Fig. 4

DNA:RNA hybrids are directly recognized by BRCA1 in vitro and in vivo. a Representative pictures of super-resolution imaging analysis of BRCA1 (cyan) and DNA:RNA hybrids (yellow) colocalization in S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. b Dot plot shows the normalized number of overlaps relative to random of BRCA1 and DNA:RNA hybrids signals in S-phase U2OS cells treated with DSMO or NCS. At least n = 40 events were counted from three independent experiments. Lines represent mean ± s.e.m. c Electrophoretic mobility shift assay (EMSA) of purified recombinant human BRCA1 or BRCA1-BARD1 with end-labeled (*) double-stranded DNA or DNA:RNA substrates. d Graph showing the percentage of protein-bound substrate at respective protein concentrations. Error bars represent s.e.m. (n = 2 independent experiments). e Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells over-expressing GFP or GFP-RNase H1 (GFP-RH1). Scale bar: 5 μm. f Dot plot shows the number of foci in e. At least n = 80 cells were counted from at least three independent experiments. Lines represent mean ± s.e.m. g Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells treated with RNase H prior to fixation. Scale bar: 10 μm. h Dot plot shows the number of foci in g. At least n = 80 cells were counted from three independent experiments. Lines represent mean ± s.e.m. ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001 (two-tailed Student’s t test). Source data are provided as a Source Data file