Fig 2: Interactions of nGRB2 with DNA damage repair factors.(A) MS identifies DDR protein associated with GRB2. Mean of coverage and unique peptides from three independent data sets are shown. (B) Strep-GRB2 precipitated from HEK293T cells followed by immunoblotting with indicated antibodies. (C) Strep-GRB2 expressed HEK293T cells treated with or without 5-Gy IR were lysed immediately followed by Strep-Tactin precipitation and immunoblotting with indicated antibodies. (D) HEK293T cells were cotransfected with red fluorescent protein (RFP)–GRB2 and green fluorescent protein (GFP)–NBS1 or GFP alone as control, precipitated with GFP-trap beads, and immunoblotted with indicated antibodies. (E) HEK293T cells expressing Flag-RAD50 or Flag-Ctrl were cotransfected with RFP-GRB2 for 24 hours. Unperturbed cells were immunoprecipitated with Flag-M2 beads and immunoblotted with indicated antibodies. (F) HEK293T cells expressing Flag-MRE11 were cotransfected with Strep-GRB2 or Strep-Ctrl for 24 hours. Unperturbed or IR-treated (5 Gy) cells were immunoprecipitated with Flag-M2 beads and immunoblotted with indicated antibodies. (G) Strep-Tactin precipitation of WT and K109R mutant Strep-GRB2 from HEK293T cells that were unperturbed or IR treated (5 Gy) and lysed, followed by Western blot detection with the indicated antibodies. (H) MST isotherms of 100 nM Atto488–labeled WT, K109R, or K109A mutant to titrating concentrations of human MRE11 (residues 1 to 411). All data are representative of three independent experiments.

Fig 3: MRNIP condensation promotes DNA end resection.a, b MRNIP condensates promoted MRE11/RAD50-mediated dsDNA degradation in vitro. c The impact of PEG-8000 on MRNIP participated degradation of dsDNA by MRE11/RAD50 complex. For (b, c), the final concentration of GFP, MRNIP-GFP or ?IDR1-GFP was 10 µM (b) and 2 µM (c). d, e ssDNA fiber length was examined via IF assay with anti-BrdU antibody. Data are presented as means ± SEM. Two-tailed unpaired Student’s t test. d, n = 40 (WT), n = 59 (KO-MRNIP). e n = 41 (EV), n = 52 (MRNIP-GFP), n = 50 (?IDR1). f In situ detection of ssDNA in cells confirmed that MRNIP condensates promote radiation-induced ssDNA formation. Data are presented as means ± SEM. Two-tailed unpaired Student’s t test. Middle panel: n = 3 biological replicates; Right panel: n = 91 (EV), n = 95 (MRNIP-GFP), n = 78 (?IDR1). g Restoration of MRNIP in HeLa-MRNIP-KO cells increased RAD51 foci. Data are presented as means ± SEM. Two-tailed unpaired Student’s t test. Middle panel: n = 3 biological replicates; Right panel: n = 205 (EV), n = 211 (MRNIP-GFP), n = 167 (?IDR1). h MRNIP knockout reduced radiation-induced RPA1 foci. Data are presented as means ± SEM; n = 3 biological replicates. Two-tailed unpaired Student’s t test. i Restoration of MRNIP in HeLa-MRNIP-KO cells increased RPA1 foci. Data are presented as means ± SEM, n = 3 biological replicates. Two-tailed unpaired Student’s t test. For (d–i), cells were treated with 10 Gy X-rays and recovered for 8 h (f), 4 h (g) or 1 h (h, i) before analysis. For (e–g, i), HeLa-KO-MRNIP cells stably expressing sgRNA-resistant MRNIP-GFP, MRNIP-?IDR1-GFP (?IDR1) and empty vector (EV) were used. ns, no significance.

Fig 4: shRNA-mediated CHK1 knockdown reduces G2/M arrest and HR repair in K562 cells. (A) The cell cycle distribution of K562 cells transduced with shNC or shCHK1 (shKD) was detected following treatment with DMSO (control) or VP16. (B) Statistical analysis of the G2/M phase percentages (%) shown in A (*P<0.05, **P<0.01). (C) Western blot analysis of the G2/M arrest markers pS216CDC25c, pY15CDK1 and total CDC25c and CDK1 in K562 cells infected with lentivirus carrying shVector, shNC or shCHK1 (shKD) under normal conditions and in the presence of DMSO or VP16. (D) Western blot analysis of the mitotic marker pS10-H3 and total H3 in K562 cells infected with lentivirus carrying shVector, shNC or shCHK1 (shKD) under normal conditions and in the presence of DMSO or VP16. (E) Western blot analysis of the NHEJ pathway-related proteins Ku70, Ku80 and ligase 4 (Lig4) in shVector-, shNC-, or shCHK1-transduced (shKD) K562 cells under normal conditions and after treatment with DMSO or VP16. (F) Relative quantification of Ku70 (a), Ku80 (b) and Lig4 (c) expression shown in E by densitometric analysis; target band intensity was normalized to that of GAPDH. (G) Western blot analysis of the HR pathway-related proteins BRCA1, Rad50, MRE11 and Rad51 in shVector-, shNC-, or shCHK1-transduced (shKD) K562 cells under normal conditions and after treatment with DMSO or VP16. (H) Relative quantification of BRCA1 (a), Rad50 (b), MRE11 (c) and Rad51 (d) expression shown in G by densitometric analysis; band intensity was normalized to that of GAPDH (*P<0.05). NS, not significant. CHK1, checkpoint kinase 1; HR, homologous recombination; VP16, etoposide.

Fig 5: PDZ-7 induces DNA damage response and is selectively cytotoxic towards DNA repair-deficient cells(A) Induction of DNA-damage response over the course of PDZ-7 treatment. A549 cells were treated with 250 nM PDZ-7 for 1 hour and post-incubated in drug-free medium up to 72 hours. Activation of MRN complex was determined by Western Blotting, using antibodies towards Nbs1 phosphorylated at Ser343 (p-Nbs1), Mre11 at Ser676 (p-Mre11) and total Rad50. (B) CHO AA8-derived UV sensitive mutants with defects in nucleotide excision repair pathway displayed specific sensitivity to PDZ-7. Nuclease-deficient variants UV41 (XPF-) and UV135 (XPG-) were 4.0 and 2.2 times more sensitive (IC50 value), compared to cells with defects in damage recognition (UV61, CSB-) and in helicase activity (UV5, XPD-). Loss of Ku80 protein involved in NHEJ repair in xrs6 cells amounted to 2.2-fold increase in sensitivity in comparison to parent CHO-K1 cells. V-C8 cells deficient in DNA damage repair via HR (BRCA2-) had 53-fold lower PDZ 7 IC50 value in comparison to parent V79 cells. Statistical significance was determined using two-tailed Student t-test.