Fig. 6

BRCA2 controls DNA:RNA hybrid levels at DSBs by interacting with RNase H2 and mediating its recruitment to DSBs. a Dot plot showing the normalized number of overlaps relative to random of γH2AX and RNASEH2A signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. b Representative images of PLA between RNASEH2A and γH2AX in not irradiated (no ir) or irradiated (2 Gy) HeLa-FUCCI cells knocked-down for BRCA2. Scale bar: 10 μm. Dot plot shows the number of signals per nucleus of PLA between RNASEH2A and γH2AX in cells knocked-down for BRCA2. At least n = 150 cells were counted from three independent experiments. Lines represent mean ± s.e.m. c Co-immunoprecipitation of endogenous RNASEH2A from not irradiated (no ir) or irradiated 5 Gy (ir) HEK293T cell extracts, prepared in the presence of benzonase to avoid contaminant nucleic acids. Asterisks indicate specific band. This experiment was repeated three times independently with similar results. d Immunoblot of GST pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to GST-tagged BRCA2 fragments. This experiment was repeated two times independently with similar results. e Immunoblot of streptavidin pull-down assessing binding of recombinant Histidin (His)-tagged RNase H2 to biotinylated BRC repeats. This experiment was repeated two times independently with similar results. f Dot plot showing the normalized number of overlaps relative to random of γH2AX and DNA:RNA hybrid signals in S-phase cells knocked-down for BRCA2 and treated with NCS. At least n = 50 events were counted from two independent experiments. Lines represent mean ± s.e.m. g DRIP-qPCR at 1.5 kb on the right from the I-PpoI cut site within DAB1 gene in S/G2-phase-sorted HeLa-FUCCI cells knocked-down for BRCA2 and transfected with the I-PpoI nuclease. The bar graph shows the average fold induction of cut samples relative to uncut from n = 3 independent experiments. Error bars represent s.e.m. ^*P < 0.05, ^***P < 0.001 (two-tailed Student’s t test). (RH2 is RNase H2; RH2A is RNASEH2A). Uncropped blots are shown in Supplementary Fig. 8. Source data are provided as a Source Data file