A Role for the MRN Complex in ATR Activation via TOPBP1 Recruitment Anja M. Duursma, Robert Driscoll, Josh E. Elias, Karlene A. Cimprich  Molecular Cell  Volume 50, Issue 1, Pages 116-122 (April 2013) DOI: 10.1016/j.molcel.2013.03.006 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 50, 116-122DOI: (10.1016/j.molcel.2013.03.006) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 MRN Binds an ATR-Activating Structure (A) M13-based structures used in this study consist of 7 kb M13 phage circular ssDNA and annealed oligonucleotides. Both structures contain a double-biotinylated oligonucleotide of 50 bases. The ATR-activating structure has in addition three oligonucleotides of 80 bases biotinylated on the 3′ end and with a free 5′ end. Biotinylated primer ends are blocked with streptavidin, as represented by dots. (B) Model structures induce ATR-dependent phosphorylation of CHK1 in Xenopus NPE, as detected by western blot analysis. ATRi, ATR inhibitor (ATR-45). (C) Similar to the experiment shown in (B), but here the effect of an ATM inhibitor (ATMi: KU-55933) was assessed. (D) Western blot analysis of a pull-down showing specific binding to the ATR-activating structure. o, M13 only. (E) Silver-stained gel showing proteins isolated from NPE, with the structures shown. Arrows indicate the bands isolated for mass spectrometry analysis and the proteins subsequently identified in those bands. (F) Western blot analysis of proteins associated with control and ATR-activating structures isolated from NPE confirms enriched binding of the xMRN complex to the ATR-activating structure. Five percent of flowthrough (FT) is shown. See also Figure S1. Molecular Cell 2013 50, 116-122DOI: (10.1016/j.molcel.2013.03.006) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 The xMRN Complex Is Required for ATR Checkpoint Activation (A) Western blot analysis of mock- or MRE11-depleted NPE incubated with the structures shown. (B) Rescue of ATR signaling in xMRE11-depleted NPE by human recombinant MRN complex (hMRN). Samples were prepared and analyzed as in (A). (C) Western blot analysis of mock- or MRE11-depleted LSE supplemented with sperm chromatin and aphidicolin. See also Figure S2. Molecular Cell 2013 50, 116-122DOI: (10.1016/j.molcel.2013.03.006) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 xMRN Recruits xTOPBP1 to ATR-Activating Structures (A) Proteins isolated with the control structure or ATR-activating structure from mock- or xMRE11-depleted NPE were analyzed by western blotting. Five percent of FT is shown. (B) Western blot analysis of proteins isolated with control or ATR-activating structures before and after addition of the xRAD9 C-terminal tail (C-tail) to NPE. (C) Western blot of NPE incubated with or without the xRAD9 C-terminal tail and with the structures shown. (D) Pull-down of control and ATR-activating structures from NPE supplemented with recombinant protein of TOPBP1 BRCT 1 and 2, 3–6, or 6–8. GST, glutathione S-transferase. See also Figure S3. Molecular Cell 2013 50, 116-122DOI: (10.1016/j.molcel.2013.03.006) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Targeting the TOPBP1 AAD to ssDNA Rescues MRN Depletion (A) CHK1 phosphorylation was monitored by western blot in mock- or MRE11-depleted NPE containing the indicated structure and, where indicated, OB-AAD. The control (C) is water. (B) Experiment similar to that in (A). Where indicated, xRAD9 C-terminal tail and OB-AAD were added to NPE. (C) Model for MRN function in ATR activation (see Discussion for details). See also Figure S4. Molecular Cell 2013 50, 116-122DOI: (10.1016/j.molcel.2013.03.006) Copyright © 2013 Elsevier Inc. Terms and Conditions