RETRACTED: DNA-PKcs-PIDDosome: A Nuclear Caspase-2-Activating Complex with Role in G2/M Checkpoint Maintenance

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).This article has been retracted at the request of the Authors.Our paper reported the identification of a nuclear protein complex comprising DNA-PKcs, PIDD, and caspase-2 and characterization of its role in G2/M checkpoint maintenance, thereby providing insight into the functional significance of nuclear caspase-2. We recently identified errors affecting several figure panels where original data were processed inappropriately such that the figure panels do not accurately report the original data. We believe that the most responsible course of action is to retract the paper. We sincerely apologize to the scientific community for any inconvenience this might cause

The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex.

BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response

Working model for activation of BRIT1 deubiquitination by BRUCE UBC.

<p>Following exposure to IR, the E2/E3 activity of BRUCE increases and catalyzes ubiquitination of its substrate(s), yet to be identified, towards deubiquitination of BRIT1 and enhancement of DNA repair. The putative substrate of BRUCE could be USP8 or the E3 ligase for BRIT1 (blue lines), or another unknown protein X (red lines). In any case, assertion of BRUCE E2/E3 activity is expected to tip the balance towards deubiquitination of BRIT1 by promoting USP8 Dub function or/and by inhibiting the E3 ligase activity, thereby enhancing deubiquitination of BRTI1 and promoting DNA damage signaling and repair (see text for details).</p

BRUCE UBC domain is required for pATM repair foci formation.

<p>U2OS-shBRUCE cells with stable expression of BRUCE or BRUCE mutants as indicated to the left were treated with DOX to knockdown endogenous BRUCE. After irradiation, cells were fixed and immunofluorescence stained for pATM (<b>A</b>) and quantified for cells with more than five nuclear foci (<b>B</b>). Bars, 10 μm. Error bars represent standard deviation from a triplicate of a representative experiment.</p

BRUCE UBC domain is required for BRIT1 deubiquitination and its repair foci formation post the formation of the BRUCE-USP8-BRIT1 nuclear complex.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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. (<b>D</b>) 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. (<b>E</b>) 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.</p

BRUCE UBC domain is required for RAD51 foci formation and HR repair.

<p>(<b>A</b>) BRUCE UBC domain is required for RAD51 foci formation. U2OS-shBRUCE cells with stable expression of BRUCE or BRUCE mutant as indicated to the left were treated with DOX to knock down endogenous BRUCE. Cells were irradiated, fixed and immunofluorescence stained for RAD51 (green) and counterstained with DAPI. Bars, 10 μm. (<b>B</b>) Quantification of RAD51 foci in (<b>A</b>). Cells with positive RAD51 foci (more than five foci) were quantitated and a total of more than two hundred cells were counted for each sample. (<b>C</b>) BRUCE UBC domain is needed for HR repair. U2OS stable cell lines as indicated were treated with DOX and transfected with the DR-GFP HR reporter and I-<i>Sce</i>I. Following 48 hrs of continued culture, cells were collected and subject to flow cytometry analysis. Percentage of GFP+ cells in control (no DOX treatment) were set as 1. Bars represent standard error of the mean (S.E.M) from four independent experiments. P value is calculated by Student’s <i>T</i>-test, two tailed. (<b>D</b>) Immunoblot showing expression level of HA-tagged I-<i>Sce</i>I in the samples presented in (<b>C</b>).</p

BRUCE UBC domain is required for MDC1 repair foci formation.

<p>U2OS-shBRUCE cells with stable expression of BRUCE or BRUCE mutants as indicated to the left were treated with DOX to knockdown endogenous BRUCE. After irradiation, cells were fixed and immunofluorescence stained for MDC1 (<b>A</b>) and quantified for cells with more than five nuclear foci (<b>B</b>). Bars, 10 μm. Error bars represent standard deviation from a triplicate of a representative experiment.</p

shBRUCE-U2OS cell lines with expression of exogenous BRUCE variants.

<p>(<b>A</b>) Diagram of a DOX-inducible shBRUCE construct under H1 promoter (tandem eight repeats of shBRUCE sequences, top) and five BRUCE constructs expressing wild type BRUCE, mutant BRUCE with BIR or UBC domain mutated at the active site as indicated by the respective number of amino acid residue, or with the entire domain deleted (Δ). These BRUCE constructs are resistant to shBRUCE by introducing wobble mutation; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144957#sec002" target="_blank">Methods</a> for details. (<b>B</b>) U2OS cells with stable expression of the H1-shBRUCE construct (U2OS-shBRUCE) were transfected with each of the BRUCE constructs in (A). The resultant samples were selected for stable cell lines expressing each BRUCE variant as indicated on the top. These stable cell lines were left untreated or treated with DOX for 4 days to knockdown BRUCE. RT-PCR analysis of each cell line showing levels of endogenous BRUCE mRNA were significantly decreased after DOX treatment (upper arrow), whereas that of exogenous BRUCE mRNA remained the same across the cell lines (lower arrow); <i>Actin</i> as loading control. (<b>C</b>) Cell lysates of the above cell lines were immunoblotted with FLAG antibody to examine expression of exogenous BRUCE variants; α-Tubulin blotting as loading control.</p

BRUCE UBC domain is required for NBS1 repair foci formation.

<p>U2OS-shBRUCE cells with stable expression of BRUCE or BRUCE mutants as indicated to the left were treated with DOX to knockdown endogenous BRUCE. After irradiation, cells were fixed and immunofluorescence stained for NBS1 (<b>A</b>) and quantified for cells with more than five nuclear foci (<b>B</b>). Bars, 10 μm. Error bars represent standard deviation from a triplicate of a representative experiment. DOX treated shBRUCE-U2OS cells were irradiated and the cell lysates subject to immunoblotting with the indicated antibodies showing the protein levels of ATM, MDC1 and NBS1 are not reduced post BRUCE knockdown (<b>C</b>).</p

Truncated N-BRUCE or C-BRUCE cannot support BRIT1 foci formation in irradiated U2OS cells.

<p>(<b>A</b>) 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>B</b>) BRUCE restores formation of IR-induced BRIT1 foci. DOX-inducible shBRUCE-stable-expression U2OS cells (U2OS-shBRUCE, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144957#pone.0144957.ref024" target="_blank">24</a>]) 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. (<b>C</b>) 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. (<b>D</b>) 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. (<b>E</b>) 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; α-tubulin blotting as loading control.</p