NBA1 accumulates to DNA damage sites in response to IR. (A) NBA1 localizes to IRIF. U2OS cells that stably express a GFP-NBA1 fusion were treated with 10 Gy IR followed by a 4-h incubation at 37°C. Cells were then extracted with 0.5% Triton X-100, fixed with 3.7% formaldehyde, and stained with an anti-GFP antibody and Hoechst dye. (B) GFP-NBA1 forms IRIF that colocalize with BRCA1 foci. U2OS cells stably expressing GFP-NBA1 protein were treated with 10 Gy IR. After 2h incubation of cells post-IR, cells were fixed and stained with antibodies against GFP and BRCA1. (C) GFP-NBA1 accumulates to laser-induced DNA damage regions that colocalize with γ-H2AX. U2OS cells stably expressing GFP-NBA1 were treated with a laser for microirradiation.

(A) NBA1 is required for proper G2–M regulation in response to IR. U2OS cells were untreated or treated with 3 Gy IR, then incubated for 1 h at 37°C before fixation and p-H3 antibody staining. Two independent experiments were performed with two different pairs of siRNAs to NBA1 and BRCC36. (B) NBA1 is required for optitmal BRCA1 and Rap80 foci formation in response to IR. U2OS cells were transfected with control or siRNAs against NBA1, BRCC36, or BRE for 2 d, then irradiated with 10 Gy IR, incubated for 2 h, fixed and immunostained with the indicated antibodies. (C) The defect in BRCA1 IRIF formation in cells expressing shRNA to NBA1 can be rescued by expression of an shRNA-resistant NBA1 cDNA. U2OS cells expressing NBA1 shRNA#1 were infected with virus expressing NBA1 cDNA lacking the target sequence of shRNA#1, irradiated with 10 Gy IR, incubated for 4 h, fixed, and stained with Brca1 antibodies.

Members of the BRCA1 A complex contain ubiquitin-binding domains. (A) Alignment of the VWA domain of NBA1 and PSMD4/Rpn10. (B) Alignment of the MPN domains from various species of Abra1, Abro1, and other MPN domain-containing proteins. Residues outlined in red are required for enzymatic activity. (C) Alignment of the UEV domain from BRE with the UEV domain of human E2-conjugating enzyme Rad6a and UbcH5. (D) Protein domains present in the components of BRCA1 A complex. (UIM) Ubiquitin-interacting motif; (AIR) Abraxas-interacting region; (MPN−) Mpr1/Pad1 N-terminal region lacking catalytic activity; (MPN+) Mpr1/Pad1 N-terminal region with an intact protease catalytic site; (CC) coiled coil; (UEV) ubiquitin-conjugating enzyme variant domain; (VWA) von Willebrand factor type A domain.

NBA1 is required for radioprotection. (A) Overview of the pool-based shRNA screen for regulators of the DNA double-strand break response in mammalian cells. (B) Identification of NBA1 as a gene required for IR resistance. shRNAs are graphed based on their mean normalized log2 Cy5/Cy3 ratio, with a ratio less than −1 indicating a decrease in abundance greater than twofold. Two shRNAs targeting distinct regions of the NBA1 sequence scored in the screen. Two unrelated shRNAs that were considered unchanged are also shown for comparison. Error bars represent standard deviation across triplicates. (C) Validation of NBA1 shRNAs that lead to increased IR sensitivity. shRNAs were individually packaged into retroviruses and infected into cells in independent triplicates. Cells were either treated with 3 Gy IR or left untreated and cell viability was monitored ∼9 d later. An ATM shRNA that also scored in the screen and led to IR sensitivity is shown for comparison. (D) Validation of the NBA1 IR sensitivity phenotype using siRNAs. Three different pairs of siRNAs against NBA1 gene and BRCC36 gene were transfected into U2OS cells. Cell survival was measured by colony-forming assay. (E) NBA1 cDNA resistant to shRNA rescues IR sensitivity. U2OS cells expressing a control firefly shRNA or NBA1 shRNA#1 was infected with virus expressing a shRNA#1-resistant NBA1 cDNA or a control vector construct and cells were tested for sensitivity to IR.

NBA1 is a BRCA1 A complex component. (A) TAP purification of Flag-HA-NBA1. (B) NBA1 interacts with BRCA1. Immunoprecipitated Flag-HA-tagged NBA1 was fractionated in the gel and probed with antibodies against BRCA1. (C) TAP purification of Flag-HA-Abra1. (D) Endogenous NBA1 interacts with Rap80 and Abra1. Lysates from 293T cells treated with 10 Gy IR follwed with 2 h incubation or untreated were used for immunoprecipitation with antibodies against Abra1 or Rap80, franctionated with SDS-PAGE gel and Western blot was carried with antibodies against Abra1, Rap80, BRE, or NBA1. (E) NBA1 controls BRE and Abra1 abundance. U2OS cells were infected with control firefly (FF) hairpin or shRNA#1 targeting the 3′UTR region of NBA1 or 2 d later infected again with NBA1 cDNA lacking the 3′UTR region. Cell extracts were immunoblotted for Abra1, BRE, and NBA1 protein levels. (F) GFP-tagged NBA1 interacts with each member of the BRCA1-A complex. 293T cells stably expressing GFP-NBA1 or GFP-BRE were used to prepare lysates for the immunoprecipitation with antibodies against GFP. The immunoprecipitates were then separated in SDS-PAGE gel and tested for binding to Rap80, Abra1, and BRCC36 using Western blot and antibodies against Rap80, Abra1, BRCC36, BRE, and NBA1.

NBA1 serves as an adaptor protein for the BRCA1 A complex. (A) NBA1 and BRE proteins interact with the N terminus of Abra1. Cell lysates from 293T cells stably expressing GFP-tagged mutants of Abra1 were immunoprecipitated with GFP antibodies and immunoblot analysis was carried out with antibodies against NBA1, BRE, or BRCC36. (B) A diagram for Abra1 deletion mutants. (C) NBA1–Rap80 interaction, similar to BRCC36–Rap80 interaction, depends on the AIR domain of Rap80 that is required for Rap80–Abra1 interaction. Cell lysates from 293T cells stably expressing GFP-tagged mutants of Rap80 were immunoprecipitated with GFP antibodies and Western blot was carried out with antibodies against NBA1 or BRCC36. (D) A diagram for Rap80 deletion mutants. (E) NBA1 and BRE interaction with BRCC36 is mediated through Abra1. Cell lysates from 293T cells stably expressing GFP-tagged NBA1 or BRE protein were immunoprecipitated with GFP antibodies and Western blot was carried out with antibodies against Abra1, BRE, or BRCC36. (F) A diagram mapping the regions of Abra1 that interacts with different members of the BRCA1-A complex.

BRCA1 A complex components Rap80, Abra1, BRE, and BRCC36 bind to polyubiquitin chains. (A) In vitro assay to test the ability of each member of the BRCA1-A complex to bind ubiquitin chains. The indicated GST-tagged proteins were purified from bacteria and incubated with either K63-linked polyubiquitin chains or K48-linked polyubiquitin chains. After 1 h incubation, the pulled-down proteins were fractionated using SDS-PAGE gel, either stained with Coomassie blue for visulization of the expression of proteins, or immunoblotted with antibodies against ubiquitin. (B) A comparison of protein domains between the BRCA1 A, 19S proteasome, CSN, and eIF3 complexes. (PCI) Proteasome/CSN/IF3 domain.