1. Volume 53, Issue 1, Pages 7-18 (January 2014)
DNA Double-Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities 
Atsushi Shibata, Davide Moiani, Andrew S. Arvai, Jefferson Perry, Shane M. Harding, Marie-Michelle Genois, Ranjan Maity, Sari van Rossum-Fikkert, Aryandi Kertokalio, Filippo Romoli, Amani Ismail, Ermal Ismalaj, Elena Petricci, Matthew J. Neale, Robert G. Bristow, Jean-Yves Masson, Claire Wyman, Penny A. Jeggo, John A. Tainer 
Molecular Cell 
Volume 53, Issue 1, Pages 7-18 (January 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 53, 7-18DOI: (10.1016/j.molcel.2013.11.003)
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3. Figure 1
Development of Inhibitors against MRE11 Exo- or Endonuclease Activity
(A) Derivation by modification of mirin.
(B) TmMre11 structures in complex with exo- and endonuclease inhibitors. The top left panel shows an overlay of cocrystal structures of TmMre11 (cartoon, colored brown) in complex with the exonuclease inhibitors, mirin (sticks, carbon atoms colored brown), and PFM39 (green). The bottom left panel shows an overlay of the cocrystal structures of TmMre11 (cyan) in complex with the endonuclease inhibitors PFM01 (purple) and PFM03 (dark green). Each inhibitor type has a distinct binding site in proximity to the active site metal ions. The right hand panel shows an overlay of the unliganded TmMre11 (cartoon, colored pink) with mirin (brown) and PFM03 (cyan). The relative position of each inhibitor is highlighted by a dotted representation of its van der Waals surface. The endonuclease and exonuclease inhibitor binding sites are adjacent, but respectively positioned to distinctly disrupt dsDNA (light green tubes) end opening by His61 or the ssDNA (violet tube) pathway adjacent to the loop containing Asn93–Phe102, leading toward the active site metal ions. Nitrogen, oxygen, and sulfur atoms are colored blue, red, and yellow, respectively. Active site metal ions are shown as magenta spheres.
(C) Mirin and PFM39, but not PFM03, inhibit 5 nM MRN exonuclease activity. Labeled DNA (100 nM) were incubated with human MRN for 30 min at 37°C.
(D) PFM03, but not mirin or PFM39, inhibits human MRE11 endonuclease activity. Analysis using MRN in place of MRE11 is shown in Figure S1E. ϕX174 circular ssDNA was incubated with purified human MRE11 and DMSO or inhibitor at the indicated concentration. The inhibition assay was established in a time and protein concentration-dependent manner to cause loss of ∼70% circular DNA. The figure shows the percent of circular ssDNA degraded relative to the control (DMSO-treated) sample. c, circular DNA; ∗, degraded DNA. PFM01 was excluded from inhibitor analysis due to inefficient solubility in vitro. Error bars represent SEM from greater than three experiments. Figures S1G–S1I show the impact of the inhibitors on MRN DNA binding and ATPase activity. See also Figure S1.
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4. Figure 2 MRE11 Inhibitors Prevent DSB End Resection
(A) MRE11 inhibitors reduce chromatin-bound RPA levels after IR. IR-induced RPA retention in A549 cells was monitored by FACS analysis at 2 hr after 10 Gy IR. G2 cells were gated using FACSDiva software (Figure S2A).
(B) Quantification of RPA-FACS analysis using two different concentrations of inhibitor.
(C) Either exo- or endonuclease inhibitor treatment reduces IR-induced RAD51 foci formation in G2 cells. RAD51 foci were quantified in A549 cells 2 hr after 3 Gy IR. Knockdown efficiency of CtIP and BRCA2, included as controls, is shown. Representative images are shown in Figure S2C.
(D) MRE11 recruitment to DNA damage sites is unaffected by the MRE11 inhibitors. GM05757 primary human fibroblasts were subjected to a UV microbeam. Protein recruitment was analyzed 1 hr after the addition of each inhibitor. γH2AX is used to identify damage sites. The scale bar is 10 μm. For G2 foci analysis, 4 μM aphidicolin (APH) was added after irradiation. APH does not affect γH2AX, RPA, RAD51 foci formation, NHEJ, or HR in G2 phase cells. Full controls for using APH in the context of repair and signaling have been undertaken (Shibata et al., 2010, 2011). Error bars represent SEM from greater than three independent experiments. PFM01 (100 μM), PFM03 (100 μM), PFM39 (100 μM), or mirin (500 μM) were added 30 min before irradiation in (A), (C), and (D). For further analysis of ATM activation after inhibitor treatment, see Figures S2D and S2E. See also Figure S2.
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5. Figure 3 Distinct Phenotypes of MRE11 Inhibitors in DSB repair
(A and B) Exonuclease, but not endonuclease, inhibitor treatment causes a DSB repair defect in G2. DSB repair in G2 (CENPF+) cells was investigated by γH2AX foci analysis. 1BR3 (WT) hTERT were fixed and stained at 8 hr after 3 Gy IR. Inhibitors were added 30 min before IR. The full time course for DSB repair is shown in Figure S3A.
(C) DSB repair defects following inhibitor addition are consolidated by chromosomal break analysis. Irradiated G2 cells were collected at 12–16 hr after 6 Gy IR.
(D) Depletion of the HC building factor, KAP-1, does not alleviate the DSB repair defect in exonuclease inhibitor-treated cells. 1BR3 hTERT cells were subjected to KAP-1 siRNA and irradiated with 3 Gy. KAP-1 knockdown efficiency is shown. To prevent progression of S phase cells into G2 during analysis, we added replicative polymerase inhibitor APH in all foci assays (A, B, and D). The dots in the box plot were created using SigmaPlot 12.0 and represent outliers (>95% or <5%) of samples. A line in each box represents the median. Error bars represent SEM from greater than three experiments. See also Figure S3.
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6. Figure 4
MRE11 Endonuclease Activity Commits to Homologous Recombination
(A) Addition of MRE11 endonuclease inhibitors rescues the repair defect in HR-defective cells. γH2AX foci were enumerated in 48BR (WT) and HSC62 (BRCA2-defective) primary cells at 8 hr after 3 Gy IR. The full time course for DSB repair and cell cycle profiles is shown in Figure S4.
(B) The chromosome break defect observed in BRCA2 siRNA-treated 1BR3 (WT) hTERT was alleviated following treatment with endonuclease inhibitor.
(C) RAD51 foci formation is diminished in HSC62 (BRCA2-defective) cells with or without inhibitor treatment. RAD51 foci formation was analyzed 2 hr after 3 Gy IR.
(D) Treatment with an endonuclease inhibitor causes an additive repair defect in XLF, but not control, cells, demonstrating NHEJ usage. γH2AX analysis was performed in XLF-defective hTERT cells after 2 Gy IR. Cells were irradiated with 2 Gy (rather than 3 Gy) to enhance accuracy in scoring the greater number of DSBs remaining in XLF-defective cells.
(E) Inhibitor treatment impairs HR, but endonuclease inhibitor addition only enhances NHEJ usage. PFM39 (50 μM), PFM01 (50 μM), or PFM03 (50 μM) was added 8 hr after I-SceI transfection until cell fixation. The frequency of DSB repair usage was measured using the reporter cell lines U2OS DR-GFP (HR) or H1299 dA3 (NHEJ). The percentage of repair was normalized to the DMSO-treated control. Error bars represent SEM from greater than three experiments. See also Figure S4.
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7. Figure 5
MRE11 Endonuclease Functions Upstream of Exonuclease Activity in Homologous Recombination
(A and B) Endonuclease inhibitors alleviate the repair defect conferred by the exonuclease inhibitors. DSB repair was analyzed by γH2AX foci in G2 cells at 2 and 8 hr after irradiation with 3 Gy. The repair defect is only observed at 8 hr, consistent with the fact that at 2 hr, DSB repair occurs by MRE11-independent NHEJ.
(C) A defect in IR-induced RAD51 foci formation was confirmed in the presence of double-inhibitor-treated 1BR3 (WT) hTERT cells.
(D) Cells treated with MRE11 exonuclease and endonuclease inhibitors repair DSBs by NHEJ, similar to the result in cells treated with MRE11 endonuclease inhibitor alone.
(E) HR and NHEJ repair frequency following single or combined endonuclease and exonuclease inhibitor treatment was measured by the U2OS DR-GFP (HR) or H1299 dA3 (NHEJ) assay, as described in Figure 4E. Error bars represent SEM from greater than three experiments. For further evidence showing that inhibition of MRE11 endo- and not exonuclease activity allows NHEJ usage, see Figure S5.
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8. Figure 6
Analysis of DSB Repair Pathway Choice in MRE11-Defective ATLD Cells
(A) ATLD2 cells show significantly reduced MRN levels, causing impaired IR-induced ATM activation and downstream signaling. Cells were harvested 30 min after 3 Gy IR (right panel).
(B) Depletion of KAP-1 alleviates the DSB repair defect in ATLD2 cells, confirming that the repair defect is caused by impaired chromatin modification (see also Figure S6). DSB repair in G2 cells is examined by γH2AX foci analysis after 3 Gy IR. Box plot with statistical analysis is shown in the right panel.
(C) Impaired RPA and RAD51 foci formation in ATLD2 G2 cells is not rescued by KAP-1 siRNA, demonstrating that MRE11 is essential for resection.
(D) Exo- or endonuclease inhibitor treatment shows no additive impact on DSB repair in ATLD + KAP-1 siRNA cells.
(E) The fraction rescued by KAP-1 siRNA is prevented by a DNA-PKi. DNA-PKi is added at 3.5 hr after 3 Gy IR, when most NHEJ has been completed in G2. Error bars represent SEM from greater than three experiments.
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9. Figure 7
Combined MRE11 Exonuclease 3′-5′ and EXO1/BLM 5′-3′ Loss Allows Repair Switch to NHEJ
(A) MRE11 3′-5′ exonuclease and EXO1/BLM 5′-3′, which digest in opposite directions, both contribute to IR-induced RPA foci formation. 1BR3 (WT) hTERT cells were fixed and stained with RPA (a resection marker) and 53BP1 (a DSB marker) at 2 hr after 3 Gy IR. RPA signal intensity was measured by ImageJ. The quantified signal intensity of RPA foci at 53BP1 foci is shown in the right panel. For each sample, 200–300 foci were analyzed.
(B) MRE11 exonuclease and EXO1/BLM activities redundantly contribute to RPA Ser4/Ser8 phosphorylation. A549 cells are harvested at 2 hr after 30 Gy IR. The pRPA signal was quantified by ImageJ. Since phosphorylated RPA migrates distinctly to unmodified RPA migration, the pRPA signal was normalized to Ku80 instead of total RPA.
(C) Inhibition of MRE11 exonuclease activity in EXO1/BLM-depleted cells allows normal DSB repair kinetics. γH2AX foci analysis was performed in A549 cells following EXO1/BLM siRNA with or without inhibitors after 3 Gy IR.
(D) HR and NHEJ repair frequency following EXO1/BLM siRNA alone or EXO1/BLM siRNA + PFM39 was measured by the U2OS DR-GFP (HR) or H1299 dA3 (NHEJ) assay. Error bars represent SEM from greater than three experiments.
(E) Model for repair pathway choice at two-ended DSBs in G2. (1) In WT cells, ssDNA regions are expanded by MRE11 3′-5′ exonuclease and EXO1/BLM activity. (2) Loss of either MRE11 3′-5′ exonuclease or EXO1/BLM results in a DSB repair defect since neither HR nor NHEJ can ensue. (3) The DSB repair pathway can be switched from HR to NHEJ in the absence of CtIP, MRE11 endonuclease, or combined loss of MRE11 exonuclease + EXO1/BLM activity. See also Figure S7.
Molecular Cell  , 7-18DOI: ( /j.molcel )
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