Journal: iScience

Article Title: Regulation of DNA damage response by trimeric G-proteins

doi: 10.1016/j.isci.2023.105973

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-pS957 SMC , Cell Signaling Technology , 5D11G5 (Clone); Cat# 4805, RRID: AB_2192322.

Techniques: Recombinant, Electron Microscopy, Labeling, Magnetic Beads, Protease Inhibitor, Membrane, CRISPR, Plasmid Preparation, Expressing, Software

Journal: Cell reports

Article Title: Competition between NBS1 and ATMIN controls ATM signaling pathway choice.

doi: 10.1016/j.celrep.2012.11.002

Figure Lengend Snippet: Figure 4. Antagonism and Redundancy of ATMIN and NBS1 for ATM Signaling and Function (A)H&E stainingonrepresentativeintestinesofmice with the indicated genotypes, 5 days after Villin- creERT-mediated deletion of Atmin and Nbs1. (B) High-magnification representation of H&E- stained crypts. (C)RepresentativeMCM6 IHCon intestines of mice with the indicated genotypes, 5 days after Villin- creERT-mediated deletion of Atmin and Nbs1. (D) Quantification of (C); **p = 0.0093. (E) Cells were treated with 20 Gy irradiation and fixed at the indicated time points post treatment. The percentage of subG1 cells was determined by flow cytometry analysis. In (D) and (E), error bars show the SD of at least three independent experiments, and Student’s t test was used for statistical analysis. (F) Four days after Villin-creERT-mediated dele- tion of Atmin and Nbs1, mice were treated with the indicated doses of irradiation (n = 5 mice per genotype per dose). (G) H&E staining on representative intestines of mice with the indicated genotypes, 24 hr after treatment with 14 Gy irradiation. (H) Representative 53BP1 IHC in intestines of mice with the indicated genotypes, 24 hr after treatment with 14 Gy irradiation. Arrowheads in- dicate cells with 53BP1 foci. (I) Mice were culled 2 hr after 10 Gy irradiation, and proteinlysateswere prepared from intestinal tissue. Lysates were resolved on a 6% SDS-PAGE gel and the membrane was probed for pS1987-ATM, pS957-SMC1, SMC, pS15-p53, p53, and actin. See also Figure S4.

Article Snippet: The following antibodies were used: pS1981-ATM (10H11.E12; Cell Signaling), ATM (2C1; Santa Cruz), pS824-Kap1 (Bethyl Laboratories), Kap1 (Bethyl Laboratories), b-actin (A5060; Sigma), pS957-SMC1 (5D11G5; Millipore), SMC1 (AB3908; Millipore), pS966-SMC1 (A300-050A; Bethyl Laboratories), p53 (2524; Cell Signaling), NBS1 (NB100-143, Novus Biologicals), DHX9 (gift from Professor F. Grosse), pS15-p53 (9284 and 9286; Cell Signaling), pATM (2152-1, Epitomics), horseradish-peroxidase-conjugated goat anti-mouse/rabbit immunoglobulin G (Jackson).

Techniques: Staining, Irradiation, Cytometry, SDS Page, Membrane

Journal: Nucleic Acids Research

Article Title: ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

doi: 10.1093/nar/gkv754

Figure Lengend Snippet: ATM-dependent phosphorylation of MRE11, its dependence on the MRN complex and identification of specific sites using invitro kinase assays. ( A ) Detection of MRE11 phosphorylation in cells treated with 10 Gy (+) or mock treated (−) by western blotting (WB) with a pSQ/pTQ antibody of immunoprecipitated (IP) MRE11 from both control and A-T cells. ( B ) Detection of MRE11 phosphorylation and MRN complex members in cells treated with 10 Gy IR (+) or mock treated (−) by western blotting (WB) with a pSQ/pTQ antibody and antibodies to the MRN complex members from control, A-T and NBS patient cells. ( C ) Schematic of putative ATM phosphorylation sites denoted by the target residues (↓), serine glutamine (SQ) or threonine glutamine(TQ) within the MRE11 protein with the three characterized domains of MRE11 shown, the nuclease domain and the two DNA binding domains (DBD) 1 and 2. The three overlapping GST tagged MRE11 fragments are shown. GST-MRE11A: 1-273aa; GST-MRE11B: 223-525aa; GST-MRE11C: 487-708aa and the SQ and TQ sites they each contain are specified. ( D ) ATM phosphorylates GST-MRE11C containing four putative SQ sites. Invitro kinase assays of immunoprecipitated ATM incubated with MRE11 GST fragments A, B and C with 10 Gy IR (+) or without (−) in both control cells and A-T cells. Autoradiographs and Coomassie staining are shown for each of the GST's spanning MRE11 A–C. ( E ) Alanine mutation of S676 and S678 cause a loss of GST-MRE11C phosphorylation. GST-MRE11C, containing four putative ATM SQ phosphorylation sites, were each mutated to alanine and used in ATM kinase assays to identify specific sites of phosphorylation after 10 Gy (+) or mock treatment (−).

Article Snippet: Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Control, Binding Assay, Incubation, Staining, Mutagenesis

Journal: Nucleic Acids Research

Article Title: ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

doi: 10.1093/nar/gkv754

Figure Lengend Snippet: Investigation of MRE11 phosphorylation kinetics at MRE11S676S678. ( A ) MRE11pS676pS678 antibody immunoprecipitate the phosphorylated form of MRE11 after ionizing radiation (IR). Western blot (MRE11) of immunoprecipitated MRE11 using an antibody against MRE11S676S678 (nonP) site and the corresponding phosphorylated MRE11pS676pS678 (pSpS) sites from control and ATLD2 lymphoblastoid cells treated with 0 or 10 Gy IR. Unbound sample from each immunoprecipitation was run in parallel and also immunoblotted for total MRE11 (GeneTex). ( B ) Dose-dependent increase of MRE11 phosphorylation following IR. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control and A-T lymphoblastoid cells treated with 0, 2, 5 or 10 Gy IR. ATLD2 cells were also run as a negative control (0 and 10 Gy). Western blot for the MRN complex proteins, MRE11, RAD50 and NBS1. ( C ) Increasing MRE11 phosphorylation over 2 h post 5 Gy. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control lymphoblastoid cells either untreated (−) or harvested at 0.6, 2, 4 or 10 h post 5 Gy IR. ATLD2 lymphoblastoid cells were either untreated (−) or harvested at 2 and 10 h post 5 Gy IR and run as a negative control. ( D ) MRE11 phosphorylation in response to DNA double strand break inducing agents. Western blot of immunoprecipitated MRE11 using an antibody against MRE11pS676pS678 (pSpSMRE11) sites from control cells treated with either IR, camptothecin (CPT), hydrogen peroxide (H2O2), etoposide (ETOP), cisplatin (CISP) or methlymethanesulfonate (MMS).

Article Snippet: Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Control, Negative Control

Journal: Nucleic Acids Research

Article Title: ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

doi: 10.1093/nar/gkv754

Figure Lengend Snippet: Investigation of ATM signalling, cell survival and chromosomal aberrations in the cell line expressing non-phosphorylatable MRE11S676AS678A. ( A ) Stable MRN complex in WT and non-phosphorylatable mutant MRE11 corrected cell lines. Western blot of immunoprecipitated MRE11 from control (MCR5) and ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) cell lines. Also immunoblotted for NBS1 and RAD50. ( B ) ATM signalling in the non-phosphorylatable mutant MRE11 cell line is comparable to the WT corrected cell line. Total cell extracts from ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) cell lines were extracted 30 min post 5 Gy (+) or left as unirradiated controls (−) and western blotted for ATM S1981 autophosphorylation and total ATM, as well as the ATM kinase substrates, SMC1 and KAP1. Tubulin was immunoblotted as loading control. ( C ) The non-phosphorylatable mutant MRE11 cell line displays increased cellular sensitivity to IR. Plot of percent survival after 0, 1, 2, 3, 4 and 5 Gy IR. NFF (control), ATLDMRE11 (WT), ATLDS676AS678A (MUT), ATLDVEC (VEC), A-T, ATLDS676A (S676A) and ATLDS678A (S678A) cell lines were treated with increasing doses of IR and percent survival assessed by clonogenic cell survival assay. The mean ± standard deviation is plotted from 3 independent experiments. ( D ) Non-phosphorylatable mutant MRE11 cell line is unable to correct radiation induced chromosomal aberrations. Aberrations (chromatid breaks, chromosome breaks and interchanges) were scored from Giemsa stained metaphases in 2 Gy irradiated NFF (control), A-T, ATLDMRE11 (WT), ATLDS676AS678A (MUT), ATLDVEC (VEC), ATLDS676A (S676A) and ATLDS678A (S678A) cell lines. Induced chromosomal aberrations (ICA) were totalled and divided by number of metaphases as indicated in parentheses.

Article Snippet: Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).

Techniques: Expressing, Mutagenesis, Western Blot, Immunoprecipitation, Control, Clonogenic Cell Survival Assay, Standard Deviation, Staining, Irradiation

Journal: Nucleic Acids Research

Article Title: ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

doi: 10.1093/nar/gkv754

Figure Lengend Snippet: Phosphorylation status does not affect recruitment of YFP tagged MRE11 to sites of breaks or its loss from these sites but DNA repair is defective. ( A ) YFP tagged MRE11 (green) co-localized with γH2AX (red) to carbon ion tracks of damage in U2OS cells. YFP-MRE11 (WT), YFP-MRE11S676AS678A (MUT) or phosphomimetic YFP-MRE11S676DS678D (DD) were transfected into U2OS cells, fixed 10 min post damage and counterstained with DAPI (blue). ( B ) Real time quantitation of YFP tagged MRE11 localizing to sites of damage. YFP-MRE11 (WT), YFPMRE11S676AS678A (MUT) or phosphomimetic YFP-MRE11S676DS678D (DD) in U2OS cells being recruited to sites of carbon ion damage during the first 10 min post damage. ( C ) Dissociation of YFP tagged MRE11 (green) from carbon ion tracks of damage in U2OS cells. YFP-MRE11 (WT), YFP-MRE11S676AS678A (MUT) or phosphomimetic YFP-MRE11S676DS678D (DD) were transfected into U2OS and samples fixed as unirradiated (UNIR), 10 min, 4 or 12 h post damage then immunostained with γH2AX (red) and counterstained with DAPI (blue). The bar represents 10 μm. ( D ) Real time quantitation of YFP tagged MRE11 localizing to sites of damage. YFP-MRE11 (WT), YFPMRE11S676A (S676A) or YFP-MRE11S678A (S678A) in U2OS cells being recruited to sites of uranium ion damage during the first 10 min post damage. ( E ) Reduced repair efficiency of DSBs in the non-phosphorylatable mutant and vector cell lines. The formation and repair of DSBs marked by γH2AX immunostaining in response to 2 Gy IR. Foci were counted from three independent experiments and the mean and standard deviation plotted.

Article Snippet: Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).

Techniques: Phospho-proteomics, Transfection, Quantitation Assay, Mutagenesis, Plasmid Preparation, Immunostaining, Standard Deviation

Journal: Nucleic Acids Research

Article Title: ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

doi: 10.1093/nar/gkv754

Figure Lengend Snippet: Defect in ATM phosphorylation of Exonuclease 1 in the MRE11S676AS678A mutant cell line. ( A ) Immunostaining of γH2AX (red) and EXO1pS714 foci (green) in response to 5 Gy IR for each cell line ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) as well as NFF (control) and A-T cell lines after 30 min. The merged image including DAPI staining is shown and the boxed cell has been enlarged in the far right panel. ( B ) Quantitation of Exonuclease 1 (EXO1pS714) foci in unirradiated (UNIR) and 5 Gy irradiated cells for each cell line ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) as well at NFF (control) and A-T cell lines after 30 min. The mean and standard deviation of three independent experiments is plotted. ( C ) Western blot of total cell extracts from ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) cell lines for phosphorylated (S714) Exonuclease 1 (pEXO1) and total Exonuclease 1 (EXO1) from unirradiated (−) and 5 Gy irradiated cells (+). PCNA is shown as loading control. ( D ) Western blot of total cell extracts from ATLDMRE11 (WT), ATLDS676AS678A (MUT) and ATLDVEC (VEC) cell lines for Exonuclease 1 (EXO1) with (+) and without (−) knockdown of Exonuclease1 by esiRNA (Sigma) after 48 h. SMC1 is shown as loading control. ( E ) Effect of Exonuclease 1 knockdown on the proportion of G2 phase cells with RPA foci for ATLDMRE11, ATLDS676AS678A and ATLDVEC cell lines. Cells were transfected with esiRNA (WT siRNA, MUT siRNA and VEC siRNA) or mock transfected (WT, MUT and VEC) for 24 h. Cells were irradiated with 5 Gy and immunostained with CENPF and RPA then G2 positive cells counted containing >5 foci after 2 h. The mean and standard deviation of three independent experiments is plotted. ( F ) Schematic model showing the role of MRE11 phosphorylation by ATM in response to DSB induction. The occurrence of DSB activates the DSB signalling cascade involving autophosphorylated ATM (ATMP) to initiate its repair. After the induction of resection ATMP phosphorylates MRE11 at S676 and S678 (WT MRE11PP) which acts as an adaptor for ATMP-dependent phosphorylation of Exonuclease 1 (EXO1P), controlling the extent of resection. This enables RAD51 to catalyze strand invasion and subsequent completion of HR repair or subsequent repair by the other pathways requiring end processing like microhomology-mediated non-homolgous recombination (mNHEJ) and SSA. If MRE11 is not phosphorylatable (MRE11S676AS678A) ATM is still normally autophosphorylated (ATMP) leading to induction of end processing, but in the absence of MRE11 phosphorylation there is no subsequent ATM-dependent phosphorylation of Exonuclease 1 (EXO1) leading to over extended resection by EXO1. Although RAD51 is recruited it largely fails to catalyze efficient strand invasion leading to a failure of the less error prone HR. But the SSA pathway is still functional as it can tolerate the extensively resected single stranded DNA unlike the other forms of homology directed repair.

Article Snippet: Whole cell extracts or immune complexes were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and proteins transferred to nitrocellulose membranes using Towbin's buffer (20% methanol, 50 mM Tris, 40 mM glycine and 0.02% SDS) at 100 V for 1 h. Blots were incubated with antibodies against MRE11 (12D7; GeneTex), Phospho-SQ/TQ (Cell Signaling Technologies), RAD50 (Upstate), NBS1 (Novus Biologicals), ATM (2C1; GeneTex), ATM pS1981 (GeneTex), SMC1 and SMC1 pS957 (GeneTex), Kap1 and Kap1 p824 (Novus Biologicals), GAPDH (GeneTex) and GFP (Abcam).

Techniques: Phospho-proteomics, Mutagenesis, Immunostaining, Control, Staining, Quantitation Assay, Irradiation, Standard Deviation, Western Blot, Knockdown, esiRNA, Transfection, Functional Assay

Journal: Cell reports

Article Title: Competition between NBS1 and ATMIN controls ATM signaling pathway choice.

doi: 10.1016/j.celrep.2012.11.002

Figure Lengend Snippet: Figure 4. Antagonism and Redundancy of ATMIN and NBS1 for ATM Signaling and Function (A)H&E stainingonrepresentativeintestinesofmice with the indicated genotypes, 5 days after Villin- creERT-mediated deletion of Atmin and Nbs1. (B) High-magnification representation of H&E- stained crypts. (C)RepresentativeMCM6 IHCon intestines of mice with the indicated genotypes, 5 days after Villin- creERT-mediated deletion of Atmin and Nbs1. (D) Quantification of (C); **p = 0.0093. (E) Cells were treated with 20 Gy irradiation and fixed at the indicated time points post treatment. The percentage of subG1 cells was determined by flow cytometry analysis. In (D) and (E), error bars show the SD of at least three independent experiments, and Student’s t test was used for statistical analysis. (F) Four days after Villin-creERT-mediated dele- tion of Atmin and Nbs1, mice were treated with the indicated doses of irradiation (n = 5 mice per genotype per dose). (G) H&E staining on representative intestines of mice with the indicated genotypes, 24 hr after treatment with 14 Gy irradiation. (H) Representative 53BP1 IHC in intestines of mice with the indicated genotypes, 24 hr after treatment with 14 Gy irradiation. Arrowheads in- dicate cells with 53BP1 foci. (I) Mice were culled 2 hr after 10 Gy irradiation, and proteinlysateswere prepared from intestinal tissue. Lysates were resolved on a 6% SDS-PAGE gel and the membrane was probed for pS1987-ATM, pS957-SMC1, SMC, pS15-p53, p53, and actin. See also Figure S4.

Article Snippet: The following antibodies were used: pS1981-ATM (10H11.E12; Cell Signaling), ATM (2C1; Santa Cruz), pS824-Kap1 (Bethyl Laboratories), Kap1 (Bethyl Laboratories), b-actin (A5060; Sigma), pS957-SMC1 (5D11G5; Millipore), SMC1 (AB3908; Millipore), pS966-SMC1 (A300-050A; Bethyl Laboratories), p53 (2524; Cell Signaling), NBS1 (NB100-143, Novus Biologicals), DHX9 (gift from Professor F. Grosse), pS15-p53 (9284 and 9286; Cell Signaling), pATM (2152-1, Epitomics), horseradish-peroxidase-conjugated goat anti-mouse/rabbit immunoglobulin G (Jackson).

Techniques: Staining, Irradiation, Cytometry, SDS Page, Membrane