Fig 2: Identification and in vitro characterisation of TOPBP1 binding phosphorylation sites in 53BP1.(A) The TOPBP1/Rad4-binding motif matches only four potential sites out of over two hundred phosphorylation sites documented for 53BP1 (Hornbeck et al., 2004; Sharma et al., 2014). (B) Fluorescence polarisation (FP) analysis shows no substantial interaction of fluorescently-labelled phospho-peptides derived from the putative phosphorylation sites centred on 53BP1-Thr344, Ser366 or Ser380 with the BRCT0,1,2 segment of TOPBP1. (C) A fluorescently-labelled phospho-peptide centred on 53BP1-Thr670 binds to the BRCT0,1,2 segment of TOPBP1 with high affinity in FP assays. Treatment with ?-phosphatase abolishes binding, confirming that the interaction is specific for the phosphorylated peptide. (D) Charge-reversal mutation of Lys155, which is implicated in phospho-binding in BRCT1 has little effect on binding of the pThr670 peptide to BRCT0,1,2, whereas mutation of the equivalent residue, Lys250 in BRCT2 substantially decreases the affinity. Mutation of both sites completely abolishes the interaction. (E) No binding of 53BP1-derived phospho-peptides centred on Thr670 or Thr334 was detected with the BRCT4,5 segment of TOPBP1. (F) Fluorescently-labelled phospho-peptides centred on 53BP1-Ser366 and Ser380 bind with modest affinity to the TOPBP1 BRCT4,5 segment. (G) Treatment of the 53BP1-Ser366 phosphopeptide with ?-phosphatase or charge-reversal mutation of Lys704, which is implicated in phospho-binding in BRCT5, abolishes interaction of the phosphopeptide to BRCT4,5.

Fig 3: (A) siRNA knockdown of CK2a and/or CK2a'. Scr control siRNA was used in lanes 1 and 5. The top panels demonstrate the input proteins that were used in the immunoprecipitation (IP) with pS23-Ab (an antibody raised against an E2 peptide containing a phosphorylated serine 23). Please note the CK2a blot is independent of the other inputs but is run with the same protein extracts. The IP was blotted for E2 which is clearly detected in the Scr control (lane 5), but not in the CK2 knockdown cells (lanes 2 to 4). (B) The indicated cells were treated with DMSO (lanes 1 and 2) or the CK2 inhibitor CX4945 (lanes 3 and 4), and Western blotting details the levels of TopBP1 and E2 in the treated cells (top blots). Immunoprecipitation (IP) with TopBP1 demonstrates a pulldown of TopBP1 and E2 (lane 2) that is abrogated by CX4945 (lane 4) (middle blots). IP with pS23-Ab pulled down E2 in control cells (lane 2) that was abolished by CX4945 (lane 4). (C) CK2a siRNA knockdown (lane 4) disrupted the E2-TopBP1 interaction as there was a reduced co-IP of E2 in the absence of CK2a (lane 7). (D) CK2a' knockdown (lane 3) disrupted the E2-TopBP1 interaction as there was a reduced co-IP of E2 in the absence of CK2a' (lane 5). Please note that lanes 1 to 3 are from the same gel and the same exposure with a lane removed. (E) Staining of U2OS E2-WT (top panels) and U2OS E2-S23A (bottom panels) with pS23-Ab. Left-hand panels are antibody only, right-hand panels are antibody plus DAPI. There was no signal generated with secondary antibody only, and no signal detected in U2OS-Vec control when the primary antibody was included. HPV16 E2 B9 monoclonal antibody was used for Western blotting in panels A and B. E2-specific antibody TVG261 (ab17185) was used for Western blotting in panels C and D. Figure S2A and S2B summarizes quantitation for repeat experiments of panels C and D, respectively. An asterisk indicates an antibody band.

Fig 4: TOPBP1 is a novel TOP2A interacting protein that localises to UFBs(a) Proteins identified in IP/MS analysis of GFP-Trap purification from HEK 293FT cells expressing GFP-TOP2A. (b) Endogenous TOP2A co-immunoprecipitates with GFP-tagged TOPBP1. U2OS control cells or U2OS cells stably expressing GFP-TOPBP1 were untreated or treated with nocodazole (0.1 µg ml-1 for 16h) as indicated and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (c) Endogenous TOPBP1 co-immunoprecipitates with GFP-tagged TOP2A. U2OS control cells or U2OS cells stably expressing GFP-TOP2A were treated with nocodazole (0.1 µg ml-1 for 16h) and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (d and e) Immunofluorescence microscopy in U2OS cells with antibodies against TOPBP1; BLM serves as a positive control. DAPI serves as a DNA stain. (f) Immunofluorescence microscopy of HeLa cells with antibodies against TOPBP1. DAPI serves as a DNA stain. (g) Immunofluorescence in human primary cells (human dermal fibroblasts) using antibody against TOPBP1. DAPI serves as a DNA stain. Scale bar = 6.4 µm.

Fig 5: TOPBP1 physically couples 9-1-1 and 53BP1 complexes.(A) Schematic of domain architecture of TOPBP1 and interactions. The selective phosphorylation-dependent interactions of 9-1-1 and 53BP1 with different BRCT domains allow for the possibility of their simultaneous interaction with a single TOPBP1 molecule and their collaborative participation in ATR interaction. (B) 53BP1 and RAD9 immunofluorescence foci partially co-localise in irradiated RPE1 cells. Scale bar, 5 µm. (C) Proximity ligation assay (PLA) events (red) for RAD9 and 53BP1 demonstrating the occurrence of RAD9 and 53BP1 molecules within 30–40 nM of each other within the nuclei of irradiated RPE1 cells. Scale bar representing 50 µm and 5 µm are indicated. (D) Scatter plot of PLA events per nucleus for RAD9 – 53BP1 proximity as a function of nuclear Hoechst signal for irradiated (top) and non-irradiated (bottom) RPE1 cells. The PLA signal is predominantly seen in G1 cells (lower Hoechst staining) and is significantly increased by irradiation of the cells. (E) Statistical analysis of PLA events per nucleus in irradiated RPE1 cells shown in D,) showing a significant increase in PLA signals on irradiation. More than 500 nuclei were counted per case. Median, mean (+), 10–90 percentiles and outliers are represented in boxplots. p values for the irradiated versus non-irradiated cells were calculated by a Mann-Whitney test. (F) Statistical analysis of PLA events per nucleus in irradiated RPE1 cells transfected either with a control scrambled siRNA (SCR) or an siRNA directed against TOPBP1 (Figure 6—figure supplement 1C). A very significant decrease in PLA signal between 53BP1 and RAD9 when TOPBP1 is knocked down. More than 200 nuclei were counted per case. Median, mean (+), 10–90 percentiles and outliers are represented in boxplots. p values for the irradiated versus non-irradiated cells were calculated by a Mann-Whitney test. Comparable data for U2OS cells is presented in Figure 6—figure supplement 1B. (G) Statistical analysis of PLA events per nucleus between RAD9 and eYFP, in irradiated U2OS cells with siRNA knockdown of endogenous 53BP1, transfected with either wild-type eYFP-53BP1 or eYFP-53BP1 with S366A and/or T670A mutations. More than 200 nuclei were counted per case. Median, mean (+), 10–90 percentiles and outliers are represented in boxplots. p values for the mutant versus wild-type eYFP-53BP1 constructs were calculated by a Mann-Whitney test.