Fig 2: WIP1 delays recruitment of BRCA1 and dephosphorylation of 53BP1 at T543. (A) Co-immunoprecipitation of WIP1 and 53BP1. HEK293 cells were transfected with either empty GFP or GFP-WIP1, subjected to immunoprecipitation using GFP-Trap 24 h after transfection and by Western blotting with 53BP1 antibody. Ponceau staining with indicated positions of GFP (empty arrowhead) and GFP-WIP1 (full arrowhead) are shown. (B) HEK293 cells transfected with EGFP or EGFP-WIP1 were exposed to 3 Gy of IR, collected at indicated times and proteins were immunoprecipitated by GFP Trap. (C) Quantification of 53BP1 pT543 signal intensity in replicating (EdU+) cells after irradiation. U2OS parental and WIP1 knockout cell lines were pulse-labeled with EdU for 30 min before irradiation. Cells were fixed after pre-extraction at indicated time-points after IR and stained with p53BP1 T543 antibody. Click chemistry was used to visualize EdU. Mean of median total intensity +/- SD is plotted. (D) Western blot analysis of whole cell lysates of U2OS cells transfected with GAPDH or PP4C siRNA in response to irradiation and/or WIP1 inhibitor. (E) Quantification of 53BP1 pT543 signal intensity in replicating (EdU+) cells after irradiation. U2OS parental and WIP1 knockout cell lines were transfected with control or PP4C siRNA 2 days before irradiation. Cells were processed and analyzed as in C. (F) Quantification of RPA2 foci in replicating (EdU+) cells after irradiation. U2OS parental cell lines with or without combined treatment with WIP1i were pulse-labeled with EdU for 30 minutes before irradiation. Cells were fixed after pre-extraction at indicated time-points and stained with RPA2 and RAD51 antibodies. Click chemistry was used to visualize EdU. Mean of median foci number +/- SD is plotted. (G) Quantification of RAD51 foci in replicating (EdU+) cells after irradiation as in F.

Fig 3: RIF1 inhibits replication initiation after exposure to high IR doses but not after low IR doses. (A) DNA synthesis after exposure to 3 Gy IR in siCtrl and siRIF1 cells. Incorporation of EdU was measured 1 h after irradiation using a flow cytometer. (B) Schematic illustration of pulse-labeling and PCNA staining. HeLa cells synchronized in mid-S phase were pulse-labeled with EdU and stained with anti-PCNA antibodies after exposure to 0.5 Gy or 3 Gy of IR. (C,D) Time course of PCNA and EdU foci formation in non-irradiated HeLa cells. PCNA foci, scored as either colocalizing with EdU foci (>0.05 µm2 overlap area, PCNA with EdU) or not (<0.05 µm2 overlap area, PCNA w/o EdU), were quantified in each nucleus. Data are the mean±s.d. Boxes in C indicate regions shown in magnified images. Scale bar: 10 µm. (E) Transient replication block after exposure to 3 Gy of IR. After pulse-labeling, HeLa cells were irradiated with 0.5 Gy or 3 Gy and were fixed 1 or 3 h after IR exposure. Cells were stained for EdU and PCNA foci formation. PCNA foci without EdU foci were counted. (F) Restoration of the replication block after RIF1 depletion. HeLa cells transfected with siCtrl or siRIF1 were irradiated with 3 Gy and were fixed 1 h after IR exposure. PCNA foci without EdU foci were counted. Data are the mean±s.d. of three independent experiments. P values indicated were calculated using a Chi-squared test. (G,H) DNA synthesis at DSB sites was measured by the colocalization of EdU with 53BP1. HeLa cells transfected with either siCtrl or siRIF1 were irradiated with 3 Gy and fixed 1 h after IR exposure. Cells were stained for EdU and 53BP1 foci formation. Frequency of 53BP1 foci colocalized with EdU were measured. Data in H are mean±s.d. and the P value indicated was calculated using a Student's t-test. Scale bar: 10 µm.

Fig 4: ATM kinase is a potential therapeutic target for host directed TB therapy.(a) Graphical representation depicting Rv load at day 1 (n = 4) and day 56 (n = 5) of infection in lungs and spleen of mice. Lung homogenates prepared at indicated time points were plated on 7H11 plates to enumerate CFU per lung or per spleen (b) Lung and spleen lysates were prepared from uninfected and Rv infected mice 56 days p.i. 100 µg lung lysates were subjected to immunoblotting with a-?H2AX, a-pATM(S1981) a-ATM, a-pCHK2(T68), a-CHK2, a-pAkt (S473), a-Akt, a-p53 and a-ß-actin antibodies. (c) PF were infected with Rv at high MOI (1:10) as described above. 24 h p.i, cells were treated with either 3.6 µM isoniazid (INH) or 10 µM ATM-I alone or INH + ATM-I together. CFUs were enumerated at 24, 48 and 72 hr post treatment. (d) Schematic representation of the mice infection and drug treatment protocol used. (e–f) CFUs were enumerated in lungs of mice at Day 1, and in the lungs (e) and spleen (f) on day 15 and 30 of infection. Number of mice in each batch was 6 (n = 6) except in Rv at Day 1(n = 5). Error bar, SD. *, p=0.05; **, p=0.005; ***, p=0.0005. (g) Model depicting the findings. Rv induces genotoxicity and causes deleterious DSBs in the host genome through SecA2 secretome. Host cell in response to the occurrence of DSBs activate ATM kinase and is recruited at the site of damage by the sensor, MRN complex. Activated ATM autophosphorylates itself and phosphorylates H2AX in the chromatin flanking the sites of DNA damage, which becomes the foundation for the recruitment of mediator protein MDC1, thus amplifying DDR. pATM promote recruitment of 53BP1 at the damage site. pATM as a part of DDR also activates downstream effectors, Chk2 and p53, which are responsible for alterations in the host cell cycle. pATM in a parallel pathway also activates Akt, which is known inhibit apoptosis and promote cell survival. Activation of ATM and Akt and subsequent inhibition of apoptosis provides survival advantage to Rv. Inhibition of ATM or Akt activation through inhibitors, ATM-I or Akt-I, respectively, promote host cell apoptosis, which impedes the bacilli growth. Phosphorylation status of proteins is depicted with a P in a circle.Figure 7—source data 1.ATM kinase is a potential therapeutic target for host directed TB therapy.Source Data for Figure 7a, c, e and f.

Fig 5: BRCA1 antagonizes ATM-dependent RIF1 accumulation at low IR doses. (A,B) Marginal formation of 0.5 Gy-induced foci of RIF1 and phospho-53BP1 in S-phase cells. HeLa cells were synchronized with a double thymidine block in G1 or S phases and were irradiated with 0.5 Gy or 3 Gy of IR. Cells were fixed and stained with anti-RIF1 (A) or anti-phospho-53BP1 (B) antibodies 0.5 h after irradiation and the number of foci per nucleus was quantified. (C) Phosphorylation of 53BP1 and CHK2 after irradiation. HeLa cells synchronized in G1 or S phase were irradiated and collected 1 h after irradiation for western blotting with the indicated antibodies. ß-actin is shown as a loading control. (D,E) Phospho-53BP1 (D) or RIF1 (E) foci formation after irradiation in BRCA1-depleted and BRCA1-overexpressing cells. HeLa cells transfected with siCtrl, siBRCA1 or a BRCA1-expressing vector were stained with anti-phospho-53BP1 or anti-RIF1 antibodies 0.5 h after IR exposure of 0.5 Gy or 3 Gy. (F) Enhanced RAD51 focus formation after exposure to 3 Gy with BRCA1 overexpression. HeLa cells transfected with empty or BRCA1-expressing vectors were stained for RAD51 focus formation 1 h after exposure to 0.5 Gy or 3 Gy. Data in A,B,D–F are mean±s.d., and the P values indicated were calculated by two-way ANOVA followed by Tukey's multiple comparisons test.