Extended Data Figure 8. TONSL-MMS22L recruitment to damaged DNA.

a (left) ChIP-qPCR analysis of GFP-TONSL recruitment to site-specific DSBs induced by AsiSI, as shown in Fig. 4d but with additional controls. Note that the colours have been changed for clarity. Mean of technical duplicates is shown. (right) Dot plot illustrating the relative enrichment of GFP-TONSL WT and N571A obtained in four independent ChIPs performed on two biologically independent chromatin preparations. Each experiment was normalized to GFP-TONSL WT enrichment at DSB-I_80bp. Mean is shown with two-sided Mann-Whitney test; ***, P < 0.001; n.s., P > 0.05; n=24. Two-sided Mann-Whitney analysis of individual experiments gave similar results. b, U-2-OS cells conditional for GFP-TONSL were laser-microirradiated. 53BP1 and Cyclin B staining was used as markers of DNA damage and cells in S/G2 phase, respectively. Representative of 3 experiments as quantified Fig. 4e. Full arrowheads, GFP-TONSL recruitment; empty arrowheads, no recruitment. Scale bars, 10 μm c, U-2-OS cells transiently transfected with GFP-TONSL WT or the indicated mutants were laser-microirradiated and processed for γH2A.X immunofluorescence. Representative cells are shown (n=200 cells per condition from two independent experiments). d, U-2-OS cells conditional for GFP-TONSL were laser-microirradiated. γH2A.X and RPA staining was used as markers of DNA damage and cells undergoing resection in S/G2 phase, respectively. The % of GFP-TONSL cells with recruitment to RPA positive (+) and RPA negative (-) laser tracks is indicated. Data are representative of 2 independent experiments, a total of 118 cells were counted. e, (top) U-2-OS cells conditional for GFP-TONSL WT and N571A were laser-microirradiated. γH2A.X and EdU staining was used as markers of DNA damage and S phase cells, respectively. (bottom) Quantification of GFP-TONSL cells with recruitment to laser tracks. Mean with individual data points are shown (n=2, a total of 138 (WT) and 174 (N571A) cells were counted). f, H4K20 methylation levels measured by mass spectrometry in synchronized TIG3 cells as in Extended Data Fig. 6c. Cell were released into S phase for 3 hours and treated with HU (3 mM) or CPT (1μM) for 3 hours or left untreated (6 hours). Mean with individual data points are shown (n=2). g, Colony formation in cells treated with control or TONSL siRNA and induced to express GFP-TONSL. As shown in Fig. 4f, but including additional mutants. Two cell concentrations in technical triplicate from two (E568A, D559A) or four (WT, N571A) biological replicates are shown. h, Representation of the complementation analysis from Fig. 4f in a single panel including both CPT treated and untreated cells. This illustrates that the toxicity of the TONSL ARD mutant is comparable to CPT treatment of cells expressing WT TONSL. i, Analysis of GFP-TONSL and MMS22L by cellular fractionation in cells inducible for GFP-TONSL ARD WT and mutant. Representative experiment of the quantification shown in Fig. 4i.