Volume 64, Issue 3, Pages 580-592 (November 2016) Mre11 Is Essential for the Removal of Lethal Topoisomerase 2 Covalent Cleavage Complexes  Nguyen Ngoc Hoa, Tsubasa Shimizu, Zhong Wei Zhou, Zhao-Qi Wang, Rajashree A. Deshpande, Tanya T. Paull, Salma Akter, Masataka Tsuda, Ryohei Furuta, Ken Tsutsui, Shunichi Takeda, Hiroyuki Sasanuma  Molecular Cell  Volume 64, Issue 3, Pages 580-592 (November 2016) DOI: 10.1016/j.molcel.2016.10.011 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 64, 580-592DOI: (10.1016/j.molcel.2016.10.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Mre11-Nuclease Activity Is Required for Repairing DSBs Induced by Etoposide, the Top2 Poison, but Not ICRF193, the Top2 Catalytic Inhibitor The diagrams of “Exchanged,” “Isochromatid,” and “Chromatid” breaks are shown in the graph legends (also see Figure S1). (A) The number of spontaneously arising mitotic chromosome breaks in mitotic chromosome spreads of DT40 cells carrying the indicated genotype. MRE11−/− and KU70−/−/MRE11−/− mutants were treated with doxycycline (100 ng/mL) for 3 days to inactivate the wild-type MRE11 transgene (Yamaguchi-Iwai et al., 1999). Note that MRE11−/H129N DT40 cells are able to proliferate. (B) ICRF193-induced mitotic chromosome breaks. The number of chromosome breaks in ICRF193-treated cells is subtracted by that in untreated cells. (C) Etoposide-induced mitotic chromosome breaks. The number of chromosome breaks in etoposide-treated cells is subtracted by that in untreated cells. (D) The number of spontaneously arising mitotic chromosome breaks in mitotic chromosome spreads of TK6 cells carrying the indicated genotype. MRE11−/− and MRE11−/H129N TK6 cells were generated conditionally by having exposed the cells to 4-OH tamoxifen (4-OHT) for the indicated days. (E) ICRF193-induced mitotic chromosome breaks in the indicated TK6 cells having treated with 4-OHT for 3 days. (F) Etoposide-induced mitotic chromosome breaks in the indicated TK6 cells having treated with 4-OHT for 3 days. In the above data, error bars indicate SD from three independent experiments. At least 100 mitotic chromosome spreads were analyzed in the three experiments. Molecular Cell 2016 64, 580-592DOI: (10.1016/j.molcel.2016.10.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Mre11-Nuclease Activity Is Required for the Repair of DSBs Induced by Etoposide in G1 (A and B) DSB-repair kinetics of TK6 cells during the G1 phase following 30 min pulse exposure to ICRF193 (A) and etoposide (B). Cells were treated with either 20 μM ICRF193 or 10 μM etoposide for 30 min and then released to fresh medium. DSB repair was evaluated by counting the number of γH2AX foci over time. The pulse exposure was done between the time 0 and 0.5 hr on the x axis, and γH2AX foci were examined at 1, 2, 4, and 6 hr on the x axis. The percentage of γH2AX-positive cells, those displaying at least four γH2AX foci per nucleus, is shown on the y axis. Error bars were plotted for SD from three independent experiments. At least 100 G1 cells were analyzed for each experiment. (C) The doses that reduce percent colony survival to 50% (LD50) following continuous exposure of the indicated genotypes to etoposide. Error bars represent SD of the mean from three independent experiments. Molecular Cell 2016 64, 580-592DOI: (10.1016/j.molcel.2016.10.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 The Nuclease Activity of Mre11 Is Required for Eliminating Top2cc Formed during the Physiological Cell Cycle (A) The number of spontaneously arising chromosome breaks in the mitotic chromosome spreads of the indicated genotypes after addition of 4-OHT at time zero. At least 100 mitotic chromosome spreads were analyzed. Error bars represent the SD of three independent experiments. (B) Schematic of the in vivo Top2cc measurement by immunodetection with anti-Top2 antibody. A total of 200 μg (for TK6, ATLD2, and GM7166) and 250 μg (for mouse brain) of genomic DNA was subjected to sedimentation by the CsCl-gradient ultracentrifugation. Genomic DNA of wild-type TK6 cells having treated with 10 μM for 2 hr was included as a control in every western blot analysis. The treatment reduced cellular survival only by ∼1% relative to untreated cells. Individual fractions were blotted to PVDF filters followed by western blot using anti-Top2 antibody. The top two fractions include free Top2, while the other right fractions include Top2cc. (C) Western blot analysis of Top2 in the indicated TK6 cells having treated with 10 μM etoposide (+) or DMSO (−) for 2 hr. (D) Western blot analysis of Top2 in MRE11−/H129N cells having treated with 4-OHT for the indicated days. (E) Quantification of Top2cc in the indicated genotypes of (C) and (D) relative to the amount of Top2cc in wild-type TK6 cells having treated with 10 μM etoposide for 2 hr. All western blot analyses included the etoposide-treated wild-type cells as a control. Every experiment was done independently at least three times, and the error bars represent SD. Molecular Cell 2016 64, 580-592DOI: (10.1016/j.molcel.2016.10.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Overexpression of Tdp2 Reverses the Mortality Associated with Severe Genome Instability of MRE11−/− and MRE11−/H129N Cells (A) Western blot analysis of the TDP2 transgene expression. Clone #1 was further analyzed. (B) The proliferation kinetics of the indicated genotypes. The middle and right panels are the proliferation kinetics after addition of 4-OHT at time zero. The y axis indicates cumulative cell numbers. (C) The number of spontaneously arising chromosome breaks in the mitotic chromosome spreads of the indicated genotypes after addition of 4-OHT at time zero. At least 100 mitotic chromosome spreads were analyzed. (D) DSB-repair kinetics of TK6 cells during the G1 phase following 30 min pulse exposure to etoposide. The data are shown as in Figure 2B. (E) The amount of Top2cc in the indicated genotypes. The days indicate time after addition of 4-OHT. The data are shown as in Figures 3C and 3D. (F) Quantification of Top2cc in Figure 4E. The data are shown as in Figure 3E. Error bars represent the SD of three independent experiments. Molecular Cell 2016 64, 580-592DOI: (10.1016/j.molcel.2016.10.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 The Endogenous Accumulation of Top2cc in Mre11-Deficient ATLD Cells, Nbs1-Deficient Nijmegen Syndrome Cells, and the Brains of Nbs1-Deficient Mice (A) The amount of Top2cc in ATLD cells, those reconstituted with a MRE11 transgene, Nijmegen syndrome cells, and those reconstituted with a NBS1 transgene. The lower panel is quantification, shown as in Figure 3E. (B) The amount of Top2cc in AT cells and those reconstituted with an ATM transgene. The lower panel is quantification. (C) The amount of Top2cc in the brain of the indicated genotypes. The analyzed mice are embryonic day 15.5 embryos, 2 day neonatal mice, and 173-day-old adult mice. The lower panel is quantification. Molecular Cell 2016 64, 580-592DOI: (10.1016/j.molcel.2016.10.011) Copyright © 2016 Elsevier Inc. Terms and Conditions