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.