Journal: bioRxiv

Article Title: Genome-scale mapping of DNA damage suppressors identifies GNB1L as essential for ATM and ATR biogenesis

doi: 10.1101/2022.09.23.508845

Figure Lengend Snippet: (A) A radar plot showing the ranking of GNB1L in all five γ-H2AX screens. (B) Representative flow cytometry analysis of RKO TP53 −/− cells expressing the indicated sgRNA. Cells were treated with the indicated replication inhibitor for 24 h or left untreated (UT), and then fixed, stained with a γ-H2AX antibody and DAPI. Red numbers indicate the percentage of γ-H2AX positive cells. (C) Quantification of the experiment shown in (B). Bars represent the mean ± s.d. (n=3 independent experiments). Comparisons are made to the sg AAVS1 control within each treatment condition, using an unpaired t-test (***p<0.001; n.s., not significant). (D) Quantitative image-based cytometry (QIBC) analysis of γ-H2AX and chromatin-bound RPA2 signal intensities in RKO TP53 −/− cells. Cells were treated with 200 μM HU or 250 nM CD437 for 24 hours, then extracted, fixed and stained with antibodies to γ-H2AX and RPA2. Red numbers indicate the percentage of cells with both high γ-H2AX and high RPA2 signal intensities for each condition. A.U., arbitrary units.

Article Snippet: The GNB1L open reading frame (ORF) was inserted into the pLVU/GFP lentiviral plasmid vector (Addgene, 24177) encoding a C-terminal GFP tag.

Techniques: Flow Cytometry, Expressing, Staining, Control, Cytometry

Journal: bioRxiv

Article Title: Genome-scale mapping of DNA damage suppressors identifies GNB1L as essential for ATM and ATR biogenesis

doi: 10.1101/2022.09.23.508845

Figure Lengend Snippet: Related to . (A) Left, representative flow cytometry analysis of RPE1-hTERT TP53 −/− cells expressing sg AAVS1 control or sg GNB1L . Cells were treated with 200 μM HU or 250 nM CD437 for 24 hours, then fixed and stained with a γ-H2AX antibody and DAPI. Red numbers indicate the percentage of γ-H2AX positive cells. Right, quantification of the experiment shown on left. Bars represent the mean ± s.d. (n=3 independent experiments independent experiments). Comparisons are made to the sg AAVS1 control within each treatment condition, using an unpaired t-test (*** p<0.001). (B) Quantitative image-based cytometry (QIBC) analysis of γ-H2AX and chromatin-bound RPA2 signal intensities in RPE-hTERT TP53 −/− cells, as described in (A). Cells were treated with 200 μM HU or 250 nM CD437 for 24 hours, then extracted, fixed and stained with antibodies to γ-H2AX and RPA2. Red numbers indicate the percentage of cells with both high γ-H2AX and high RPA2 signal intensities. A.U., arbitrary units.

Article Snippet: The GNB1L open reading frame (ORF) was inserted into the pLVU/GFP lentiviral plasmid vector (Addgene, 24177) encoding a C-terminal GFP tag.

Techniques: Flow Cytometry, Expressing, Control, Staining, Cytometry

Journal: bioRxiv

Article Title: Genome-scale mapping of DNA damage suppressors identifies GNB1L as essential for ATM and ATR biogenesis

doi: 10.1101/2022.09.23.508845

Figure Lengend Snippet: (A) Mass spectrometry results of the TurboID-based proximal labeling experiment with GNB1L-miniTurbo and AP-MS with 3xFlag-GNB1L experiments. High-confident hits with a SaintScore ≥ 0.99 and average spectral counts ≥ 3 are shown for both experiments. Color indicates the number of average spectral counts minus the control counts. (B) Anti-Flag immunoprecipitation in 293T cells expressing 3xFlag-GNB1L or 3xFlag alone (EV). Bound proteins were examined by immunoblot analysis with the indicated antibodies. (C) Immunoblot analysis of PIKK proteins in lysates from RKO TP53 −/− and RPE-hTERT TP53 −/− cells expressing sg AAVS1 control or sg GNB1L . α-actinin, loading control. Asterisk besides GNB1L immunoblots indicate non-specific bands. (D) Immunoblot analysis of PIKK proteins in lysates from RKO TP53 −/− cells expressing the indicated sgRNA. α-actinin, loading control. (E) Quantitative RT-PCR to detect mRNA of ATM, ATR, DNA-PKcs using TaqMan assays. Bars represent the mean ± s.d. (n=3 independent experiments). Results of an unpaired t-test between sg AAVS1 and sg GNB1L conditions are shown (*p<0.05).

Article Snippet: The GNB1L open reading frame (ORF) was inserted into the pLVU/GFP lentiviral plasmid vector (Addgene, 24177) encoding a C-terminal GFP tag.

Techniques: Mass Spectrometry, Labeling, Protein-Protein interactions, Control, Immunoprecipitation, Expressing, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Genome-scale mapping of DNA damage suppressors identifies GNB1L as essential for ATM and ATR biogenesis

doi: 10.1101/2022.09.23.508845

Figure Lengend Snippet: Related to . (A) Streptavidin pull-down was performed using lysates of 293T cells expressing miniTurbo-GNB1L or miniTurbo alone. 50 μM biotin was added to the cell culture medium 40 min before harvesting. Immunoblot analysis was performed with the indicated antibodies. (B) NanoBRET assay to assess the in vivo interaction between GNB1L and TELO2. Bars represent the mean ± s.d. (n=3 independent experiments). Results of unpaired t-test between unfused HaloTag and HaloTag TELO2 conditions are shown (*** p<0.001). (C) RKO TP53 −/− cells were transduced with lentiviruses expressing the indicated sgRNA and cell lysates were subjected to immunoblot analysis. α-actinin, loading control. Asterisk besides GNB1L immunoblots indicate non-specific bands. (D) Flow cytometry analysis of RKO TP53 −/− cells expressing the indicated sgRNA. Cells were treated with 250 nM CD437 for 24 hours, and then fixed, stained with a γ-H2AX antibody and DAPI. Left, representative plots. Right, quantification of γ-H2AX positive cells. Bars represent the mean ± s.d. (n=3 independent experiments). Comparisons are made to the sg AAVS1 control, using an unpaired t-test (***p<0.001; **p<0.01). A.U., arbitrary units. (E) Substrate phosphorylation of ATM, ATR, mTOR in RKO TP53 −/− cells expressing sg AAVS1 control and sg GNB1L . To test ATM activity, cells were exposed to 15 Gy ionizing radiation (IR) and allowed to recover for 1 h before harvesting. To test ATR activity, cells were exposed to 40 J of ultraviolet (UV) and allowed to recover for 5 h. mTOR inhibitor Torin 1 was added to cells at a final concentration of 200 nM for 24 hours as a control for decreased mTOR activity. (F) Proliferation curves of the cells described in (E) grown in the presence of 100 μM HU, or 5 nM gemcitabine, or in the untreated condition. Data is presented as mean ± s.d. (n=3 independent experiments).

Article Snippet: The GNB1L open reading frame (ORF) was inserted into the pLVU/GFP lentiviral plasmid vector (Addgene, 24177) encoding a C-terminal GFP tag.

Techniques: Expressing, Cell Culture, Western Blot, In Vivo, Transduction, Control, Flow Cytometry, Staining, Phospho-proteomics, Activity Assay, Concentration Assay

Journal: bioRxiv

Article Title: Genome-scale mapping of DNA damage suppressors identifies GNB1L as essential for ATM and ATR biogenesis

doi: 10.1101/2022.09.23.508845

Figure Lengend Snippet: (A), (B) Lentiviral transduction of 293T cells were used to introduce a construct encoding sgRNA-resistant FKBP mut -GNB1L resis ; degradation of this protein can be induced by addition of the dTAG-v1 molecule. Endogenous GNB1L was then inactivated with an sgRNA targeting an intron-exon junction. Two clones of the above-mentioned genotype were generated and were treated with 1 μM dTAG-v1 molecule for the indicated time before harvesting. (A) Immunoblot analysis of cell lysates with the indicated antibodies. (B) Flow cytometry analysis of cells treated with 250 nM CD437 for 24 hours. Cells were stained with a γ-H2AX antibody and DAPI. Red numbers indicate the percentage of γ-H2AX positive cells. (C), (D) HaloTag TMR ligand was used to label newly synthesized (C) or pre-existing (D) Halo-ATR protein. Left, cell lysates were subjected to immunoblotting with the indicated antibodies. TMR signal was measured by direct detection of fluorescence. Band intensity was quantified by ImageJ. Right, quantification of TMR relative to α-actinin and normalized to the signal at the 1 h (C) or 0 h (D) timepoint. Data is presented as mean ± s.d. (n=3 independent experiments). Results of unpaired t-test between −dTAG and +dTAG conditions are shown (**p<0.01; n.s., not significant). Asterisk besides GNB1L immunoblots indicate non-specific bands.

Article Snippet: The GNB1L open reading frame (ORF) was inserted into the pLVU/GFP lentiviral plasmid vector (Addgene, 24177) encoding a C-terminal GFP tag.

Techniques: Transduction, Introduce, Construct, Clone Assay, Generated, Western Blot, Flow Cytometry, Staining, Synthesized, Fluorescence

Journal: bioRxiv

Article Title: Genome-scale mapping of DNA damage suppressors identifies GNB1L as essential for ATM and ATR biogenesis

doi: 10.1101/2022.09.23.508845

Figure Lengend Snippet: (A) Schematic of the deep mutational scanning strategy. (B) The moving window analysis of the median functional score, median ΔΔG, and the conservation score for each residue of GNB1L, with a window of five residues. (C), (D) RKO TP53 −/− cells were infected with lentiviruses expressing sg GNB1L and sgRNA-resistant GNB1L-GFP variant constructs as indicated. WT, wildtype. (C) Immunoblot analysis of cell lysates with the indicated antibodies. α-actinin, loading control. (D) Cells were treated with 250 nM CD437 for 24 hours, then fixed and stained with a γ-H2AX antibody and DAPI. Left, representative flow cytometry plots. Right, quantification of γ-H2AX positive cells. Bars represent the mean ± s.d. (n=3 independent experiments). A.U., arbitrary units. (E) Anti-Flag immunoprecipitation (IP) in 293T cells expressing 3xFlag-tagged GNB1L (WT or mutants). WT, wildtype. EV, empty vector. Bound proteins were examined by immunoblotting (IB) with the indicated antibodies.

Article Snippet: The GNB1L open reading frame (ORF) was inserted into the pLVU/GFP lentiviral plasmid vector (Addgene, 24177) encoding a C-terminal GFP tag.

Techniques: Functional Assay, Residue, Infection, Expressing, Variant Assay, Construct, Western Blot, Control, Staining, Flow Cytometry, Immunoprecipitation, Plasmid Preparation

Journal: bioRxiv

Article Title: Genome-scale mapping of DNA damage suppressors identifies GNB1L as essential for ATM and ATR biogenesis

doi: 10.1101/2022.09.23.508845

Figure Lengend Snippet: (A) Left, Schematic of truncation/point mutation mapping in the TELO2 protein. Right, Anti-Flag immunoprecipitation (IP) in 293T cells expressing full length (FL) or mutant 3xFlag-TELO2. The GNB1L-TELO2 interaction was examined by immunoblotting with a GNB1L antibody. (B) Left, AlphaFold2-predicted structure of full-length GNB1L binding to the C3 fragment of TELO2 (460-640aa). Purple, surface structure of GNB1L. Beige, ribbon structure of the TELO2 fragment. Right, magnified view of binding surface. Amino acid residues 498-501 of TELO2 are labeled and highlighted in green. (C), (D) RKO TP53 −/− cells were infected with lentiviruses expressing sg TELO2 and sgRNA-resistant TELO2 variant constructs as indicated. WT, wildtype. (C) Immunoblot analysis of cell lysates with the indicated antibodies. α-actinin, loading control. (D) Cells were treated with 250 nM CD437 for 24 hours, and then fixed, stained with a γ-H2AX antibody and DAPI. Red numbers indicate the percentage of γ-H2AX positive cells. The results are representative of two independent experiments. A.U., arbitrary units.

Article Snippet: The GNB1L open reading frame (ORF) was inserted into the pLVU/GFP lentiviral plasmid vector (Addgene, 24177) encoding a C-terminal GFP tag.

Techniques: Mutagenesis, Immunoprecipitation, Expressing, Western Blot, Binding Assay, Labeling, Infection, Variant Assay, Construct, Control, Staining

Journal: bioRxiv

Article Title: Genome-scale mapping of DNA damage suppressors identifies GNB1L as essential for ATM and ATR biogenesis

doi: 10.1101/2022.09.23.508845

Figure Lengend Snippet: Related to . (A) Left, schematic of truncation/point mutation mapping in the TELO2 protein and the summary of mutant TELO2 phenotypes. Right, anti-Flag immunoprecipitation (IP) of lysates from 293T cells expressing full-length(FL) or mutant 3xFlag-TELO2. EV, empty vector. The GNB1L-TELO2 interaction was examined by immunoblot analysis with a GNB1L antibody. (B) NanoBRET assay to assess the in vivo interaction between GNB1L and mutant TELO2. Bars represent the mean ± s.d. (n=3 independent experiments). Comparisons were made using an unpaired t-test (***p<0.001; *p<0.05; n.s., not significant). (C) The predicted aligned error (PAE) matrix of GNB1L and the TELO2 aa460-640 fragment, generated by AlphaFold2-multimer. The off-diagonal quadrants represent inter-chain PAE scores. The blue stripes indicated with arrows reflect the set of residues that have a good confidence of relative inter-chain position. (D) Plot of the mean predicted aligned error between each TELO2 residue and GNB1L residues within 9 Å. The four residues 498 YMDS 501 are highlighted by red dashed lines. (E) Left, anti-Flag immunoprecipitation (IP) in 293T cells expressing 3xFlag-tagged wildtype (WT) or mutant TELO2. One sample expressing WT TELO2 was treated with 75 μM of the CK2 inhibitor TBB for 20 hours. The GNB1L-TELO2 interaction was examined by immunoblotting with a GNB1L antibody. Right, CK2 inhibition by TBB was confirmed by immunoblot analysis with an antibody recognizing phospho-CK2 substrates. EV, empty vector. (F), (G) RKO TP53 −/− cells were infected with lentiviruses expressing sg TELO2 and sgRNA-resistant TELO2 variant constructs as indicated. WT, wildtype. (F) Immunoblot analysis of cell lysates with the indicated antibodies. α-actinin, loading control. (G) Cells were treated with 250 nM CD437 for 24 hours, and then fixed, stained with a γ-H2AX antibody and DAPI. Red numbers indicate the percentage of γ-H2AX positive cells. A.U., arbitrary units.

Article Snippet: The GNB1L open reading frame (ORF) was inserted into the pLVU/GFP lentiviral plasmid vector (Addgene, 24177) encoding a C-terminal GFP tag.

Techniques: Mutagenesis, Immunoprecipitation, Expressing, Plasmid Preparation, Western Blot, In Vivo, Generated, Residue, Inhibition, Infection, Variant Assay, Construct, Control, Staining

Journal: bioRxiv

Article Title: Genome-scale mapping of DNA damage suppressors identifies GNB1L as essential for ATM and ATR biogenesis

doi: 10.1101/2022.09.23.508845

Figure Lengend Snippet: RKO TP53 −/− cells were transduced with lentiviruses expressing the indicated sgRNA and cell lysates were subjected to immunoblot analysis. α-actinin, loading control. Asterisk besides GNB1L immunoblots indicate non-specific bands.

Article Snippet: The GNB1L open reading frame (ORF) was inserted into the pLVU/GFP lentiviral plasmid vector (Addgene, 24177) encoding a C-terminal GFP tag.

Techniques: Transduction, Expressing, Western Blot, Control