Fig 2: Interaction of RNase A with Ku70/Ku80 heterodimer. (a,b) Structures of the complex between RNase A and Ku70/Ku80 obtained by molecular modeling using bioinformatics platforms HDOCK server and PatchDock, respectively. The ?G values of RNase A binding to Ku70/Ku80 heterodimer and corresponding dissociation constants (Kd) were calculated for each obtained structure using the PRODIGY resource. Heterodimer Ku70/Ku80 is yellow (a) or blue (b), and RNase A is orange (a) or purple (b). The arrow (a) shows the DNA-binding loop formed by Ku70/Ku80 heterodimer. Docking complexes characterized by the smallest values of ?G and Kd are highlighted with purple frames. (c,d) Accumulation of RNase A in B16 and HeLa cells, respectively, and its co-localization with Ku70/Ku80 heterodimer. B16 and HeLa cells were incubated in the presence of RNase A–biotin conjugate (20 µM) for 4 h followed by staining with streptavidin-APC, Ku70/Ku80 monoclonal antibody and secondary antibody with Alexa Fluor Plus 488 and analyzed by confocal fluorescence microscopy. Fluorescence in the blue channel (B panel) corresponds to DAPI (staining of nuclei), the green channel (G panel) corresponds to Ku70/Ku80 Alexa Fluor Plus 488 and the red channel (R panel) corresponds to RNase A–biotin conjugate stained with streptavidin-APC. Scale bars: 10 µm.

Fig 3: Knockdown of Ku86 expression in Rad52-/- B cells virtually ablates CSR and enforced expression of Rad52 in normal B cells significantly impairs CSR.Rad52-/- and Rad52+/+ B cells were transduced with pGFP-C-Ku86-shRNALenti lentiviral vector expressing Ku86-specific shRNA and GFP, or pGFP-C-scr-shRNALenti lentiviral vector expressing scrambled shRNA and GFP. Rad52+/+ B cells were activated with LPS for 12 h and transduced with empty pMIG-GFP retroviral vector or pMIG-GFP-Rad52 retroviral vector expressing recombinant Rad52. After lentiviral or retroviral transduction, B cells were cultured for 96 h with LPS plus IL-4. (a) B-cell expression of Ku86, Rad52 and ß-Actin proteins analysed by immunoblotting. (b) Proportions of surface IgG1+ B cells among pGFP-C-scr-shRNALenti or pGFP-C-Ku86-shRNALenti lentiviral vector-transduced (B220+GFP+) Rad52-/- and Rad52+/+ B cells were analysed by flow cytometry. Data are from one representative of three independent experiments. (c) Proportions of surface IgG1+ B cells among empty pMIG-GFP or pMIG-GFP-Rad52 retroviral vector-transduced (B220+GFP+) Rad52+/+ B cells were analysed by flow cytometry. Data are from one representative of three independent experiments.

Fig 4: Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends.(a) Ku70/Ku86 and Rad52 are recruited to CSR-targeted S region DNA in an AID-dependent manner in B cells undergoing CSR. Aicda+/+ and Aicda-/- B cells were stimulated with LPS or LPS plus IL-4 for 60 h. Cross-linked chromatin was precipitated using rabbit anti-Rad52 antibody or mouse anti-Ku70/Ku86 mAb. Precipitated Sµ, S?1 and S?3 DNA quantified by real-time quantitative PCR (qPCR); amounts relative to those in Aicda+/+ B cells, set as 1. Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Aicda+/+ and Aicda-/- mice (mean±s.d.). P-values determined using a paired Student's t-test. (b) Precipitated Sµ and Cµ DNA from Aicda+/+ and Aicda-/- B cells detected by PCR. Data are one representative of three independent experiments. (c) Rad52+/+ and Rad52-/- B cells were stimulated with LPS or LPS plus IL-4 for 60 h. Chromatin was cross-linked and precipitated using a mouse anti-Ku70/Ku86 mAb. The precipitated Sµ, S?1 and S?3 DNAs quantified by real-time qPCR; amounts in Rad52-/- B cells are as relative to those in Rad52+/+ B cells, set as 1. Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Rad52+/+ and Rad52-/- mice (mean±s.d.). P-values determined using a paired Student's t-test. (d) Rad52+/+ and Rad52-/- B cells were stimulated with LPS or LPS plus IL-4 for 60 h. DNA ends were labeled in situ with bio-dUTP using TdT. Chromatin was cross-linked and precipitated using mouse anti-Ku70/Ku86 mAb (left panel) or rabbit anti-Rad52 antibody (right panel). After resuspension, DNA with broken ends was pulled down with streptavidin magnetic beads. Precipitated Sµ, S?1, S?3 and Cµ DNAs quantified by real-time qPCR (this approach allowed for detection of Ku70/Ku86 or Rad52 bound to DSB-free ends); amounts of precipitated DNA are relative to those in Rad52+/+ B cells, set as 1 (left panel), or relative to respective input DNA (right panel). Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Rad52+/+ and Rad52-/- mice (mean±s.d.). P-values determined using a paired Student's t-test. (e,f) EMSA using a biotin-labelled double-stranded Sµ DNA probe and (e) recombinant human RAD52 and KU70/KU86 (constant amount) proteins at different ratios (1:1, 2:1 and 4:1), or (f) nuclear extracts (10 µg protein) from Rad52+/+ and Rad52-/- B cells (stimulated with LPS plus IL-4 for 60 h) incubated with mouse anti-Ku70/Ku86 mAb or irrelevant mouse IgG (control). The formation of protein–DNA complexes was shifted by anti-Ku70/Ku86 mAb. Numbers below gel image indicate relative density of protein–DNA complex bands normalized with the density of free probe. Shown is one representative gel of three independent experiments.

Fig 5: Aicda (AICDA)/CSR-inducing stimuli decrease expression of Rad52 (RAD52) and increase expression of Ku70/Ku86 (KU70/KU86) and Pol? (POL?) in mouse and human B cells.(a) C57BL/6 B cells were stimulated with different amounts of LPS only, or LPS or mCD154 plus IL-4 for 0, 24, 48 and 72 h. Aicda, Rad52, Pol? and Ku70/Ku86 transcripts were analysed by real-time qRT–PCR. Expression of Aicda normalized to Cd79b expression, expression of Rad52, Ku70/Ku86 and Pol? normalized to Gapdh expression and depicted relative to the expression in unstimulated B cells, set as 1. Expression of AID, Rad52, Ku70, Ku86, Pol? and ß-Actin proteins in B cells stimulated with LPS (3.0 µg ml-1) plus IL-4 were analysed by immunoblotting. (b) Human naive B cells were stimulated with different amounts of mCD154 plus human IL-4 and human IL-21 for 0, 24, 48, 72 or 96 h. AICDA, RAD52, KU70/KU86 and POL? transcripts were analysed by real-time qRT–PCR. Expression normalized to CD79b expression and depicted relative to the expression in unstimulated B cells, set as 1. P-values determined using a paired Student's t-test. Data are from one representative of three independent experiments.