Fig. 3: RNF19A functions in end resection through its catalytic activity and in a BARD1-dependent manner.

a–d RNF19A knockdown U2OS cells stably expressing Vec, WT, or C316A(CA) Flag-RNF19A were treated with IR (2 Gy, 3 h for RPA32 and 2 Gy, 5 h for RAD51). Cells were fixed and immunostained as indicated (a and c). Cyclin A was used as a marker of S/G2 phase. Quantification of focus signals per cell (each dot represents a single cell, n = 100 for group Cyclin A-; n = 150 in b; and n = 110 in d for group Cyclin A+) is shown in b and d. Scale bars, 10 μM. Error bars represent means ± s.d. of three independent experiments. e Schematic of ER-AsiSI system for quantification of DNA resection. Restriction enzyme AsiSI is fused to the estrogen receptor (ER) and can be induced to the nucleus and generate DSBs at sequence-specific sites by 4-OHT. The genomic DNA was extracted and quantification of ssDNA generated by resection was measured by qPCR. The primer pairs for DSBs are across BsrGI restriction sites. f RNF19A knockdown ER-AsiSI U2OS cells were reconstituted with Vec, WT, or C316A(CA) Flag-RNF19A and were pretreated with 300 nM 4-OHT for 4 h to induce DSBs. Genomic DNA was extracted and digested or mock digested with BsrGI overnight. DNA-end resection adjacent to indicated sites was measured by qPCR. g HEK293T cells stably expressing Vec or Flag-RNF19A were transfected with control (Ctrl) or BARD1 siRNAs for 48 h. HR efficiency was determined using the HR reporter assay. h U2OS cells stably expressing Vec or Flag-RNF19A were transfected with control (Ctrl) or BARD1 siRNAs were subjected to colony formation assay for assessment of response to Olaparib. i U2OS ER-AsiSI cells stably expressing Vec or Flag-RNF19A transfected with control (Ctrl) or BARD1 siRNAs for 48 h and were pretreated with 300 nM 4-OHT for 4 h before digest and measurement of DNA resection. Error bars represent means ± s.d. of three (f, h, and i) or four (g) independent experiments. p values are determined by unpaired two-sided t test in b, d and f–i. Source data are provided as a Source Data file.