Journal: International Journal of Molecular Sciences

Article Title: Wwox Binding to the Murine Brca1-BRCT Domain Regulates Timing of Brip1 and CtIP Phospho-Protein Interactions with This Domain at DNA Double-Strand Breaks, and Repair Pathway Choice

doi: 10.3390/ijms23073729

Figure Lengend Snippet: Wwox loss supports rapid initiation of HR through end-resection ( A ) Wwox expression delays the end-resection process. Plots of the ranges of ssDNA length vs. resected ssDNA frequency calculated by SMART assay. Long resected ssDNAs (5–10 μm) are detected in Wwox KO MEFs 1 h post-IR (10 γ) vs. the shorter ssDNA (1–2 μm) present in WT cells. The left panel shows representative images of ssDNA stained by BrdU 1 h post-IR (10 γ). ( B ) Immunofluorescence of pRPA in WT and Wwox KO MEFs show more pRPA recruitment in the KO cells at 0.5 h after 10 γ IR. Experiments were carried out using two different wild-type (WT3, WT4) and Wwox KO (KO3, KO5) clones in duplicate; ( C ) Immunoblot showing pRPA recruitment into chromatin fraction in Wwox KO vs. WT MEFs after IR. ( D ) Wwox silencing increases steady-state levels of Brca1-C complex proteins including CtIP, Rad50, Nbs1, and Mre11 in three different mouse cells (immortalized MEFs, mammary tumor (31-03) and pancreatic tumor cells (KPC-2)). ( E ) Wwox silencing enhances HR repair following ISce-1-induced DSBs in MEF/DR-GFP reporter cells. Bar graph shows % cells positive for GFP as an indication of relative HR, with corresponding immunoblots of cells carrying HR reporter, DR-GFP, in cells silenced for Wwox expression. ( F ) IR-induced Rad51 foci are increased in Wwox KO MEFs. Experiments were carried out using two different wild-type (WT3, WT4) and KO (KO3, KO5) clones in duplicate. ( G ) DSB repair in Wwox negative cells increases mutation acquisition. Frequency of mutations of the HPRT gene is increased in Wwox KO vs. wild-type WT MEFs following IR exposure. Survival data for the two WT and two KO clones are presented as averages. Error bars represent S.E.M and p -values were calculated by an unpaired t-test (* p < 0.05, ** p < 0.01, *** p < 0.001); N.S., not significant.

Article Snippet: Mre11 , Cell signaling, #4895S , 1:1000 , , .

Techniques: Expressing, Staining, Immunofluorescence, Clone Assay, Western Blot, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Wwox Binding to the Murine Brca1-BRCT Domain Regulates Timing of Brip1 and CtIP Phospho-Protein Interactions with This Domain at DNA Double-Strand Breaks, and Repair Pathway Choice

doi: 10.3390/ijms23073729

Figure Lengend Snippet: Antisera Information.

Article Snippet: Mre11 , Cell signaling, #4895S , 1:1000 , , .

Techniques: Magnetic Beads

Journal: Nature Communications

Article Title: MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts

doi: 10.1038/s41467-019-12271-w

Figure Lengend Snippet: Conserved RNaseH-dependence and R-loop accumulation in human cells depleted for MRN. a Cell viability of RNaseH2A normal and knockout cells depleted for RAD50 , MRE11, or NBS1 as measured by crystal violet stain ( N = 4; * P < 0.05 by t test; mean ± SEM) showed observed viability values were significantly lower than expected values after depletion of RAD50 and NBS1 . b Relative S9.6 staining intensity per nucleus in MRN complex depleted cells. N = 3; **** P < 0.0001 by ANOVA; mean ± SD. c Representative images (left) and quantification (right) of S9.6 staining per nucleus in HeLa cells treated with si-Cont or si-RAD50. Cells were transfected with either a control vector (GFP) or one expressing GFP-RNaseH1 (GFP-RNH1). N = 3; **** P < 0.0001 by t test; mean ± SD. d DRIP-qPCR analysis of R-loop accumulation at BTBD and TFPT loci in control and si-RAD50 cells with or without RNaseH treatment before precipitation. N = 3; * P < 0.05 by t test; mean ± SEM. e and f RNaseH1-dependent DNA damage phenotypes in RAD50-depleted cells shown by γ-H2AX staining and neutral comet assay for DNA breaks. e γ-H2AX staining ( N = 4; **** P < 0.0001 by Fisher’s exact test; mean ± SD) and f neutral comet assay ( N = 3; *** P < 0.001 and **** P < 0.0001 by t test; mean ± SEM). For c – f and some panels in subsequent Figs. ( , , , and cartoon schematics of an R-loop illustrate the effects being tested by the experiment are shown

Article Snippet: Equivalent amounts of protein were resolved by SDS-PAGE and transferred to polyvinylidene fluoride microporous membrane (Millipore), blocked with 5% skim milk in TBS containing 0.1% Tween-20 (TBS-T), and membranes were probed with the following antibodies: RAD50 (1:1000, [13B3/2C6]ab89, abcam), γH2AX (1:5000, [EP854(2)Y]ab81299 abcam), FANCM (1:1000, ab95014,abcam), NBS1 (1:1000, A300-187A-T, Bethyl laboratories), MRE11 (1:1000, A300-181A-T, Bethyl laboratories), RNaseH2A (1:1000, A304-149A, Bethyl laboratories), AQR (1:1000, A302-547A, Bethyl laboratories), H2AX (1:500, [D17A3]XP #7631, cell signaling), p-ATM (1:500, (10H11.E12) sc-47739, Santa Cruz), p-CHK2 (Th468) (1:1000, C13C1, cell signaling), p-ATR (Ser428) (1:1000, #2853, cell signaling), p-Chk1 (Ser345) (1:1000, 133D3, cell signaling), FANCD2 (1:1000, NB100-182SS, Novus), GFP Tag (GF28R) (1:4000, MA5-15256, ThermoFisher Scientific), GAPDH (GA1R) (1:3000, MA5-15738, ThermoFisher Scientific), and α-tubulin (1:3000, B-5-1–2, ThermoFisher Scientific).

Techniques: Knock-Out, Staining, Transfection, Control, Plasmid Preparation, Expressing, Neutral Comet Assay

Journal: Nature Communications

Article Title: MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts

doi: 10.1038/s41467-019-12271-w

Figure Lengend Snippet: Transcription–replication conflicts impair fork progression in MRN-depleted cells. a Regulation of transcription–replication conflicts by RAD50. Proximity ligation assay targeting the replisome (anti-PCNA) and RNA polymerase II (anti-RNA Pol II) is shown with representative images (left) and quantification (right). N = 3; **** P < 0.0001 by t test; mean ± SEM. b Transcription-dependent replisome slowing in RAD50-depleted cells. Cells were treated with 50 μ m cordycepin (CORD) for 2 h before IdU labeling. c R-loop-dependent replisome slowing in RAD50-depleted cells. For b and c , an experimental scheme (left), representative DNA fibers from the indicated conditions (middle), and quantified CldU track lengths are shown (right). N = 3; **** P < 0.0001 by t test; mean ± SD. d and e SIRF analysis of Mre11 binding to EdU-labeled nascent DNA during replication stress. PlaB or HU treatment promotes recruitment of Mre11 to forks. This recruitment is R-loop-dependent in PlaB-treated cells. Representative images ( d ), and quantification ( e ) are shown. N = 3 for control and PlaB, N = 2 for HU; **** P < 0.0001 by ANOVA; mean ± SD

Article Snippet: Equivalent amounts of protein were resolved by SDS-PAGE and transferred to polyvinylidene fluoride microporous membrane (Millipore), blocked with 5% skim milk in TBS containing 0.1% Tween-20 (TBS-T), and membranes were probed with the following antibodies: RAD50 (1:1000, [13B3/2C6]ab89, abcam), γH2AX (1:5000, [EP854(2)Y]ab81299 abcam), FANCM (1:1000, ab95014,abcam), NBS1 (1:1000, A300-187A-T, Bethyl laboratories), MRE11 (1:1000, A300-181A-T, Bethyl laboratories), RNaseH2A (1:1000, A304-149A, Bethyl laboratories), AQR (1:1000, A302-547A, Bethyl laboratories), H2AX (1:500, [D17A3]XP #7631, cell signaling), p-ATM (1:500, (10H11.E12) sc-47739, Santa Cruz), p-CHK2 (Th468) (1:1000, C13C1, cell signaling), p-ATR (Ser428) (1:1000, #2853, cell signaling), p-Chk1 (Ser345) (1:1000, 133D3, cell signaling), FANCD2 (1:1000, NB100-182SS, Novus), GFP Tag (GF28R) (1:4000, MA5-15256, ThermoFisher Scientific), GAPDH (GA1R) (1:3000, MA5-15738, ThermoFisher Scientific), and α-tubulin (1:3000, B-5-1–2, ThermoFisher Scientific).

Techniques: Proximity Ligation Assay, Labeling, Binding Assay, Control

Journal: Nature Communications

Article Title: MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts

doi: 10.1038/s41467-019-12271-w

Figure Lengend Snippet: A structural role for the MRN complex in mitigating R-loop associated DNA damage. a Nuclear S9.6 staining intensity after treatment with MRE11 inhibitor Mirin (50 μ m ) is shown. N = 3; t test; mean ± SD. A schematic showing the goal of testing MRE11 nuclease activity on R-loops is show. b Mean S9.6 fluorescence intensity of TK6-derived lymphoblasts of the indicated genotype after treatment with EtOH (control) or 4-OHT, to induce gene knockout. Representative FACS histograms are shown below, curve colors correspond to bar graph. N = 3; *** P < 0.001 by ANOVA; mean ± SEM. c Head-to-head comparison of dsDNA and DNA:RNA hybrid resection by purified MRN complex on 60-mer duplexes. The concentration dependent degradation of the input molecules is plotted on the right

Article Snippet: Equivalent amounts of protein were resolved by SDS-PAGE and transferred to polyvinylidene fluoride microporous membrane (Millipore), blocked with 5% skim milk in TBS containing 0.1% Tween-20 (TBS-T), and membranes were probed with the following antibodies: RAD50 (1:1000, [13B3/2C6]ab89, abcam), γH2AX (1:5000, [EP854(2)Y]ab81299 abcam), FANCM (1:1000, ab95014,abcam), NBS1 (1:1000, A300-187A-T, Bethyl laboratories), MRE11 (1:1000, A300-181A-T, Bethyl laboratories), RNaseH2A (1:1000, A304-149A, Bethyl laboratories), AQR (1:1000, A302-547A, Bethyl laboratories), H2AX (1:500, [D17A3]XP #7631, cell signaling), p-ATM (1:500, (10H11.E12) sc-47739, Santa Cruz), p-CHK2 (Th468) (1:1000, C13C1, cell signaling), p-ATR (Ser428) (1:1000, #2853, cell signaling), p-Chk1 (Ser345) (1:1000, 133D3, cell signaling), FANCD2 (1:1000, NB100-182SS, Novus), GFP Tag (GF28R) (1:4000, MA5-15256, ThermoFisher Scientific), GAPDH (GA1R) (1:3000, MA5-15738, ThermoFisher Scientific), and α-tubulin (1:3000, B-5-1–2, ThermoFisher Scientific).

Techniques: Staining, Activity Assay, Fluorescence, Derivative Assay, Control, Gene Knockout, Comparison, Purification, Concentration Assay

Journal: Journal of Virology

Article Title: The E4orf6/E1B55K E3 Ubiquitin Ligase Complexes of Human Adenoviruses Exhibit Heterogeneity in Composition and Substrate Specificity

doi: 10.1128/jvi.01890-10

Figure Lengend Snippet: FIG. 3. Degradation of Mre11 and DNA ligase IV by E4orf6/ E1B55K ligase complexes from different serotypes. A study similar to that shown in Fig. 2 was conducted, except that no p53 was present and levels of endogenous Mre11 and DNA ligase IV were determined by Western blotting using appropriate antibodies. Tubulin levels were assessed by Western blotting as loading controls, and the serotype (with subgroup) has been indicated at the top. Degradation of DNA ligase IV and Mre11 was determined by the decrease of these species in the presence of both E4orf6 and E1B55K.

Article Snippet: HA epitopes were detected using anti-HA mouse monoclonal HA.11 (BabCO) or, for immunofluorescence (IF) with rat monoclonal 3F10 (Roche), FLAG epitopes with anti-FLAG M2 mouse monoclonal antibody peroxidase conjugate (Sigma), or, for IF with rabbit anti-FLAG (Sigma-Aldrich), Elongin C with mouse monoclonal antibody SIII p15 (Transduction Laboratories), Mre11 with rabbit polyclonal antiserum NB 100-142D3 (Novus Biologicals), DNA ligase IV with rabbit polyclonal antibody (AHP554) (Serotec), integrin 3A with mouse monoclonal 29A3 (Millipore), Cul5 with rabbit polyclonal anti-Cul5 antibody (H-300) (Santa Cruz Biotechnology), Cul2 with rabbit polyclonal anti-Cul2 antibody NBP1-02780 (Novus Biologicals), tubulin with rat monoclonal anti- -tubulin antibody (YOL1/34) (Abcam), and actin with mouse monoclonal anti-actin C4 (Millipore).

Techniques: Western Blot

Journal: Journal of Virology

Article Title: The E4orf6/E1B55K E3 Ubiquitin Ligase Complexes of Human Adenoviruses Exhibit Heterogeneity in Composition and Substrate Specificity

doi: 10.1128/jvi.01890-10

Figure Lengend Snippet: FIG. 5. Cullin specificity of the various E3 ligase complexes. (A and B) H1299 cells were cotransfected with plasmid DNAs encoding FLAG-E4orf6 of different serotypes and either HA-Cul5 (A) or HA-Cul2 (B). Immunoprecipitations were carried out using anti-FLAG (E4orf6) antibody, and immunoprecipitates were immunoblotted using anti-HA (Cul5 or Cul2) antibodies. Whole-cell extracts were also immunoblotted using anti-HA or anti-FLAG antibodies. (Ci) Cul5 levels in H1299 control and H1299/Cul5KD cells. The levels of endogenous Cul5 present in H1299 control cells and H1299/Cul5KD cells were analyzed by Western blotting using anti-Cul5 antibodies, as described in Materials and Methods. (Cii) Dependence of p53 and Mre11 degradation by various E3 ligase complexes on Cul5. Mre11 and p53 degradation assays were conducted as described for Fig. 2, except that levels of both exogenous p53 and endogenous Mre11 were determined by Western blotting using appropriate antibodies, and studies were conducted in both H1299 control and H1299/Cul5KD cells, as indicated. Actin levels were assessed by Western blotting as loading controls, and the serotype (with subgroup) has been indicated at the top. (Di) Cul2 levels in H1299 control and H1299/Cul2KD cells. The levels of endogenous Cul2 present in H1299 control cells and H1299/Cul2KD cells were analyzed by Western blotting using anti-Cul2 antibodies. (Dii) Dependence of p53 and Mre11 degradation by various E3 ligase complexes on Cul2. A study identical to that shown in panel Ci was conducted using H1299 control cells and H1299/Cul2KD cells.

Article Snippet: HA epitopes were detected using anti-HA mouse monoclonal HA.11 (BabCO) or, for immunofluorescence (IF) with rat monoclonal 3F10 (Roche), FLAG epitopes with anti-FLAG M2 mouse monoclonal antibody peroxidase conjugate (Sigma), or, for IF with rabbit anti-FLAG (Sigma-Aldrich), Elongin C with mouse monoclonal antibody SIII p15 (Transduction Laboratories), Mre11 with rabbit polyclonal antiserum NB 100-142D3 (Novus Biologicals), DNA ligase IV with rabbit polyclonal antibody (AHP554) (Serotec), integrin 3A with mouse monoclonal 29A3 (Millipore), Cul5 with rabbit polyclonal anti-Cul5 antibody (H-300) (Santa Cruz Biotechnology), Cul2 with rabbit polyclonal anti-Cul2 antibody NBP1-02780 (Novus Biologicals), tubulin with rat monoclonal anti- -tubulin antibody (YOL1/34) (Abcam), and actin with mouse monoclonal anti-actin C4 (Millipore).

Techniques: Plasmid Preparation, Control, Western Blot