Volume 39, Issue 1, Pages 25-35 (July 2010) Preventing Nonhomologous End Joining Suppresses DNA Repair Defects of Fanconi Anemia  Adele Adamo, Spencer J. Collis, Carrie A. Adelman, Nicola Silva, Zuzana Horejsi, Jordan D. Ward, Enrique Martinez-Perez, Simon J. Boulton, Adriana La Volpe  Molecular Cell  Volume 39, Issue 1, Pages 25-35 (July 2010) DOI: 10.1016/j.molcel.2010.06.026 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 39, 25-35DOI: (10.1016/j.molcel.2010.06.026) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Lack of FCD-2 in Crossover-Deficient Mutants Leads to Chromosome Associations (A) Representative images of diakinesis oocytes of the indicated genotype stained with DAPI. The number of DAPI-stained bodies present is indicated in the bottom right of each panel. Scale bar, 2 μm. (B) Quantification of the number of DAPI-stained bodies in diakinesis oocytes of the indicated genotype (see color legend on top of the graph). The difference in DAPI-stained bodies between fcd-2;syp-2 and syp-2, and between fcd-2; msh-4, and msh-4 are statistically significant (Table S3). Molecular Cell 2010 39, 25-35DOI: (10.1016/j.molcel.2010.06.026) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 fcd-2 Mutants Exhibit an Increase in spo-11-Dependent RAD-51 Foci (A) Representative images of early pachytene nuclei (indicated by circles) stained with anti-RAD-51 antibodies (red) and DAPI (blue). (B) Quantification of RAD-51 foci in germlines of the indicated genotypes. Statistical analyses are reported in Table S2. Molecular Cell 2010 39, 25-35DOI: (10.1016/j.molcel.2010.06.026) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 LIG-4 Is Responsible for the Chromosome Associations Observed in Crossover-Deficient Mutants Lacking FCD-2 (A) Diakinesis nuclei of the indicated genotypes labeled with two FISH probes to chromosomes III (red) and V (green). DNA is shown in blue. Arrows in the syp-2;fcd-2 and fcd-2;msh-4 panels indicate a single DAPI body that is stained with both FISH probes, demonstrating a fusion event between chromosomes III and V. Scale bar = 2 μm. (B) Quantification of the number of DAPI-stained bodies in diakinesis oocytes of the indicated genotype (see color legend on top of the graph). Statistical analysis is shown in Table S3. Molecular Cell 2010 39, 25-35DOI: (10.1016/j.molcel.2010.06.026) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Deletion of lig-4 Suppresses the ICL-Induced Defects Observed in fcd-2 Mutants (A) Embryonic survival at different time points after treatment with 180 μM CDDP (cis-diamminedichloridoplatinum-II) for the indicated genotype. Error bars represent standard deviation. (B) Quantification of RAD-51 foci detected in the mitotic compartment of the germline at different time points after treatment with nitrogen mustard (HN2). (C) Quantification of germline apoptosis in germlines of the indicated genotypes. Apoptosis was scored using the vital dye SYTO12 before and 48 hr post treatment with 180 μM CDDP. Standard deviations were calculated from at least three independent experiments. (D) Quantification of chromosome breakages and bridges occurring in embryos of the indicated genotypes after treatment with 10 μg/ml trimethylpsoralen (TMP) and increasing doses of UVA (J). (E) Graphical representation of the frequency of postembryonic developmental abnormalities in the indicated genotypes observed before and after treatment with 180 μM CDDP. Molecular Cell 2010 39, 25-35DOI: (10.1016/j.molcel.2010.06.026) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Inhibiting NHEJ Suppresses the ICL Sensitivity of FANCD2-Deficient Human Cells (A) Western blots showing FANCD2 protein levels in siRNA control (Con) and siRNA FancD2 (D2)-treated MO59K and MO59J cells, DNA-PKcs levels in untreated cells, and Actin-loading controls. (B) Sensitivity of control and FANCD2-deficient MO59K and MO59J cells to increasing doses of mitomycin C (MMC). (C) Western blots showing FANCD2 protein levels and Actin-loading controls in control and FANCD2 siRNA-treated HeLa cells. (D) Sensitivity of control and FANCD2-deficient HeLa cells incubated without or with the DNA-PK inhibitor NU7026 (PKi) during MMC exposure. The number of experimental repeats is shown on each graph and the error bars represent the standard deviations. Molecular Cell 2010 39, 25-35DOI: (10.1016/j.molcel.2010.06.026) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Inhibiting NHEJ Suppresses the ICL Sensitivity of FANCA- and FANCC-Deficient Cells (A) Western blots showing FANCA and Actin loading controls in control and FANCA siRNA-treated HeLa cells. (B) Sensitivity of control and FANCA-deficient HeLa cells incubated without or with the DNA-PK inhibitor NU7026 (PKi) during MMC exposure. (C) Sensitivity of wild-type (WT) and FANCA-deficient mouse embryonic fibroblasts incubated without or with the DNA-PK inhibitor NU7026 (PKi) during MMC exposure. (D) Sensitivity of WT and FANCC-deficient mouse embryonic fibroblasts incubated without or with the DNA-PK inhibitor NU7026 (PKi) during MMC exposure. The WT MEF data is the same for (C) and (D) as experiments were performed in parallel. The data for FancA and FancC are presented in separate panels (C and D) to improve clarity. The number of experimental repeats is shown on each graph and the error bars represent the standard deviations. Molecular Cell 2010 39, 25-35DOI: (10.1016/j.molcel.2010.06.026) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 FANCD2 Prevents the Inappropriate Engagement of DNA-PKcs at Damaged Replication Forks (A and B) Representative images (A) and quantification (B) of phospho-Ser2056 DNA-PKcs foci in untreated control (HFF) and FANCD2-deficient (PD20) cells, and cells treated with hydroxurea (HU), mitomycin C (MMC) or ionizing radiation (IR). (C) γH2AX foci in cells treated as in (A). Cells were treated with 3 mM HU, 80 ng/ml MMC, or 5Gy IR and fixed 2 hr, 16 hr, and 30 min later, respectively. Error bars in (B) and (C) represent the standard deviation for three experiments. (D) Model illustrating the function of the FA pathway in suppressing NHEJ during ICL repair. Black and red represent WT and FA-deficient cells, respectively. The dashed red arrow represents competition between HR and NHEJ that arises in FA. Molecular Cell 2010 39, 25-35DOI: (10.1016/j.molcel.2010.06.026) Copyright © 2010 Elsevier Inc. Terms and Conditions