TRAIP/RNF206 is required for recruitment of RAP80 to sites of DNA damage

RAP80 localizes to sites of DNA insults to enhance the DNA-damage responses. Here we identify TRAIP/RNF206 as a novel RAP80-interacting protein and find that TRAIP is necessary for translocation of RAP80 to DNA lesions. Depletion of TRAIP results in impaired accumulation of RAP80 and functional downstream partners, including BRCA1, at DNA lesions. Conversely, accumulation of TRAIP is normal in RAP80-depleted cells, implying that TRAIP acts upstream of RAP80 recruitment to DNA lesions. TRAIP localizes to sites of DNA damage and cells lacking TRAIP exhibit classical DNA-damage response-defect phenotypes. Biochemical analysis reveals that the N terminus of TRAIP is crucial for RAP80 interaction, while the C terminus of TRAIP is required for TRAIP localization to sites of DNA damage through a direct interaction with RNF20–RNF40. Taken together, our findings demonstrate that the novel RAP80-binding partner TRAIP regulates recruitment of the damage signalling machinery and promotes homologous recombination.

. TRAIP foci were found in PML bodies in the absence of DNA damage (A) 293T cells were transfected with the Myc-TRAIP expression vector. After 24 hrs, transfected cells were fixed and immunofluorescence assays using anti-Myc and anti-PML antibodies. DAPI was used as an indicator for the nucleus. (B) 293T cells were transfected with indicated expression plasmid. After 24 hr, transfected cells were fixed and immunofluorescence assays using anti-Myc and -Flag antibodies. DAPI was used as an indicator for the nucleus.
Supplementary Fig. 5. TRAIP is not involved in RNF8, RNF168-mediated DNA damage response. (A and B) SFB-RNF8 or SFB-RNF168 was transfected in 293T cells with TRAIP depletion. After 48 hrs, transfected 293T cells were exposed to 10 Gy of ionizing radiation. Four hour after irradiation, cells were fixed and stained with indicated antibodies. 4′,6-Diamidino-2-phenylindole (DAPI) was used as an indicator for the nucleus. The results represent the average of two independent experiments. Error bars indicate the standard deviation for each expression plasmid transfected cells. (C) GFP-TRAIP expression vector is transfected in U2OS cells with RNF8 depletion. After 48 hours incubation, the cells were subjected to laser microirradiation assay. Laser stripes were examined at the indicated times. Error bars indicate the standard deviation (N=10).
Supplementary Fig. 6. TRAIP is important for BRCA1-A complex recruitment to the sites of DNA lesions. GFP-CCDC98 (A) or GFP-Merit40 (B) was transfected in 293T cells depleted with TRAIP. After 48 hrs, transfected 293T cells were exposed to 10 Gy of ionizing radiation. Four hours after irradiation, cells were fixed and stained with H2AX antibodies. DAPI was used as an indicator for the nucleus. The results represent the average of three independent experiments. Error bars indicate the standard deviation.  Supplementary Fig. 14.  Supplementary Fig. 11. Western blot analysis for TRAIP, RNF20 or RNF40 expression in HeLa cells transfected with the control, TRAIP, RNF20 or RNF40 siRNA. The indicated siRNAs were transfected into HeLa cells. After 48 hrs, transfected cell lysates were subjected to Western blotting analysis using the indicated antibodies. See full blots in the Supplementary Fig. 14.  Supplementary Fig. 12. The functional analysis of RNF20 on DNA damage response pathway. (A) Depletion of RNF20 leads to checkpoint defects. HeLa cells transfected with indicated siRNA were exposed to 0 or 2 Gy of ionizing radiation. Cells were incubated for 1 hr and then fixed and subjected to staining with antibody to phosphorylated histone H3 (pH3) and propidium iodide. The percentages of mitotic cells were determined by fluorescence-activated cell sorting analysis. The results represent the average of two independent experiments. Error bars indicate the standard deviation. (B) Depletion of RNF20 leads to radiation sensitivity. HeLa cells were transfected with control or RNF20 siRNAs. 48 hours after transfection, 500 cells were plated and irradiated with indicated doses of ionizing radiation. The number of surviving colonies was counted 14 days later. These experiments were performed in triplicate, and the results represent the average of three independent experiments. (C) Depletion of RNF20 reduced the homologous recombination. DR-GFP U2OS cells were transfected with indicated siRNA. 48 hours after transfection, I-SceI expression vector was transfected. 3 days later, GFP positive cells were analyzed by flowcytometry. (D-F) 293T cells were transfected with control or RNF20 siRNAs. After 48 hr transfected 293T cells were exposed to 10 Gy of ionizing radiation. Four hours after irradiation, cells were fixed and stained with anti-RAP80, -BRCA1, -53BP1or -H2AX antibodies. DAPI was used as an indicator for the nucleus. The results represent the average of three independent experiments. Error bars indicate the standard deviation. Supplementary Fig. 13. TRAIP antibody specificity for immunofluorescent assay. HeLa cells were transfected with indicated siRNA. After 48 hours, transfected HeLa cells were fixed and stained with anti-TRAIP antibody. DAPI was used as an indicator for the nucleus.
Supplementary table 1. The list of 37 positive clones with the highest-galactosidase activity from the yeast two-hybrid screen with the full-length RAP80 as bait and a human HeLa cDNA library as prey. Clones containing the TRAIP cDNA were denoted by red font.

AD-Hybrid -2
The activation domain (AD) is fused in frame to the 68 th aa of complement component 1, q subcomponent binding protein (C1QBP), nuclear gene encoding mitochondrial protein (NM_001212).

AD hybrid -9
The activation domain (AD) is fused in frame to the 3 rd aa of cleavage and polyadenylation specific factor 6, 68kDa (CPSF6) (NM_007007).

AD-Hybrid -10
The activation domain (AD) is fused in frame to the 21 st aa of cleavage and polyadenylation specific factor 6, 68kDa (CPSF6) (NM_007007).

AD-Hybrid -11
The activation domain (AD) is fused in frame to the 13 th aa of complement component 1, q subcomponent binding protein (C1QBP), nuclear gene encoding mitochondrial protein (NM_001212).

AD-Hybrid -13
The activation domain (AD) is fused in frame to the 69 th aa of complement component 1, q subcomponent binding protein (C1QBP), nuclear gene encoding mitochondrial protein (NM_001212).

AD-Hybrid -14
The activation domain (AD) is fused in frame to the 30 th aa of complement component 1, q subcomponent binding protein (C1QBP), nuclear gene encoding mitochondrial protein (NM_001212).

AD hybrid -17
The activation domain (AD) is fused in frame to the 70 th aa of TRAF interacting protein (TRAIP) (NM_005879).