ability of 53BP1 to bind efficiently also relies on chromatin reorganization mediated by ubiquitin-dependent removal of specific chromatin proteins in order to reveal dimethylated K20 of histone H4.…”

BRCA1, PARP, and 53BP1: conditional synthetic lethality and synthetic viability

“…BRCA1 interacts with multiple proteins involved in DNA repair and tumorigenesis, including, PALB2, BRIP1/BACH1, CtIP, TOPBP1, Abraxas, and RAP80 and is present in multiple distinct complexes (Wong et al, 1998;Yu et al, 1998;Cantor et al, 2001;Greenberg et al, 2006;Xia et al, 2006;Kim et al, 2007;Sobhian et al, 2007;Wang et al, 2007;Zhang et al, 2009b). Thus, BRCA1 may function partly as a scaffold protein coordinating the assembly of protein complexes involved in mediating HR-mediated DSBR and checkpoint function (Greenberg et al, 2006).…”

“…Thus, the BRCT domains of BRCA1 likely mediate critical interactions required for assembly of protein complexes required for proper DNA repair function and tumor suppression. This includes interaction of BRCA1 BRCT domains with: (i) BACH1/BRIP1 and TOBP1 (Cantor et al, 2001;Greenberg et al, 2006); (ii) Abraxas and RAP80 (Kim et al, 2007;Liu et al, 2007b;Wang et al, 2007); and (iii) CtIP and the MRE11/NBS/RAD50 complex (Wong et al, 1998;Sartori et al, 2007;Chen et al, 2008).…”