Single-Stranded DNA Orchestrates an ATM-to-ATR Switch at DNA Breaks Bunsyo Shiotani, Lee Zou  Molecular Cell  Volume 33, Issue 5, Pages 547-558 (March 2009) DOI: 10.1016/j.molcel.2009.01.024 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Double-Stranded DNA-Induced ATM Activation in Human Cell Extracts (A) dsDNA, but not ssDNA, induces the phosphorylation of ATM and Chk2 in a concentration-dependent manner. Increasing concentrations (1.25, 12.5, 125, and 1250 nM) of ssDNA (ss70) or dsDNA (ds70) was incubated in HeLa cell nuclear extracts. In all the panels in this figure, the levels of phospho-ATM (Ser1981), ATM, phospho-Chk2 (Thr68), and Chk2 were analyzed by western blotting. (B) dsDNA-induced phosphorylation of ATM and Chk2 is inhibited by ATM inhibitor. ds70 (125 nM) was incubated in extracts in the presence or absence of 10 μM KU-55933. (C) dsDNA-induced Chk2 phosphorylation depends on ATM in extracts. ds70 (125 nM) was incubated in the nuclear extracts derived from AT cells or the AT cells complemented with ATM. (D) dsDNA-induced phosphorylation of ATM and Chk2 is Nbs1 dependent. ds70 (125 nM) was incubated in the extracts derived from cells treated with Nbs1 siRNA or cells mock treated. (E) dsDNA-induced phosphorylation of ATM and Chk2 is Ku70 independent. ds70 (125 nM) was incubated in the extracts derived from cells treated with Ku70 siRNA or cells mock treated. Molecular Cell 2009 33, 547-558DOI: (10.1016/j.molcel.2009.01.024) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 The Activation of ATM by dsDNA Is Length and End Dependent (A) dsDNA activates ATM in a length-dependent manner. ds20, ds40, and ds70 were incubated in HeLa cell nuclear extracts at the indicated concentrations. The three DNA fragments were used at either equal molar concentrations (the left half) or equal DNA mass (the right half). The three panels represent the experiments done with three different sets of DNA concentrations, which increase by 10-fold from top to middle, and from middle to bottom panel. ATM and phospho-ATM were analyzed by western blotting. (B) The dsDNA proximal to breaks contributes to ATM activation. ds40, bubble (30 nt ssDNA flanked by 20 bp dsDNA on each side), and ds70 (all at 12.5 nM) were incubated in extracts and analyzed as above. (C) The binding of MRN to dsDNA is length dependent. Biotinylated ds20, ds40, and ds70 (all at 6.25 pmole) were attached to streptavidin-coated beads and incubated with purified MRN. The DNA-bound Nbs1 and Rad50 were detected by western blotting. (D) ATM activation depends on DNA ends. ds20 (left) and ds70 (right) were unmodified or biotinylated at all four DNA ends. ds20 (1.25 μM) and ds70 (12.5 nM) were incubated with or without streptavidin and added to extracts. (E) Paired DNA ends are required for ATM activation. The paired or unpaired ends of a fork structure (20 bp dsDNA and 50 nt ssDNA; 1.25 μM) were blocked with streptavidin as indicated and were analyzed in extracts as above. The DNA structures used in each experiment are depicted below the corresponding panel, with the dots representing biotin. Molecular Cell 2009 33, 547-558DOI: (10.1016/j.molcel.2009.01.024) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Regulation of ATM Activation by SSOs (A and B) SSOs of random sequences interfere with ATM activation in a manner dependent on the length of SSO but independent of the length of dsDNA. (A) ds20 (1.25 μM) and (B) ds70 (12.5 nM) with 3′ or 5′ SSOs at the indicated length were incubated in extracts. ATM, phospho-ATM, Chk2, and phospho-Chk2 were analyzed by western blotting. (C) Poly(A) SSOs interfere with ATM activation in a length-dependent manner. ds20 (1.25 μM) with 3′ or 5′ poly(A) SSOs at the indicated length was analyzed as above. (D) ssDNA interferes with ATM activation more efficiently in cis than in trans. ds70 (12.5 nM), either alone or in combination with free 25 nt random ssDNA (25 or 62.5 nM), and ds70 with 3′ or 5′ 25 nt SSOs of random sequences (12.5 nM) were incubated in extracts and analyzed as above. (E) SSOs attenuate the binding of MRN to dsDNA in extracts, but not in an assay using purified proteins. ds70 and ds70 with 25 nt 3′ SSOs were biotinylated at the 3′ end of one of the two strands. The DNA structures (1.25 pmole) were incubated with extracts or purified MRN complex and retrieved with streptavidin-coated beads. The Nbs1 and Mre11 associated with DNA were analyzed by western blotting. Molecular Cell 2009 33, 547-558DOI: (10.1016/j.molcel.2009.01.024) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 ATM Activation Requires the Junctions of ssDNA and dsDNA (A–C) ATM activation is dependent on ssDNA/dsDNA junctions but not SSO ends. ds20 with 5′ (A) or 3′ (B) 25 nt SSOs, and ds70 with 5′ 25 nt SSOs (C) were unmodified or biotinylated at the junctions or the SSO ends. These ds20- and ds70-derived DNA structures (1.25 μM and 12.5 nM, respectively) were incubated with or without streptavidin and added to extracts. ATM, phospho-ATM, Chk2, and phospho-Chk2 were analyzed by western blotting. (D) ATM is not activated by DNA nicks and gaps. A plasmid was nicked by N. BbvCI or linearized by SmaI (Figure S4A). The resulting nicked plasmid was further treated with exonuclease III for the indicated periods to generate ssDNA gaps. Uncut, linear, nicked, and gapped plasmids (4 ng/μl) were incubated in extracts and analyzed as above. Molecular Cell 2009 33, 547-558DOI: (10.1016/j.molcel.2009.01.024) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Resection of DNA Ends Promotes an ATM-to-ATR Switch In Vitro (A) Inhibition of ATM activation by resection of DNA ends. A plasmid linearized with HpaI was treated with T7 exonuclease (T7) or exonuclease III (III) to generate SSOs. The resulting DNA (12 ng/μl) was incubated in extracts. (B) Recruitment of RPA and ATRIP to SSOs. ds20 and ds20 with 5′ or 3′ 50 nt poly(A) SSOs (1.25 pmole) were attached to beads and incubated in extracts. The RPA and ATRIP associated with DNA were detected by western blotting. (C) The phosphorylation of RPA32 induced by unprocessed linear plasmid is dependent on ATM and DNA-PKcs. Linear plasmid (4 ng/μl) was incubated in extracts in the presence of KU-55933 and NU7026 (10 μM). RPA and phospho-RPA (Ser33) were analyzed by western blotting. (D) Induction of RPA phosphorylation (Ser33) by resection of DNA ends. Linear plasmid was resected as in (A) and incubated in extracts in the presence of KU-55933 and NU7026 (10 μM). (E) The SSO-induced RPA32 phosphorylation is independent of ATM and DNA-PKcs. Linear plasmid was mock treated or processed by T7 exonuclease (2 min) and exonuclease III (5 min). The resulting DNA (4 ng/μl) was incubated in extracts with KU-55933 and NU7026 (10 μM) or wortmannin (20 μM). (F) The SSO-induced RPA32 phosphorylation is ATR-dependent. Nuclear extracts were prepared from cells treated with ATR siRNA or cells mock treated. Linear plasmid was resected as in (E) and incubated in both extracts. Molecular Cell 2009 33, 547-558DOI: (10.1016/j.molcel.2009.01.024) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Consecutive Activation of ATM and ATR in Cells (A) Distinct kinetics of Chk2 and Chk1 phosphorylation after IR. HeLa cells were treated with 10 Gy of IR and were analyzed at the indicated time points. The levels of phospho-Chk2 (Thr68), Chk2, phospho-Chk1 (Ser345), Chk1, and Tubulin (a loading control) in the soluble extracts (Sol., see the Experimental Procedures) were analyzed by western blotting. (B) Transient accumulation of phosphorylated ATM on chromatin after IR. HeLa cells were treated with 10 Gy of IR and the chromatin fractions (Chr., see the Experimental Procedures) and whole-cell extracts (WCE) were prepared at the indicated time points. The levels of phospho-ATM (Ser1981), ATM, and Orc2 (a loading control) were analyzed by western blotting. (C) Immunostaining of RPA in IR-treated cells. HeLa cells were treated with 10 Gy of IR, and RPA32 was stained at the indicated time points. (D) Quantifications of the cells positive for IR-induced RPA foci in the experiment shown in (C). More than 200 cells were counted at each time point. Molecular Cell 2009 33, 547-558DOI: (10.1016/j.molcel.2009.01.024) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Regulation of the ATM-to-ATR Switch by ATM, CtIP, and Exo1 (A) ATR is not required for the attenuation of ATM activation by DSB resection. HeLa cells transfected with control siRNA or ATR siRNA were irradiated with IR (10 Gy) and analyzed at the indicated time points. The levels of ATR, phospho-Chk2 (Thr68), Chk2, phospho-Chk1 (Ser345), and Chk1 were monitored by western blotting. (B) ATM is required for DSB-induced ATR activation. HeLa cells were treated with KU-55933 (5 μM) or mock treated with DMSO for 30 min prior to the IR treatment, and analyzed as above at the indicated time points. (C) Partial inhibition of ATM leads to delayed and reduced activation of Chk2 and Chk1. HeLa cells were treated with KU-55933 (1 μM) or DMSO for 30 min prior to IR and analyzed at the indicated time points. (D) Expression of CtIP and Exo1 promotes the ATM-to-ATR switch in cells. HeLa cells were transfected with the plasmids encoding CtIP and Exo1 and were treated with IR. Chromatin and soluble fractions were prepared from cells collected at the indicated time points. The levels of phospho-ATM (Ser1981), Orc2, phospho-Chk2 (Thr68), Chk2, phospho-Chk1 (Ser345), and Chk1 in the indicated fractions were analyzed as above. (E) Expression of CtIP and Exo1 cannot bypass the requirement of ATM for IR-induced ATR activation. Cells overexpressing CtIP and Exo1 were treated with KU-55933 (5 μM) or mock treated with DMSO for 30 min prior to IR. The levels of phospho-Chk1 (Ser345) and Chk1 were analyzed at the indicated time points. (F) A model for an SSO-orchestrated ATM-to-ATR switch at DSBs. Molecular Cell 2009 33, 547-558DOI: (10.1016/j.molcel.2009.01.024) Copyright © 2009 Elsevier Inc. Terms and Conditions