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

Abstract

DNA-RNA hybrids triggered by double-strand breaks (DSBs) are crucial intermediates during DSB repair, and their timely resolution requires numbers of RNA helicases, including DEAD box 1 (DDX1). However, how these helicases are recruited to DSB-induced hybrids in time remains largely unclear. Here, we revealed that squamous cell carcinoma antigen recognized by T cells 3 (SART3) promotes DDX1 binding to DNA-RNA hybrids at DSBs for optimal homologous recombination (HR) repair. SART3 itself associates with DNA-RNA hybrids and PAR chains and accumulates at DSBs in both PARylation- and DNA-RNA hybrids-dependent fashion. SART3 also associates with DDX1 and is necessary for DDX1 enrichment at DSBs. The defective SART3-DDX1 association observed in cells expressing the cancer-associated variant SART3-R836W impairs not only the accumulation of DDX1, but also hybrid removal and HR efficiency. Moreover, SART3 promotes DNA end resection through enhancing USP15-BARD1 association and BRCA1-BARD1 retention. Together, our study reveals an role of SART3 in DSB repair, rendering SART3 a promising target for cancer therapy.

Fig. 1. SART3 is enriched at DSBs in a PARylation- and DNA‒RNA hybrids-dependent manner.

a U2OS cells stably expressing GFP-SART3 were microirradiated. Cell images were captured at different time points (left). Error bars represent mean ± SEM of 10 independent measurements. Scale bar, 10 µm. b U2OS-DR-GFP cells were transfected with Flag or Flag-SART3 and then infected with lentivirus expressing I-SceI. ChIP assays were performed. c, d U2OS cells stably expressing GFP-SART3 were pretreated with ABT-888 (c) or transfected with siRNA (d), followed by microirradiation. The proportion of cells with SART3 accumulation was measured. e Schematic representation of SART3 domains. HAT half-a-tetracopeptide repeats. CC coiled-coil. NLS nuclear localization sequences. RRM RNA recognition motif. f U2OS cells were respectively transfected with a series of SART3 truncated constructs (T1, T2, T3, and T4) or full length (FL), followed by microirradiation. Representative images are shown (left). Scale bar, 10 µm. g, h U2OS cells expressing GFP-SART3 were pretreated with transcription inhibitor prior to microirradiation. Representative images are shown (right). Scale bar, 10 µm. i, j HEK293T cells transfected with the indicated constructs were harvested for incubation with biotin-DNA‒RNA hybrids (i) or S9.6 antibody (j). The proteins pulled down were analyzed by immunoblotting. k U2OS cells expressing GFP-SART3-WT or -YA1 were microirradiated. l U2OS-DR-GFP cells were transfected with the indicated constructs, followed by ChIP-qPCR as in (b). m Schematic illustrates the positions of the primers employed for ChIP-qPCR in U2OS-DR-GFP cells (top). Primer-1 and primer-2 are denoted by black and red arrow respectively. SceGFP: I-SceI-cleaved GFP. U2OS-DR-GFP cells stably expressing Flag-SART3 were transfected with GFP or GFP-RNase H1 construct followed by ChIP-qPCR as in (b). n Recombinant GST-SART3-WT and -YA1 mutant were purified and then transferred onto PVDF membrane, followed by incubation with biotin-PAR polymers and analyzed by immunoblotting. o U2OS cells transfected with GFP-SART3-WT or -YA1 were pretreated with ABT-888 for 2 h prior to microirradiation. The proportion of cells with SART3 accumulation were measured. Error bars represent mean ± SEM, N = 3 (b–d, g–h, k–m, o) or 4 (f) independent experiments, and p values were calculated using an unpaired two-tailed Student’s t test (no adjustment for multiple comparisons). Source data are provided as a Source Data file.

Fig. 2. SART3 promotes DSB repair.

a, b U2OS cells stably expressing GFP or GFP-SART3 were transfected with siNC or siSART3-1, followed by treatment with DMSO or ETO (10 µM for 2 h, and allowed to repair 0.5 h). The levels of γH2AX were analyzed by immunoblotting (a). U2OS cells were transfected with siNC or siSART3-1/3, followed by treatment with DMSO or ETO (10 µM for 2 h) and further recovery for 0.5, 3 or 8 h. The samples were analyzed by immunoblotting using indicated antibodies. b The intensity of γH2AX was quantified by Image J software and normalized. c U2OS-DR-GFP cells stably expressing Flag-SART3 or Flag were transfected with siNC, siSART3-1 or siCtIP. 24 h later, cells were infected with I-SceI lentivirus. Percentage of GFP-positive cells was quantitated by FACS at 48 h after virus infection (top). The SART3 knockdown efficiency was verified by immunoblotting (bottom). d–g U2OS cells stably expressing either GFP-SART3 or GFP were transfected with siNC or siSART3, followed by treatment with indicated concentrations of ETO for 24 h (e), CPT for 1 h (f), HU for 2 h (g) or Olaparib for 48 h (h). The cells were further incubated for colony formation. SiSART3 indicates siSART3-1, if not specified. In (c–g), error bars represent mean ± SEM (N = 3 independent experiments), and p values were calculated using unpaired two-tailed Student’s t test (c) or one-way ANOVA analysis with Tukey test (d–g). Source data are provided as a Source Data file.

Fig. 3. SART3 promotes DNA end resection.

a–e U2OS, or U2OS cells stably expressing GFP or GFP-SART3 were transfected with siNC or siSART3. After 48 h, cells were treated with 10 µM ETO for 2 h and further recovered for 2 h. Immunofluorescence assays were performed using antibodies against BRCA1 (a), BARD1 (b), RPA32 (c), RNF8 (d), and RNF168 (e). Representative images are shown (top or left). The proportions of cells with foci were measured (bottom or right). Scale bars for overall and magnified images are respectively 50 and 10 µm. f U2OS cells stably expressing GFP or GFP-SART3 were transfected with siNC or siSART3 for 48 h, followed by treatment with 5 µM CPT for 2 h and further cultured for 3 h. Whole-cell lysates were harvested for immunoblotting with antibodies as indicated. g Cartoon illustrates a quantitative DNA resection assay (top). U2OS-ER-AsiSI cells transfected with the indicated siRNA were treated with 4-OHT. The gDNA was extracted and digested overnight followed by qPCR to measure DNA end resection (bottom). Immunoblotting verifies the knockdown efficiency of siRNAs (right). h U2OS cells were transfected with siNC or three different siRNAs targeting SART3, followed by incubation with 10 µM BrdU for 48 h. The cells were then treated with 5 µM CPT for 2 h and recovered for an additional 3 h. Immunofluorescence assays were performed using an antibody against BrdU. Denaturation was carried out using 2 M hydrochloric acid for 10 min. Representative images under native (non-denaturing) condition (left) and denatured condition (middle) are shown, and the proportion of cells with foci was quantified (right). Knockdown efficiencies of SART3 are examined by Western blotting (bottom-right). Scale bars for overall and magnified images are respectively 50 and 10 µm. In (a–e, g, h), error bars represent mean ± SEM (N = 3 independent experiments), and p values were calculated using unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.

Fig. 4. SART3 depletion upregulates DSBs-induced DNA‒RNA hybrids.

a, b U2OS cells were transfected with siNC or siSART3. After 36 h, cells were treated with 5 µM CPT for 2 h, followed by S9.6 immunostaining. The numbers represent mean S9.6 intensity per nucleus measured by Cellprofiler (a). N = 281, 391, 364, 338, 301, 302, 308, 348, 330 correspond to the nine 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. Representative images are shown (b), with the areas highlighted by circles quantified for S9.6 intensity analysis. Scale bar, 10 µm. c Cartoon deciphers DR-IP (top). U2OS-ER-AsiSI cells were transfected with siNC or siSART3. After 48 h, cells were treated with 4-OHT followed by DRIP-qPCR analysis (bottom). d DRIP-qPCR analysis was performed as in (c) supplemented with RNase H during genome cleavage. The primers spanning the AsiSI-induced HR-1 break site and the actin exon region were used for qPCR. The yellow shaded area represents RNase H treatment. e R-ChIP-qPCR analysis of DNA‒RNA hybrids. Cartoon deciphers R-ChIP (top). U2OS-DR-GFP cells stably expressing V5-RNase H1-D210N mutant were transfected with siNC or siSART3. 24 h later, cells were infected with I-SceI lentivirus. After 36 h, cells were harvested followed by R-ChIP. The levels of DNA-RNA hybrids around DSBs were detected by qPCR. In (c–e), error bars represent mean ± SEM (N = 3 independent experiments), and p values were calculated using unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.

Fig. 5. Binding to SART3 is required for optimal DDX1 accumulation at sites of damage.

a HEK293T cells transfected with the indicated constructs were lysed in the presence of RNase A, followed by immunoprecipitation and immunoblotting. b U2OS cells stably expressing GFP or GFP-SART3 were transfected with siNC or siSART3, followed by treatment with 5 µM CPT for 2 h and further recovery for 1 h. The cells were fixed and subjected to immunostaining and quantification analysis (right). The percentages of cells with more than 10 DDX1 foci were quantified. Scale bars for overall and magnified images are respectively 50 and 10 µm. c HEK293T cells transfected with GFP or GFP-SART3 were treated with CPT (5 µM, 2 h), followed by recovery and subsequent immunoprecipitation and immunoblotting. d U2OS cells stably expressing Flag or Flag-RNase H1 were transfected with siNC or siSART3. Cells were microirradiated and fixed, followed by co-immunofluorescence staining with anti-γH2AX and anti-DDX1 antibodies. Representative images are shown (left). Scale bar, 10 µm. e HEK293T cells expressing various of GFP-SART3 truncations were lysed in the presence of RNase A and Benzonase, followed by immunoprecipitation and immunoblotting. f HEK293T cells transfected with the indicated constructs were lysed for immunoprecipitation, followed by immunoblotting. g HEK293T cells transfected with the indicated constructs were harvested and incubated with biotin-DNA‒RNA hybrids. Pulled-down proteins were analyzed by immunoblotting, and GFP-SART3 levels were quantified by Image J, normalized to input SART3. h, i U2OS cells stably expressing GFP, GFP-SART3 (WT), GFP-SART3-R836W (h) or GFP-SART3-T4 (i) were transfected with siNC or siSART3. Cells were then treated with CPT, followed by co-immunofluorescence staining with anti-DDX1 and anti-γH2AX antibodies. The percentages of cells with more than 10 DDX1 foci were quantified (middle). Representative images are shown (left). Scale bars for overall and magnified images are respectively 50 and 10 µm. The knockdown efficiency of SART3 was determined by Western blotting (right). Error bars represent the mean ± SEM, N = 3 (b, h, i) or 4 (d) independent experiments, and p values were calculated using unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.

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

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.

Fig. 7. SART3 facilitates BARD1 deubiquitination via enhancing BARD1-USP15 association.

a HEK293T cells transfected with the indicated constructs were treated with CPT (5 µM, 2 h), followed by immunoprecipitation with anti-GFP agarose beads and immunoblotting with the indicated antibodies. Asterisks indicate non-specific bands. b U2OS cells transfected with siRNAs were treated with CPT and recovered for 0 h, 2 h, or 5 h, respectively. Triton-insoluble fractions (TIF) and whole-cell lysates (WCL) were harvested for immunoblotting. “R” stands for recovery. c, d HEK293T cell expressing GFP-SART3 and Myc-BARD1 were immunoprecipitated with anti-GFP beads, followed by immunoblotting with anti-Myc or anti-GFP (c). HEK293T cell lysates were immunoprecipitated with anti-BARD1 antibodies, followed by immunoblotting with antibodies against SART3 or USP15 (d). e Schematic representation of the SART3 truncated mutants (top). HEK293T cells co-transfected with Myc-BARD1 and GFP-SART3 truncations were harvested for Co-IP with anti-GFP beads, followed by immunoblotting with antibodies against Myc, GFP, or USP15. f HEK293T cells were co-transfected with GFP-USP15, Myc-BARD1, and Flag-SART3-WT or truncations. 36 h later, cells were treated with CPT, followed by Co-IP. g HEK293T cells transfected with the indicated siRNAs were co-transfected with HA-Ub, Flag-BARD1 and GFP-SART3-WT or truncations. 36 h later, cells were treated with CPT and subjected to denatured IP with anti-Flag agarose beads, followed by immunoblotting with the indicated antibodies. h, i U2OS cells transfected with siNC, siSART3, siUSP15-1, or both siSART3 and siUSP15-1 were treated with CPT (5 µM, 2 h) and further cultured for 2 h, followed by immunofluorescence with antibodies against BRCA1 or BARD1. Representative images are shown (left). Proportion of cells with BRCA1 or BARD1 foci was measured (right). Scale bars for overall and magnified images are respectively 50 and 10 µm. j U2OS-DR-GFP cells stably expressing Flag, Flag-SART3, Flag-SART3-ΔHAT4-7 or Flag-SART3-ΔRRM were transfected with either siNC or siSART3. After 24 h, cells were infected with I-SceI lentivirus. The percentage of GFP-positive cells was quantitated by FACS 48 h post I-SceI lentivirus infection. In (h–j), error bars represent mean ± SEM (N = 3 independent experiments), and p values were calculated using unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.

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.