Fig. 8: Binding to DNA-RNA hybrids is critical for the function of SART3 in DSB repair.

a, b U2OS cells stably expressing GFP, GFP-SART3, or GFP-SART3-YA1 were transfected with siSART3 and treated with CPT for 24 h followed by CCK8 assay (a), or Olaparib for 48 h followed by colony formation assay (b). c U2OS-DR-GFP stably expressing Flag, Flag-SART3, or Flag-SART3-YA1 were transfected with siSART3, then infected with I-SceI lentivirus. The percentage of GFP-positive cells was quantitated by FACS (left). The specified proteins were examined by immunoblotting (right). d U2OS cells stably expressing GFP, GFP-SART3, or GFP-SART3-YA1 were transfected with siSART3, treated with ETO (RPA32, BRCA1 and BARD1) or CPT (DDX1) and further recovery for 2 h. Immunostainings was performed. The percentages of cells with more than 10 or 20 foci were quantified (bottom). Representative images are shown (top). Scale bars for overall and magnified images are respectively 50 and 10 µm. e U2OS-ER-AsiSI cells stably expressing GFP, GFP-SART3 or GFP-SART3-YA1 were transfected with the indicated siRNAs and treated with 4-OHT, followed by qPCR to measure DNA end resection (top). Immunoblotting verifies the knockdown of SART3 and CtIP (bottom). f HEK293T cells transfected with the indicated constructs were treated with CPT (5 µM, 2 h). Chromatin fractions were isolated for immunoprecipitation with anti-GFP beads, followed by immunoblotting with indicated antibodies. Asterisks indicate none-specific bands. CF-IP stands for Chromatin Fractions-IP. g U2OS-GFP-MMEJ cells stably expressing Flag, Flag-SART3, Flag-SART3-R836W, or Flag-SART3-YA1 were transfected with the indicated siRNAs followed by infection with I-SceI lentivirus. The percentage of GFP-positive cells was quantified by FACS analysis (top). The specified proteins were examined by immunoblotting (bottom). h U2OS cells stably expressing GFP, GFP-SART3, or GFP-SART3-YA1 were transfected with siSART3 for 48 h. Cells were then exposed to 15 J/m² UVC and repaired for 4 h. The TIF and WCL were harvested and analyzed with the indicated antibodies. i Working model of how SART3 regulates HR repair. In (a–e), and (g) error bars represent mean ± SEM (N = 3 independent experiments), and p values were calculated using one-way ANOVA analysis with Tukey test (a, b) or unpaired two-tailed Student’s t test (c–e, g). Source data are provided as a Source Data file.