Volume 68, Issue 4, Pages 797-807.e7 (November 2017) Repair of UV-Induced DNA Damage Independent of Nucleotide Excision Repair Is Masked by MUTYH  Abdelghani Mazouzi, Federica Battistini, Sarah C. Moser, Joana Ferreira da Silva, Marc Wiedner, Michel Owusu, Charles-Hugues Lardeau, Anna Ringler, Beatrix Weil, Jürgen Neesen, Modesto Orozco, Stefan Kubicek, Joanna I. Loizou  Molecular Cell  Volume 68, Issue 4, Pages 797-807.e7 (November 2017) DOI: 10.1016/j.molcel.2017.10.021 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 68, 797-807.e7DOI: (10.1016/j.molcel.2017.10.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Acetohexamide Alleviates the UV Sensitivity of NER-Deficient Cells (A) Schematic representation of the experimental setup used to perform the high-throughput drug screen, where drugs were used at five times the maximal plasma concentration (CLOUD; CeMM library of unique drugs). (B) Bubble plot displaying the drugs used plotted against cell viability. Blue bubbles indicate wild-type (WT) HAP1 cells, red bubbles indicate XPA-deficient HAP1 cells (ΔXPA), and the green bubble highlights acetohexamide. The size of the bubbles indicates significance, displayed as −log10 (p value). The screen was performed in two biological replicates. Cell viability was determined using CellTiter-Glo. (C) Chemical structure of acetohexamide. (D) Dose-response curve of WT and ΔXPA cells treated with or without 0.5 mM acetohexamide for 6 hr, followed by UV radiation. Survival was assessed after 3 days, using CellTiter-Glo. Displayed is the relative viability obtained by normalizing the raw data of the DMSO control to acetohexamide-treated cells. Error bars indicate SEM (n = 3). (E) Colony formation using the same conditions indicated in (D), where cells were kept in culture for 10 days following UV radiation, after which they were fixed and stained. (F) Quantification of macroscopic colonies stained with crystal violet in (E). The experiment was performed in two biological replicates and the error bars indicate SEM (n = 2; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, Student’s t test). See also Figures S1 and S2. Molecular Cell 2017 68, 797-807.e7DOI: (10.1016/j.molcel.2017.10.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Acetohexamide Enhances the Clearance of Cyclobutane Pyrimidine Dimers in NER-Deficient Cells (A) Dose-response curve of HAP1 WT and ΔXPA cells treated with or without 0.5 mM acetohexamide for 6 hr, followed by illudin S treatment. Survival was assessed after 3 days using CellTiter-Glo. Error bars denote SEM (n = 3). (B) Colony formation of WT fibroblasts (BJ) and XPA-patient derived fibroblasts (XP12BE denoted as XPAΔ/Δ) treated with or without 0.5 mM acetohexamide for 6 hr, followed by UV radiation (as indicated) and then kept in culture for 10 days. (C) Quantification of macroscopic colonies stained with crystal violet in (B). The experiment was performed in two biological replicates and the error bars indicate SEM (n = 2; ∗p < 0.05 and ∗∗p < 0.01, Student’s t test). (D) WT BJ and XPAΔ/Δ fibroblastoid cells were treated with 0.5 mM acetohexamide for 6 hr, irradiated with 15 J/M2, and then fixed and immunostained with an anti-cyclobutane pyrimidine dimer (CPD) antibody at the indicated times. Nuclear DNA was counterstained with DAPI. Scale bar, 10 μm. (E) Scatterplot displaying the quantification of CPD intensities per nucleus of WT and XPAΔ/Δ cells in the presence or absence of 0.5 mM acetohexamide, of more than 100 cells. Black lines within each column represent median intensities (∗∗∗∗p < 0.0001, Student’s t test). See also Figures S2 and S3. Molecular Cell 2017 68, 797-807.e7DOI: (10.1016/j.molcel.2017.10.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Loss of MUTYH Mimics Acetohexamide Function (A) Bubble plot displaying the percentage of rescue that represents the difference between the raw signal of a given cell line (where “D” denotes delta) treated with acetohexamide + UV compared to the raw signal of the same cell line treated with DMSO + UV, divided by the difference between the raw signal of a given cell line treated with acetohexamide compared to the raw signal of the same cell line with DMSO (signalAcetohexamide + UV − signalDMSO + UV)/(signalAcetohexamide − signalDMSO). The biggest value of this score was set to 100%. The UV radiation used was 15 J/m2. Survival was assessed after 3 days using CellTiter-Glo. The green, red, and blue bubbles highlight ΔMUTYH, ΔXPA, and WT HAP1 cells, respectively, and black bubbles indicate the rest of the knockout cell lines. The size of the bubbles indicates the significance as −log10 (p value). BER, base excision repair; NER, nucleotide excision repair; DSBR, double-strand break repair; MMR, mismatch repair; FA, Fanconi anemia; DR, direct reversal; TLS, translesion synthesis. The screen was done in 2 biological replicates and each biological replicate consisted of three technical replicates. (B and C) Dose-response curve of WT, ΔPOLH (B), and ΔCSB (C) cells treated with or without 0.5 mM acetohexamide for 6 hr, followed by UV radiation (5 J/m2). Survival was assessed after 3 days, using CellTiter-Glo. Error bars denote the SEM (n = 3; ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, Student’s t test). (D) Survival of WT and MUTYH-deficient HAP1 (ΔMUTYH) cells with or without 0.5 mM acetohexamide treatment, followed by UV radiation, as assessed after 3 days using CellTiter-Glo. Error bars indicate the SEM (n = 3). Loss of MUTYH protein was confirmed by immunoblotting using an anti-MUTYH antibody. ACTIN was used as a loading control. (E) Deletion of MUTYH in WT HAP1 or an XPA-deficient background was confirmed by immunoblotting using an anti-MUTYH antibody. TUBULIN was used as a loading control. (F) Clonogenic survival of WT, ΔXPA, or ΔXPA-MUTYH cells irradiated with UV at the indicated doses or left untreated. Cells were fixed and stained 10 days later. The experiment was performed in two biological replicates. (G) Quantification of macroscopic colonies stained with crystal violet in (F). Error bars denote the SEM (n = 2; ∗∗p < 0.01 and ∗∗∗p < 0.001, Student’s t test). (H) WT HAP1 cells were treated with or without 0.5 mM acetohexamide for 6 hr, then released into compound-free media for the indicated time points and immunoblotted with an anti-MUTYH antibody. ACTIN was used as a loading control. (I) WT HAP1 cells were either treated with 0.5 mM acetohexamide alone or with 10 μM of the proteasome inhibitor MG132 for 6 hr and analyzed by immunoblotting using an anti-MUTYH antibody. ACTIN was used as a loading control. See also Figure S4. Molecular Cell 2017 68, 797-807.e7DOI: (10.1016/j.molcel.2017.10.021) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Loss of MUTYH, or Use of Acetohexamide, Corrects UV Sensitivity and Defective Clearance of UV-Induced Lesions in NER-Deficient Cells, without Accumulation of Chromosomal Instability (A) Left panel: colony formation of the WT, ΔXPA, or ΔXPA-MUTYH HAP1 cells treated with or without 0.5 mM acetohexamide for 6 hr followed by 2 J/M2 UV radiation and then kept in culture for 10 days. Right panel: macroscopic colonies were stained with crystal violet and quantified. This experiment was done in two biological replicates. (B) Left panel: WT, ΔMUTYH, ΔXPA, or ΔXPA-MUTYH HAP1 cells were treated with 15 J/M2 UV or left untreated, then kept in culture for the indicated recovery times, and analyzed by dot blot for the presence of CPDs within genomic DNA. DNA was counterstained with methylene blue (MB) as a loading control. Right panel: quantification of left panel is shown. The experiment was repeated twice with 3 technical replicates each time and all the data points were pooled together. (C) Left panel: WT, ΔXPA, or ΔXPA-MUTYH HAP1 cells were treated with 15 J/M2 UV or left untreated, then harvested immediately or kept in culture for 4 hr, and analyzed by dot blot for the presence of 6-4PPs within genomic DNA. DNA was counterstained with MB as a loading control. Right panel: quantification of left panel is shown. This experiment was performed in two biological replicates with 6 technical replicates for each replicate; three replicates were used for 6-4PPs and the other three for the loading control (MB). (D) WT, ΔXPA, or ΔXPA-MUTYH HAP1 cells were treated with 15 J/M2 and then released at the indicated time points and immunoblotted with the indicated antibodies. ACTIN was used as a loading control. (E) WT fibroblasts (BJ) and XPA-patient derived fibroblasts (XP12BE; XPAΔ/Δ) stably expressing HA-tagged MUTYH were exposed to UV radiation through a polycarbonate isopore membrane filter. After 1 hr cells were fixed and immunostained with anti-XPC and HA antibodies. Nuclear DNA was counterstained with DAPI. Scale bar, 10 μm. (F) MUTYH-DNA complex where the DNA exhibits a central kink where the CPD is located that results in a global 50-degree bending of the DNA. (G) Representations of the DNA-protein complex at the CPD site, for MUTYH-CPD (on the left) and T4 endonuclease V-CPD (1VAS, on the right). The CPD and the complementary adenines are highlighted in licorice and colored by atom names. The proteins are represented in gray cartoon, and the protein surfaces of the residues that interact with CPD and adenines are in green. (H) Number of chromosomal abnormalities (chromatid breaks, gaps, translocations, and deletions) per metaphase spread of ΔXPA or ΔXPA-MUTYH HAP1 3 days following UV radiation. 15 metaphases per condition were scored. Error bars throughout this figure represent mean ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, Student’s t test). See also Figure S4. Molecular Cell 2017 68, 797-807.e7DOI: (10.1016/j.molcel.2017.10.021) Copyright © 2017 Elsevier Inc. Terms and Conditions