Fig 1: FAM35A and C20ORF196 form a complex with REV7. a Silver-stained SDS-PAGE gel showing the polypeptides that were immunopurified from extracts of HEK293 cells expressing FLAG-tagged REV7, FAM35A and C20ORF196 using the anti-FLAG antibody. The major polypeptides on the gel (arrows) were identified by mass spectrometry. b–d Immunoblot showing the IP of FLAG-tagged REV7 (b), FAM35A (c) and C20ORF196 (d). IPs were performed with or without 100 µg ml–1 EtBr. The cross-reactive bands were indicated as asterisks. e–g MBP-pulldown (e, f) and GST-pulldown (g) examined the direct interactions among REV7, FAM35A and C20ORF196. The bait and prey proteins were detected with Coomassie blue staining and immunoblotting, respectively. h Schematic representation of FAM35A and POT1
Fig 2: FAM35A and C20ORF196 promote NHEJ to repair DSB. a, b Sensitivity assay of FAM35A (a) and C20ORF196 (b) knockout DT40 cells. The mean and s.d. from three independent experiments are shown. c Random integration assay of FAM35A and C20ORF196 knockout DT40 cells. Cells were transfected with the pLoxPuro plasmid and clonogenic potential was determined in the presence of puromycin. Data are expressed as the mean number of puromycin resistant colonies ± s.d. (n = 3). d Sensitivity assay of FAM35A-/- cells re-expressing GFP-fused wild-type or mutated chicken FAM35A. Protein expression levels of GFP-chFAM35A were shown by immunoblotting on the bottom
Fig 3: FAM35A and C20ORF196 block resection in the BRCA1-deficient cells. a, b The absence of FAM35A (a) or C20ORF196 (b) suppressed the olaparib sensitivity of the BRCA1-deficient cells. The mean and s.d. from three independent experiments are shown. c Chromosome aberration of various DT40 cells. Cells were treated with or without 1 µM olaparib for 8 h and then incubated with 100 ng ml–1 colcemid for 4 h before fixing. The mean and s.e.m. are shown. d, e Immunofluorescence (d) and its quantification (e) showing that FAM35A and C20ORF196 block the end resection in the BRCA1-deficient cells. The DT40 cells were treated with 4 Gy X-ray and released 4 h before fixing. The mean and s.d. from three independent experiments are shown. Scale, 2 µm. f A scatter diagram shows RAD51 foci numbers in DT40 cells after X-ray treatment. The mean and s.d. are shown. g A model shows that REV7–FAM35A–C20ORF196 complex blocks resection through binding the 5'-recessed DSB ends. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Statistics was performed by two-tailed t-test
Fig 4: FAM35A prefers to bind ssDNA. a, b Gel-shift assay (a) and its quantification (b) shows that MBP-FAM35A_C (381–904 aa) prefers to bind ssDNA. Reactions contained 5 nM of the indicated 32P-labeled substrates and 0, 50, 100, 200, 400 or 800 nM purified MBP-FAM35A_C or 800 nM MBP. c, d Competition experiments between 60 nt ssDNA and 30 nt ssDNA (c) or 60 bp dsDNA (d) for MBP-FAM35A_C binding
Fig 5: FAM35A and C20ORF196 are in the same pathway with RIF1 to repair DSB. a, b Genetic interaction analysis of FAM35A with C20ORF196 (a) and RIF1 (b) in DT40 cells. The mean and s.d. from three independent experiments are shown
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