Fig 2: BRUCE UBC domain is required for BRIT1 deubiquitination and its repair foci formation post the formation of the BRUCE-USP8-BRIT1 nuclear complex.(A) BRUCE UBC domain is needed for BRIT1 foci formation. U2OS-shBRUCE cells expressing BRUCE variants as indicated to the left were treated with DOX to knockdown endogenous BRUCE. Cells were irradiated (5 Gy), fixed and immunofluorescence stained with BRIT1 antibody. Bars, 10 μm. (B) Quantification of the BRIT1 foci results in (A). Cells with positive BRIT1 foci (more than five nuclear foci) were quantitated and a total of more than two hundred cells were counted for each sample. (C) UBC mutation or deletion does not affect the binding of BRUCE with USP8 and BRIT1. U2OS-shBRUCE cell lines with stable expression of WT, UBC C4666A mutant, or ΔUBC BRUCE (all FLAG tagged) as indicated on to top were co-transfected with GFP-BRIT1 and Myc-USP8 constructs. BRUCE was isolated from the cell lysates by IP of FLAG followed by immunoblotting of the IP complex with antibodies indicated to the right. (D) UBC domain is required for BRIT1 deubiquitination. U2OS-shBRUCE cells were untreated or treated with DOX followed by transfection with GFP-BRIT1 and My-ubiquitin (K48R). After irradiation (5 Gy), cells were subject to immunoprecipitation with anti-Myc antibody to pulldown total ubiquitinated proteins, among which ubiquitinated BRIT1 products were detected by immunoblotting with an anti-GFP antibody. (E) UBC domain is needed for chromatin relaxation induced by DNA damage. U2OS-shBRUCE cells with expression of exogenous BRUCE and BRUCE variants as indicated to the top were treated with DOX to knock down endogenous BRUCE. After irradiation (5 Gy), cells were subject to micrococcal nuclease digestion assay. Chromatin relaxation was monitored by the release of nucleosomes. Mono- (*), di- (**) and tri-nucleosomes (***) are indicated.

Fig 3: Impaired cytokinesis induced by MKLP1 depletion do not impair DNA repair.(A) Immunofluorescence staining of pATM (green) and centrosomes by ?-tubulin antibody (red) in U2OS-shBRUCE cells before and after DOX treatment with cell nucleus counterstained with DAPI. Bars, 10 µm. (B) and (C) Quantification of defective cytokinesis in irradiated shBRUCE-U2OS cells (5 Gy) with or without BRUCE depletion induced by Dox treatment, scored by counting bi- and multi-nucleated cells (B) and centrosome amplification by ?-tubulin staining (C). (D) Western blotting showing knockdown of MKLP1 by siRNA at 48 hrs post transfection of U2OS cells. (E) and (F) Quantification of cytokinesis defects in cells depleted of MKLP1 scored by bi- and multi-nucleated cells (E) and centrosome amplification (F). (G) and (H) immunofluorescence staining of pATM (green) and centrosomes (red) with cell nucleus counterstained with DAPI (blue) of shBRUCE-U2OS cells treated with siRNA targeting MKLP1 (G) and the pATM foci results were quantified in (H). Bars, 10 µm. Error bars represent standard deviation from a triplicate of a representative experiment. (I) and (J) immunofluorescence staining of BRIT1 (green) and centrosomes (red) with cell nucleus counterstained with DAPI (blue) of U2OS cells treated with the siRNA targeting MKLP1 (I) and the BRIT1 foci results were quantified in (J). Bars, 10 µm. Error bars represent standard deviation from a triplicate of a representative experiment.

Fig 4: Truncated N-BRUCE or C-BRUCE cannot support BRIT1 foci formation in irradiated U2OS cells.(A) Diagram depicting plasmid constructs expressing human full length (FL) BRUCE (4857 amino acids), N-BRUCE (aa 1–2025), and C-BRUCE (aa 2024–4857) with BIR and UBC domains indicated. (B) BRUCE restores formation of IR-induced BRIT1 foci. DOX-inducible shBRUCE-stable-expression U2OS cells (U2OS-shBRUCE, [24]) were transiently transfected with a FLAG vector or a construct expressing FLAG fused with BRUCE (FLAG-BRUCE). Cells were treated with DOX to induce expression of shBRUCE and followed by exposure to IR (5 Gy). 1 hr post IR, cells were immunofluorescence stained with antibodies specific for FLAG (red) and endogenous BRIT1 (green) with cell nucleus counterstained with DAPI (blue). Bars, 10 µm. (C) N-BRUCE cannot support BRIT1 foci formation. U2OS cells with endogenous BRUCE depleted by siBRUCE were transiently transfected with an expression construct of FLAG fused with N-BRUCE (aa 1–2025, depicted in Fig 1A). At 1 hr post IR (5 Gy), cells were immunofluorescence stained with antibodies against FLAG (red) and endogenous BRIT1 (green) with cell nucleus counterstained with DAPI (blue). Bars, 10 µm. (D) C-BRUCE cannot support BRIT1 foci formation. DOX-inducible shBRUCE-U2OS cells described in (B) was transiently transfected with a construct expressing FLAG fused C-BRUCE (aa 2024–4857, depicted in Fig 1A). Cells were treated with DOX to induce expression of shBRUCE followed by exposure to IR (5 Gy). 1 hr post exposure, cells were immunofluorescence stained with antibodies against FLAG (red) and endogenous BRIT1 (green) with cell nucleus counterstained with DAPI (blue). Bars, 10 µm. (E) BRIT1 protein levels remain unchanged post BRUCE knockdown. BRUCE expression was depleted by siRNA as indicated. Whole cell lysates (WCL) were examined by immunoblotting for BRUCE and BRIT1; a-tubulin blotting as loading control.