Journal: Nature cell biology

Article Title: TRIP13 Regulates DNA Repair Pathway Choice through REV7 Conformational Change

doi: 10.1038/s41556-019-0442-y

Figure Lengend Snippet: a. Silver-stained gel of FLAG-HA empty vector (EV) and FLAG-HA-REV7 (R7) purifications. b. Table of high confidence REV7-interacting proteins and average abundance of proteins recovered from 3 biologically independent experiments. c. Western blot of FLAG IP from HEK293T cells transfected with empty vector or FLAG-TRIP13 with or without DNA damage. d. 14-day clonogenic survival assay of USOS wild-type, REV7−/−, and TRIP13−/− cell lines treated with varying Olaparib doses, n=3 independent experiments, wild-type vs. TRIP13−/− #3: p = 0.0001, wild-type vs. TRIP13−/− #7: p = 0.002 (2-Way ANOVA). e. 14-day clonogenic survival assay of wild-type or TRIP13−/− cells with or without expression of TRIP13 cDNA upon treatment with Olaparib, n=3 biologically independent experiments, wild-type vs. TRIP13−/− + Empty vector: p = 0.002, TRIP13−/− + Empty vector vs. TRIP13−/− + TRIP13: p = 0.002 (2-Way ANOVA). f. 14-day clonogenic survival assay of U2OS wild-type and REV7−/− with or without TRIP13 overexpression treated with varying Olaparib doses, n=3 biologically independent experiments, Wild-type + Empty vector vs. wild-type + TRIP13: p = 0.005, REV7−/− + Empty vector vs. REV7−/− + TRIP13: p = 0.31 (2-Way ANOVA). g. 5-day cytotoxicity analysis of HCC1937 SHLD2-deficient cells infected with pBabe-empty vector or pBabe-TRIP13 treated with the indicated doses of Olaparib. n=5 biologically independent experiments, p=0.58 (2-Way ANOVA). All error bars indicate SEM. Immunoblots are representative of at least 2 independent experiments. Statistical source data are provided in Source Data Fig. 1. Unprocessed blots are provided in Unprocessed blots Fig. 1.

Article Snippet: 15 N-labelled HSQC NMR The 15 N-HSQC spectra of 15 N-labeled REV7 proteins were acquired in 20 mM HEPES, 100 mM NaCl, 10 mM DTT, pH 7.4 with 10% D2O at 25°C, on a 600 MHz Bruker Avance II spectrometer with a Prodigy cryoprobe.

Techniques: Staining, Plasmid Preparation, Western Blot, Transfection, Clonogenic Cell Survival Assay, Expressing, Over Expression, Infection

Journal: Nature cell biology

Article Title: TRIP13 Regulates DNA Repair Pathway Choice through REV7 Conformational Change

doi: 10.1038/s41556-019-0442-y

Figure Lengend Snippet: a. Quantification of resected ssDNA in U2OS wild-type, TRIP13−/− (two clones) and REV7−/− cells measured by SMART assay, ~100 fibers were counted per experiment, Wild Type vs. TRIP13−/− #3: p < 0.0001, Wild Type vs. TRIP13−/− #7: p = 0.004, Wild Type vs. REV7−/−: p < 0.0001 (Mann-Whitney test, two-tailed), bars indicate mean and SEM. b. Representative images for (a), with BrdU in exposed ssDNA tracts labeled red. (Scale bar: 1 μm) c. Proportion of U2OS cells with greater than 10 p-RPA32(S33) foci 6 hours following IR treatment. n=3 biologically independent experiments, Wild Type vs. TRIP13−/− #3: p = 0.001, Wild Type vs. REV7−/−: p = 0.01 (Student’s t-test, two-tailed). d. Representative images of data in (c) (Scale bar: 10 μm). e. Proportion of U2OS cells with greater than 10 RAD51 foci 6 hours following IR treatment. n=6 biologically independent experiments, Wild Type vs. TRIP13−/− #3: p = 0.01, Wild Type vs. REV7−/−: p = 0.02 (Student’s t-test, two-tailed). f. Representative images of data in (e) (Scale bar: 10 μm). g. Percentage of GFP-positive cells following infection of U2OS DR-GFP cells with I-SceI adenovirus with knockdown of BRCA2 or TRIP13. n=3 biologically independent experiments, siCtrl vs siBRCA2: p = 0.0004, siCtrl vs. siTRIP13–1: p = 0.004, siCtrl vs. TRIP13–6: p = 0.01 (Student’s t-test, two-tailed), all error bars indicate SEM. Statistical source data are provided in Source Data Fig. 4.

Article Snippet: 15 N-labelled HSQC NMR The 15 N-HSQC spectra of 15 N-labeled REV7 proteins were acquired in 20 mM HEPES, 100 mM NaCl, 10 mM DTT, pH 7.4 with 10% D2O at 25°C, on a 600 MHz Bruker Avance II spectrometer with a Prodigy cryoprobe.

Techniques: Clone Assay, MANN-WHITNEY, Two Tailed Test, Labeling, Infection

Journal: Nature cell biology

Article Title: TRIP13 Regulates DNA Repair Pathway Choice through REV7 Conformational Change

doi: 10.1038/s41556-019-0442-y

Figure Lengend Snippet: a. 14-day clonogenic survival assay of U2OS wild-type expressing pBabe-empty vector or pBabe-TRIP13 and REV7−/− cell lines treated with the indicated mitomycin C (MMC) doses. n=4 biologically independent experiments (Except for REV7−/−: n=3), Wild-type + Empty vector vs. Wild-type + TRIP13: p = 0.02, Wild-type + Empty vector vs. REV7−/−: p < 0.0001 (2-Way ANOVA). b. Average number of chromosomal aberrations per cell in baseline condition or following treatment with 20 ng/mL of MMC, with or without TRIP13 overexpression. n=3 biologically independent experiments. c. Percentage of cells with radial chromosome formation in baseline condition or following treatment with 20 ng/mL of MMC, with or without TRIP13 overexpression. n=3 biologically independent experiments. d. 14-day clonogenic survival assay of U2OS wild-type expressing pBabe-empty vector or pBabe-TRIP13 and REV7−/− cell lines treated with the indicated UV doses. n=4 biologically independent experiments, Wild-type + Empty vector vs. Wild-type + TRIP13: p < 0.0001, Wild-type + Empty vector vs. REV7−/− : p = 0.002 (2-Way ANOVA). e. Relative UV-induced mutation frequencies in HEK293T cells transfected with nontargeting, REV7- or TRIP13-targeting siRNAs as measured by the SupF assay. n=3 biologically independent experiments (Except for REV7−/−: n=2), siCtrl vs siTRIP13–6: p = 0.0006 (Student’s paired t-test, two-tailed). f. Western blot of GFP IP from U2OS wild type, TRIP13−/−, pBabe and pBabe-TRIP13 cells transfected with GFP-tagged REV3 binding domain (REV3-BD). g. Quantification of western blot in (f). All error bars represent SEM. All immunoblots are representative of at least 2 independent experiments. Statistical source data are provided in Source Data Fig. 5. Unprocessed blots are provided in Unprocessed blots Fig. 5.

Article Snippet: 15 N-labelled HSQC NMR The 15 N-HSQC spectra of 15 N-labeled REV7 proteins were acquired in 20 mM HEPES, 100 mM NaCl, 10 mM DTT, pH 7.4 with 10% D2O at 25°C, on a 600 MHz Bruker Avance II spectrometer with a Prodigy cryoprobe.

Techniques: Clonogenic Cell Survival Assay, Expressing, Plasmid Preparation, Over Expression, Mutagenesis, Transfection, Two Tailed Test, Western Blot, Binding Assay

Journal: Nature cell biology

Article Title: TRIP13 Regulates DNA Repair Pathway Choice through REV7 Conformational Change

doi: 10.1038/s41556-019-0442-y

Figure Lengend Snippet: a. Crystal structures of C-REV7 and O/C-MAD2 with the dynamic seatbelt region in green. Purple indicates the REV7-binding fragment of REV3. Constructed from 3VU7 and 3GMH. b. (Top) UV absorbance trace at 280 nm showing elution of purified REV7 upon AEC. (Bottom) Coomassie-stained gel of the corresponding fractions. c. 15N-labelled HSQC NMR spectrum of REV7-F1 (blue) and REV7-F2 (red). d. Western blot of AEC fractions of purified REV7R124A and REV7R124A, Δseatbelt. e. Schematic showing the requirement of TRIP13 ATPase activity for the C-REV7 to O-REV7 transition in cells upon seatbelt binding motif (SBM) binding. f. Western blot of AEC fractions of REV7R124A upon overnight incubation at 4 °C (top) or 37 °C (bottom) with or without SBM. g. Pulldown of C-REV7 and O-REV7 by GST-SHLD3 at the indicated time points. Coomassie stained gels and immunoblots are representative of at least 2 independent experiments. Unprocessed blots are provided in Unprocessed blots Fig. 2.

Article Snippet: 15 N-labelled HSQC NMR The 15 N-HSQC spectra of 15 N-labeled REV7 proteins were acquired in 20 mM HEPES, 100 mM NaCl, 10 mM DTT, pH 7.4 with 10% D2O at 25°C, on a 600 MHz Bruker Avance II spectrometer with a Prodigy cryoprobe.

Techniques: Binding Assay, Construct, Purification, Staining, Western Blot, Activity Assay, Incubation

Journal: Nature cell biology

Article Title: TRIP13 Regulates DNA Repair Pathway Choice through REV7 Conformational Change

doi: 10.1038/s41556-019-0442-y

Figure Lengend Snippet: a. Schematic showing the remodeling and release of REV7 from SHLD3 complexes upon action of the TRIP13 ATPase. b. Western blot showing glutathione bead-bound GST-SHLD3 and the release of REV7 into the unbound fraction over time in the absence or presence of ATP or the non-hydrolysable ATP analog AMP-PNP. c. Quantification of ATP-dependent release of REV7 from SHLD3 over time by TRIP13. n=3 independent experiments, -ATP vs +ATP: p = 0.006, +ATP vs. +AMP-PNP: p = 0.002 (2-way ANOVA). d. Western blot of GFP IP from U2OS wild type, TRIP13−/−, pBabe and pBabe-TRIP13 cells transfected with GFP-tagged SHLD3. e. Quantification of western blot in (d). f. Schematic for TRIP13 counteraction of REV7 seatbelt closure, preventing the formation of functional Shieldin complex. g. Focus formation of REV7 6 h after irradiation in U2OS wild-type, REV7−/− and TRIP13−/− cells (Scale bar: 10 μm). h. Quantification of REV7 foci for (g). n=6 biologically independent experiments (Except for TRIP13−/− #7, n=3), Wild Type vs. TRIP13−/− #3: p = 0.01, Wild Type vs. TRIP13−/− #7: p = 0.02, Wild Type vs. REV7−/−: p = 0.002, (Student’s t-test, 2-tailed), error bars represent SEM. i. Gel filtration elution profiles of REV7 from U2OS wild-type and three TRIP13−/− clones. All immunoblots are representative of at least 2 independent experiments. Statistical source data are provided in Source Data Fig. 3. Unprocessed blots are provided in Unprocessed blots Fig. 3.

Article Snippet: 15 N-labelled HSQC NMR The 15 N-HSQC spectra of 15 N-labeled REV7 proteins were acquired in 20 mM HEPES, 100 mM NaCl, 10 mM DTT, pH 7.4 with 10% D2O at 25°C, on a 600 MHz Bruker Avance II spectrometer with a Prodigy cryoprobe.

Techniques: Western Blot, Transfection, Functional Assay, Irradiation, Filtration, Clone Assay