Extended Data Fig. 9: The role of DSB-targeted SHLD2 in the suppression of homologous recombination and the mapping of the SHLD2-C–SHLD1 complex binding to ssDNA.

a, Representative micrographs of RPE1 BRCA1^KO53BP1^KO cells transduced with the indicated GFP-fusion proteins, pre-extracted, fixed and stained for RAD51 and GFP 3 h after ionizing radiation (10 Gy). Protein expression was induced for 24 h before exposure to ionizing radiation using 1 µg ml⁻¹ doxycycline. Data relates to Fig. 4b. Note that owing to the pre-extraction required for visualization of RAD51 foci, the visualization of non-FHA-tagged SHLD2 is lost. b, SDS–PAGE analysis of purified SHLD2-C–SHLD1 complexes. Strep/HA–SHLD2(421–904)–Flag-SHLD1 complexes were purified from transiently transfected 293T cells. Concentrations of purified proteins were estimated by Coomassie staining and comparison to a standard curve of known BSA concentrations visualized by fluorescence at 700 nm. SHLD2-C m1 and SHLD2(S)-C denote SHLD2-C constructs carrying the OB-fold m1 mutation and the internal deletion (Δ655–723) corresponding to the naturally occurring splice variant of SHLD2, respectively. Open and filled arrowheads mark the bands corresponding to SHLD2-C and SHLD1, respectively. EV refers to empty Strep/HA vector. A representative stained gel from two independent experiments is shown. c, Representative image of the ³²P-labelled ssDNA EMSA with SHLD2-C–SHLD1 for K_d determination shown in Fig. 4e. d, Model of the SHLD2 OB-fold domains and the engineered mutations (red spheres, point mutations; red ribbons, splice variant deletion). Model relates to Fig. 4b, d.