USP5 is necessary for cell survival after DNA damage.

<p>A: Knockdown of USP5 expression by siUSP5 treatment. <b>B</b>: Colony forming assay after treatment with Bleocin, hydroxyurea, or methyl-methanesulfonate, with or without siUSP5 treatment. Filled square indicates without siUSP5 treatment and filled circle indicates with siUSP5 treatment; error bars, ± SED. The <i>P</i>-value was calculated using Student's <i>t</i>-test.</p

USP5 co-localizes with DNA DSBs.

<p>A: EGFP-tagged USP5 was expressed in HeLa cells, and cells were irradiated with the laser at a dose of 100 frames with 100 µM of 8-MOP. 8-MOP sensitizes laser light to produce DNA base damage and strand breaks. <b>B</b>: EGFP-tagged USP5 co-localizes with DNA DSBs produced by a restriction enzyme. Plasmid DNAs for expression of USP5-EGFP and Cherry-TA were introduced in U2OS SceI cells with or without NLS-SceI expression plasmid DNA by Lipofect amine 2000. After overnight incubation, cells were fixed and stained by anti-γH2AX antibody. <b>C</b>: Endogenous USP5 co-localizes with DNA DSBs produced by a restriction enzyme. Plasmid DNA for expression of NLS-SceI was introduced in U2OS SceI cells by Lipofect amine 2000. After overnight incubation, cells were fixed and stained by anti-USP5 antibody and anti-γH2AX antibody. At least 150 cells were counted in each sample. <b>D</b>: Damage response of USP5-EGFP after laser micro-irradiation with or without ATM inhibitor. Cells were irradiated with the laser at a dose of 100 frames with 100 µM of 8-MOP and with or without 10 µM of ATM inhibitor. The results are averages obtained from two independent experiments, and more than 52 cells were irradiated and analyzed for the damage response. <b>E</b>: Domain analysis of USP5 with regard to the damage response. Schematic presentation of domains in USP5 and the GFP-tagged mutant constructs. zf-UBP, Zinc-finger in ubiquitin-hydrolases and other proteins; UBA, Ubiquitin Associated domain. EGFP-tagged USP5 full length or deletion mutant was expressed in HeLa cells, and cells were irradiated with the laser at a dose of 100 frames with 100 µM of 8-MOP. The results are averages obtained from at least two independent experiments and more than 33 cells were irradiated and analyzed.</p

USP5 interacts with RAD18 and USP5 depends on RAD18 in DSB repair.

<p>A: Interaction between expressed proteins. We transiently expressed EGFP-tagged RAD18 in cells expressing FLAG-His-tagged USP5 and pulled down by the His-tag and pull downs were detected with anti-GFP antibody. <b>B</b>: Interaction between EGFP-tagged RAD18 and endogenous USP5. Cells were treated with or without Bleocin for 2 hr and then cells were extracted. Extracts were pulled down with anti-GFP antibody and detected by anti-USP5 antibody. <b>C</b>: Damage response of USP5-EGFP after laser micro-irradiation in RAD18-proficient or -deficient cells. EGFP-tagged USP5 and DsRed-tagged RAD18 WT or each mutant were co-expressed in RAD18-deficient cells, and the damage response after laser micro-irradiation was analyzed. Cells were irradiated with the laser light for 100 ms. <b>D</b>: HR frequencies in cells depleted of USP5 and/or RAD18. Results of western blot analysis after siRNA treatment are shown on the top. The GFP-positive cell fraction in cells depleted of USP5 and/or RAD18 was determined and compared with that in cells treated with siCont or siBRCA1 for determination of frequencies; error bars, ± SED. The <i>P</i>-value was calculated using Student's <i>t</i>-test.</p

USP5 is required for disassembly of polyubiquitin chains at sites of DNA damage.

<p>A: EGFP-tagged ubiquitin was expressed in HeLa cells with or without siUSP5 treatment. Cells were irradiated with the laser light for 100 ms. The intensity of EGFP-Ub at the irradiated site was analyzed and summarized in the graph. The results are averages obtained from three independent experiments (n = 5). <b>B</b>: Cells were irradiated with the laser for 10 frames with 100 µM of 8-MOP and then fixed and stained by anti-ubiquitin antibody (FK1; left panel, FK2; right panel) and anti-RAD18 antibody. Anti-RAD18 antibody is used for showing irradiated sites.</p