Fig. 6: SART3 recruits DDX1 to resolve DNA-RNA hybrids formed at DSBs. | Nature Communications

Fig. 6: SART3 recruits DDX1 to resolve DNA-RNA hybrids formed at DSBs.

From: SART3 promotes homologous recombination repair by stimulating DNA-RNA hybrids removal and DNA end resection

Fig. 6

a U2OS cells were transfected with siNC, siSART3, siDDX1, or siSART3 and siDDX1, followed by CPT treatment. The samples then were performed immunostaining using S9.6 antibody. The numbers represent mean S9.6 intensity per nucleus measured by Cellprofiler. N = 263, 560, 570, 530, 322, 366, 410, 343, 371, 286, 332,414 correspond to the twelve groups shown on the x-axis, based on three replicates. Error bars represent mean ± SD. P values were calculated using a two-tailed Mann–Whitney U test. b U2OS-ER-AsiSI cells transfected with indicated siRNAs were treated with 4-OHT. The gDNA was extracted for DRIP-qPCR. The yellow shaded area represents RNase H treatment. c Purified DDX1 (15 µg) and increased amounts of SART3 (3.75, 7.5, 15, 30 µg) were incubated with 30 picomoles of biotin-DNA-RNA or biotin-dsDNA, followed by streptavidin-bead incubation. d The various amounts of DDX1 (0.05, 0.1, and 0.15 µg) were respectively incubated with FAM-labeled DNA-RNA substrates in the absence or presence of purified SART3 (0.2 µg). The samples were then examined by 12% native polyacrylamide gel for analysis. D 25 nt represents a DNA length of 25 nt, and R 25 nt represents an RNA length of 25 nt. The numbers represent the lane order. e U2OS-ER-AsiSI cells stably expressing GFP, GFP-SART3 (WT), GFP-SART3-R836W, GFP-SART3-YA1, or GFP-SART3-T4 were transfected with siNC or siSART3. After 48 h, cells were treated with 4-OHT followed by DRIP-qPCR analyses. f U2OS-DR-GFP cells stably expressing Flag, Flag-SART3 (WT), Flag-SART3-R836W or both Flag-SART3-R836W and Flag-RNase H1 were transfected with siNC or siSART3, followed by infection with I-SceI lentivirus. The percentage of GFP-positive cells was quantified by FACS analysis. g, h U2OS cells stably expressing GFP, GFP-SART3, or GFP-SART3-R836W were transfected with siRNA, treated with CPT for 24 h followed by CCK8 assay (g), or Olaparib for 48 h followed by colony formation assay (h). In (b), and (e–h), error bars represent mean ± SEM (N = 3 independent experiments), and p values were calculated using unpaired two-tailed Student’s t test (b, e, f) or one-way ANOVA analysis with Tukey test (g, h). Source data are provided as a Source Data file.

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