Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage

Abstract

The Brca1 A complex contains Brca1/Bard1, Abraxas, Rap80, and Brcc36; however, with the exception of the Brca1-Abraxas interaction, how the A complex is assembled is not known. The A complex is localized to sites of DNA damage through the UIM domains of RAP80, which bind K63-linked polyubiquitin chains. In this study, we identified an FHA domain RING finger E3 ubiquitin ligase, RNF8, and an E2-conjugating enzyme known to form K63-polyubiquitin chains, Ubc13, each of which is required to recruit the Brca1 A complex to sites of DNA damage. Rnf8 localizes to sites of DNA damage through an FHA-domain-containing region. We found that Rap80 contains an Abraxas interaction domain [AIR (Abraxas-interacting region)], required for association of Rap80 with Abraxas, Brca1, and Brcc36. Abraxas and Brcc36 associate through coiled-coil domains on each protein. These data suggest a model through which Ubc13 and Rnf8 are recruited to sites of DNA damage through DNA-damage-induced phosphorylation of a chromatin-associated protein and generate polyubiquitin chains that then recruit Rap80 and the entire Brca1 A complex to DNA-damage foci. This sequential E3 ubiquitin ligase recruitment constitutes a ubiquitin ligase cascade required for DNA repair and checkpoint signaling.

Fig. 1.

UBC13 and RNF8 are responsible for DNA-damage-induced foci formation of the Brca1 A complex. (A) U2OS cells were transfected with control or siRNA oligos against UBC13 or RNF8 for 48 h, irradiated with 10 Gy, and incubated at 37°C for 2 h before being fixed and immunostained with antibodies against Rap80, Abraxas, ubiquitin (FK2), 53BP1, and Brca1. Three different siRNA oligos against each gene were used, and similar results were obtained. (B) U2OS cells were untreated or treated with 10 Gy IR. Two hours later, cells were fixed and immunostained with antibodies against Rnf8 and Brca1. (C) Mutants of RNF8 were generated as a GFP-SV40 NLS N-terminal Rnf8 fusion protein. Plasmids expressing these deletion mutants were transiently expressed in U2OS cells for 48 h. Cells were then irradiated at 10 Gy and incubated for 2 h before being fixed and stained with antibodies against GFP.

Fig. 2.

Rap80 interacts with Abraxas through the AIR domain, and both UIM and AIR domains of Rap80 are required for optimal Rap80 foci formation. (A) The indicated Rap80 mutants were generated in a GFP–SV40 NLS N-terminal Rap80 fusion construct and used to examine foci formation as well as Abraxas and Brcc36 binding. (B) U2OS cells stably expressing GFP-tagged Rap80 and mutant proteins were either untreated or treated with IR, incubated at 37°C for 2 h, fixed, and stained with antibodies against GFP. At least 400 cells were counted for quantification. Cells that contained >10 foci were counted as positive. (C) 293T cells were transiently transfected with plasmids expressing the GFP-tagged Rap80 fusion proteins in A. After 40 h, cell lysates were prepared and immunoprecipitated (IP) with antibodies against Abraxas. WB indicates Western blot.

Fig. 3.

Rap80 and Abraxas are mutually dependent for DNA-damage-induced foci formation. (A) U2OS cells were transfected with control or siRNA oligos against Rap80 or Abra1 for 48 h, irradiated with 10 Gy, and incubated at 37°C for 2 h before being fixed and immunostained with antibodies against Rap80, Abraxas, and Brca1. Three different siRNA oligos against each gene were used, and similar results were obtained. (B) Abra1 WT and mutants D1–4 were generated in a GFP–N-terminal Abra1 fusion construct, and mutants D5–10 and CC were generated in a GFP–SV40 NLS N-terminal Abra1 fusion construct and used to examine foci formation and Rap80 and Brcc36 binding. The SV40 NLS signal is indicated as a black rectangle. (C) 293T cells were transiently transfected with plasmids expressing cDNAs for GFP-tagged Abraxas and its mutants from B. After 40 h, cell lysates were prepared and immunoprecipitated with antibodies against Rap80. The asterisk indicates the correct size for the expressed fusion proteins.

Fig. 4.

Abraxas mediates the interaction of Brcc36 with Rap80. (A) BRCC36 affects IRIF formation of Rap80, Abraxas, and BRCA1 proteins. U2OS cells were transfected with control oligos or siRNA oligos against BRCC36 for 48 h, irradiated with 10 Gy, and incubated at 37°C for 2 h before being fixed and immunostained with antibodies against Rap80 and Brca1. Three different siRNA oligos against BRCC36 were used, and similar results were obtained (data not shown). (B) Brcc36 associates with Rap80 and Abraxas. Cell lysates from 293T cells treated or not treated with 10 Gy IR were incubated for 2 h and then immunoprecipitated with antibodies against Rap80, Abraxas, or control rabbit IgG. These immunoprecipitates were then fractionated by SDS/PAGE and immunoblotted with anti-Brcc36, Rap80, or Abraxas antibodies. (C) The AIR domain of Rap80 and the coiled-coil domain of Abraxas are required for binding to Brcc36. 293T cells were transiently transfected with plasmid constructs expressing GFP-fused mutant proteins of Rap80 and Abraxas from Figs. 2 and 3. After 40 h, cell lysates were prepared and immunoprecipitated with antibodies against GFP. Immunoblots were carried out with antibodies against Brcc36 or GFP. The asterisk indicates the GFP fusion proteins. (D) The Brcc36 and Rap80 interaction is decreased in Abra1 siRNA-treated cells. 293T cells were transfected with control oligos or siRNA oligos against Abra1 for 48 h; cells lysates were then prepared and immunoprecipitated with antibodies against Rap80. The immunoprecipitates were then fractionated in SDS/PAGE gel and blotted with antibodies against Brcc36 or Rap80. The input protein level was also measured for Brcc36 and Abraxas by using WB. (E) The coiled-coil domain of Brcc36 is required for binding to Abraxas. Plasmid constructs expressing control empty vector or fusion proteins GFP–Brcc36 (1–316 aa) or GFP–Brcc36ΔC (1–249 aa) were transiently transfected into 293T cells for 36 h. Cell lysates were prepared and immunoprecipitated with antibodies against Abraxas and immunoblotted with antibodies against GFP.

Fig. 5.

A model describing the role of Ubc13 and Rnf8 in the generation of ubiquitin chains that recruit the Brca1 A complex to sites of DNA damage. See the text for details. Question marks indicate uncertainty in the target of the Rnf8 FHA domain and the K63 polyubiquitin chain receptor responsible for Rap80 binding.