Fig 2: Identification of BRE as a genetic interactor of BRCA2 using a MSCV-based insertional mutagenesis screen. a Schematic representation of MSCV-mediated insertional mutagenesis in Brca2 conditional mouse ES cells. Brca2KO/KO cells generated after PGK-CRE-mediated deletion of the conditional allele are not viable. Mutagenesis by MSCV-CRE can generate viable Brca2KO/KO ES cells. b Southern blot analysis of HAT-resistant ES cell colony that lost conditional Brca2 allele (Brca2KO/KO; Clone 3d) after MSCV-CRE transduction. Upper band: conditional allele (CKO); lower band: knock-out allele (KO). c Quantification of Bre expression by real-time RT-PCR in Brca2 conditional mutant (Brca2CKO/KO) and Brca2KO/KO ES cells with viral insertion at chr: 5qB1 (Brca2KO/KO;Clone 3d). Data are represented as mean ± s.d. Top panel shows the distance between viral insertion and start of first exon of Bre. d Expression of HA-tagged BRE from MSCV LTR in Brca2CKO/KO clones. Two independent clones Clone #1 and Clone #2 analyzed by western blot analysis were used further. Left panel shows the scheme of relevant alleles of Brca2CKO/KO; MSCV-BRE ES cells. e Southern blot analysis of HAT-resistant ES cell colonies after CRE-mediated deletion of conditional allele in Brca2CKO/KO; MSCV-BRE ES cells to identify Brca2KO/KO clones (marked with solid stars), upper band: conditional allele (CKO); lower band: knock-out allele (KO). Schematic diagram of CRE-induced loss of conditional Brca2 allele in Brca2CKO/KO; MSCV-BRE cells is shown at the top. f Upper panel shows western blot for BRCA2 knockdown by two different shRNAs (#1 and #2) and a non-specific (NS) control and HA-BRE expression in MCF7 cells that were stably expressing either empty vector (MCF7Neo) or vector expressing HA-BRE (MCF7BRE). GAPDH was used as a loading control. Growth of MCF7 cells after BRCA2 knockdown in the presence or absence of HA-BRE expression represented in the lower panel. Fold growth was calculated by dividing cell counts on particular day with cell count on day 1. P values are shown in Supplementary Table 2. P values were calculated using paired two-tailed t-test

Fig 3: Physiological relevance of BRE overexpression. a Immunohistological staining of 15 BRCA2-deficient tumors using BRE and CDC25A antibodies. Numbers represent different tumor numbers. Control represents staining with control IgG. H: high expression, L: low expression. b Allograft tumor growth of KB2P1.21 (Brca2 negative) cells with and without stable expression of HA-BRE. Values are represented as mean ± s.e.m. P values were calculated using ANCOVA test. c Western blot showing the expression of HA-BRE and CDC25A in tumors from allograft experiment. GAPDH was used as control. d Proposed model explaining the survival of BRE overexpressed BRCA2-deficient cells. CDC25A protein stabilization is regulated by the balanced action of ubiquitylation and deubiquitylation mediated by β-TRCP and DUB3, respectively. In cells with normal BRE expression level, loss of BRCA2 induces DNA damage that leads to β-TRCP-mediated degradation of CDC25A and cells undergo cell cycle arrest and apoptosis. BRCA2 loss in BRE overexpressing cells results an increase in BRE/USP7/CDC25A complex that perturbs the balance of CDC25A ubiquitylation/deubiquitylation and leads to an increase in CDC25A even in presence of DNA damage. This elevated level of CDC25A leads to the survival of cells with damaged DNA

Fig 4: Position of BRCA2 variants. Schematic representation of the position of class 1/2 variants (green dots), class 4/5 variants (red dots), and BRCA2 variants of uncertain significance (VUS) (gray dots) in the BRCA2 gene. Ta transcriptional activation domain, P phosphorylation site, H helical domain, DBD DNA binding domain, OB oligonucleotide binding fold, T Tower domain, TR2 C-terminal RAD51 binding site, NLS nuclear localization signal, aa amino acids19,39,40

Fig 5: Representative images of complementation phenotypes and controls. Brca2-/loxPPim1DR-GFP/WT cells expressing WT BRCA2 or BRCA2 variants were transfected with a Cre-GFP expression plasmid to induce loss of the conditional Brca2 allele and restore the Hprt gene. Upon Cre-recombinase expression cells become Brca2 deficient, which is lethal unless complemented by the expression of a (partially) functional BRCA2 variant. Untransfected cells that still contain the conditional Brca2 allele lack Hprt expression and will subsequently not survive HAT selection as shown in the –Cre-recombinase control. Thirteen days post Cre-GFP transfection culture dishes were stained with methylene blue