Tatsukawa K et al. (2024), Resection of DNA double-strand breaks activates...

XB-ART-60566

Nucleic Acids Res 2024 Apr 12;526:3146-3163. doi: 10.1093/nar/gkae082.

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Resection of DNA double-strand breaks activates Mre11-Rad50-Nbs1- and Rad9-Hus1-Rad1-dependent mechanisms that redundantly promote ATR checkpoint activation and end processing in Xenopus egg extracts.

Tatsukawa K , Sakamoto R , Kawasoe Y , Kubota Y , Tsurimoto T , Takahashi TS , Ohashi E .

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Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11-Rad50-Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection activates the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9-Hus1-Rad1 (9-1-1) clamp serve as the DNA structure sensors. ATR activation depends also on Topbp1, which is loaded onto DNA through multiple mechanisms. While different DNA structures elicit specific ATR-activation subpathways, the regulation and mechanisms of the ATR-activation subpathways are not fully understood. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9-1-1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for ATM, which, in turn, stimulates Dna2- and Topbp1-loading. Nevertheless, MRN promotes Dna2-mediated end processing largely independently of ATM. 9-1-1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9-1-1. ATR facilitates Mre11 phosphorylation and ATM dissociation. These data uncover that long-range end resection activates two redundant pathways that facilitate ATR checkpoint signaling and DNA processing in a vertebrate system.

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Species referenced: Xenopus laevis
Genes referenced: atm atr chek1 dna2 exo1 hus1 mre11 msh2 nbn nr2e1 orc2 rad1 rad50 rad9a rbbp8 rfc1 rps3a topbp1

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	Figure 1. Linear DNA activates ATR through a 9–1–1-independent pathway in Xenopus egg extracts. (A) 0.2 μl each of mock-treated (lane 1) and Rad9-depleted NPE (lane 2) were analyzed by immunoblotting with indicated antibodies. Rfc1 was used as a loading control. () Cross-reacting band. (B) Singly-primed circular ssDNA (lanes 3–12), primed linear ssDNA (lanes 13–22), or linear dsDNA (lanes 23–32) was incubated in NPE shown in (A), and Chk1 phosphorylation was analyzed by immunoblotting with phosphorylated-Chk1 (S344) and Chk1 antibodies. Rfc1 was used as a loading control. The top panel represents a superimposed image (magenta, pChk1; green, Chk1). () indicates the heavy chain of rabbit IgG leaked out from IgG beads used for immunodepletion. Note that although the pre-immune serum was used for the ‘mock’ control, the amount of IgG leakage varies between pre- and post-immune sera. (C) Relative intensities of pChk1 signals were quantified and normalized to that of the 20-min time-point of the mock-treated samples. Mean ± one SD, n = 3. (D) Immunodepletion of Topbp1 with a rescue experiment. 0.2 μl each of mock-treated (lane 1) and Topbp1-depleted NPE supplemented with either buffer (lane 2) or 150 nM of recombinant Topbp1 (lane 3) were analyzed by immunoblotting with indicated antibodies. Rfc1 was used as a loading control. () IgG. (E) Singly-primed circular ssDNA (lanes 4–15) or linear dsDNA (lanes 16–27) was incubated in NPE shown in (D), and Chk1 phosphorylation was analyzed by immunoblotting. () IgG. (F) Quantification of Chk1 phosphorylation for (E), normalized to the 20-min time-point of the mock-treated samples. Mean ± one SD, n = 3.
	Figure 2. Bypass of the requirement of MRN in the initiation of DSB end resection by 3′-ssDNA overhangs. (A) Schematic diagram of the DNA substrate and the DSB end resection assay. The substrate was incubated in NPE, purified, and digested with S1 nuclease to measure the length of the dsDNA portion and with SacI to monitor end resection at two termini separately. (B) 0.2 μl each of mock-treated (lane 1) and Mre11-depleted NPE (lane 2) were analyzed by immunoblotting with indicated antibodies. Rfc1 was used as a loading control. (C) The tailless substrate (lanes 2–12) or the tailed substrate (lanes 13–23) was incubated in NPE shown in (B), processed as described in (A), and analyzed by agarose gel electrophoresis. The DNA fragment of approximately 6.4 kb corresponds to the major head-to-tail NHEJ product. (D) The tailed substrate was incubated in NPE shown in (B), and Chk1 phosphorylation was analyzed by immunoblotting. (*) IgG.
	Figure 3. Redundant roles of 9–1–1 and MRN in ATR activation and DSB end resection. (A) 0.2 μl each of mock-treated (lane 1), Rad9-depleted (lane 2), Mre11-depleted (lane 3), and Rad9/Mre11-doubly-depleted NPE (lane 4) were analyzed by immunoblotting with indicated antibodies. Rfc1 was used as a loading control. () Cross-reacting band. (B) The tailed substrate was incubated in NPE shown in (A), and Chk1 phosphorylation was analyzed by immunoblotting. () IgG. (C) Quantification of Chk1 phosphorylation for (B), normalized to the 20-min time-point of the mock-treated sample. Mean ± one SD, n = 3. See Supplementary Figure S3A for individual experiments. (D) The tailed substrate was incubated in NPE shown in (A) and analyzed as described in Figure 2A. (E) Quantification of long-range end resection for (D). Relative intensities of the DNA signals within the 3- to 5-kb range were quantified and normalized to the intensity of the input DNA. Mean ± one SD, n = 3. (F) Relative intensities of pChk1 signals in response to tailed DNA in Mre11- and Rad9/Mre11-depleted NPE with a rescue experiment with recombinant 9–1–1, processed as described in (C). Mean ± one SD, n = 3. See Supplementary Figure S3D for a representative image and S3E for individual experiments. (G) Quantification of long-range end resection in Mre11- and Rad9/Mre11-depleted NPE with a rescue experiment with recombinant 9–1–1, processed as described in (E). Mean ± one SD, n = 3. See Supplementary Figure S3F for a representative image. (H) Relative intensities of pChk1 signals in response to tailed DNA in mock-, Rad9-, Nbs1- and Rad9/Nbs1-depleted NPE, processed as described in (C). Mean ± one SD, n = 3. See Supplementary Figure S4B for a representative image and S4C for individual experiments. (I) Quantification of long-range end resection in mock-, Rad9-, Nbs1- and Rad9/Nbs1-depleted NPE, processed as described in (E). Mean ± one SD, n = 3. See Supplementary Figure S4D for a representative image.
	Figure 4. ATM is an essential component of the MRN-dependent ATR activation pathway. (A) 0.2 μl each of mock-treated (lane 1), Rad9-depleted (lane 2), Mre11-depleted (lane 3), ATM-depleted (lane 4), Rad9/Mre11-doubly-depleted (lane 5), Rad9/ATM-doubly-depleted (lane 6), and Mre11/ATM-doubly-depleted NPE (lane 7) were analyzed by immunoblotting with indicated antibodies. Rfc1 was used as a loading control. (B) The tailed substrate was incubated in NPE shown in (A), and Chk1 phosphorylation was analyzed by immunoblotting. (*) IgG. (C) Quantification of Chk1 phosphorylation for (B). Data were processed and presented as described in Figure 1C. Mean ± one SD, n = 3. See Supplementary Figure S5A for individual experiments. (D) The tailed substrate was incubated in NPE shown in (A) and analyzed as described in Figure 2A. (E) Quantification of long-range end resection for (D). Data were processed and presented as described in Figure 3E.
	Figure 5. Recruitment of checkpoint and end-resection factors on tailed DNA. (A) A diagram of the substrate for the DNA pull-down assay. A 3-kb plasmid was linearized with BsaI, and a hairpin adaptor carrying Cy5- and biotin-modified bases was ligated to one terminus. Another terminus was cleaved with PstI to make a 4-nt 3′-overhang, and a poly-dA tail was appended to this terminus with TdT. The resulting DNA was immobilized on biotin-Sepharose through tetrameric streptavidin as a linker. (B) The substrate shown in (A) was immobilized on beads, incubated with GST-S-LacI in NPE depleted of mock, Rad9, Mre11, or both, purified, treated with the Nb.BbvCI nickase to separate two strands, and analyzed by alkaline agarose gel electrophoresis. The DNA was visualized by SYBR-Gold (middle), and the resected DNA strand was monitored by Cy5 fluorescence (bottom). The top panel represents a superimposed image (magenta, Cy5; green, SYBR-Gold). () indicates substrates escaped from Nb.BbvCI digestion. (C) Immunoblotting for Chk1 phosphorylation. () IgG. (D) The immobilized substrate was recovered and bound proteins were analyzed by immunoblotting with indicated antibodies. LacI was used as a loading control. (E) Relative intensities of DNA-bound Dna2 (Top), Exo1 (middle), and Topbp1 signals (bottom) were quantified and normalized to that of the 20-min time-point of the mock-treated samples. Mean ± one SD, n = 3. (F) Bar graphs representing 20-min time points in (E). Filled circles represent individual data. Statistical significance was evaluated using the one-way analysis of variance with Dunnett's test.
	Figure 6. Requirement of the serine 1131 phosphorylation site in Topbp1 in MRN- and 9–1–1-dependent ATR activation subpathways. (A) Immunodepletion of Topbp1 with a rescue experiment. 0.2 μl each of mock-treated (lane 1) and Topbp1-depleted NPE supplemented with either buffer (lane 2) or 150 nM of recombinant Topbp1 (WT: wild-type, lane 3; SA: S1131A, lane 4) were analyzed by immunoblotting with indicated antibodies. Orc2 was used as a loading control. (B) Poly(dA)70–(dT)70 oligonucleotides, the tailless, or the tailed substrate were incubated in NPE shown in (A), and Chk1 phosphorylation was analyzed by immunoblotting. () IgG. (C) Quantification of pChk1 signals for (B), normalized to the 20-min time-point of the mock-treated samples. Mean ± one SD, n = 3. Filled circles represent individual data. Statistical significance was evaluated using paired t-test. (D) Immunodepletion of Topbp1 and Rad9 with a Topbp1 rescue experiment. 0.2 μl each of untreated (lane 1), Rad9-depleted (lane 2) and Rad9/Topbp1-doubly-depleted NPE (lanes 3–5) supplemented with either buffer (lane 3) or 150 nM of recombinant Topbp1 (WT: wild-type, lane 4; SA: S1131A, lane 5) were analyzed by immunoblotting with indicated antibodies. Msh2 was used as a loading control. () IgG. (E) The tailed substrate was incubated in NPE shown in (D), and Chk1 phosphorylation was analyzed by immunoblotting. (*) IgG. (F) Quantification of pChk1 signals for (E), normalized to the 20-min time-point of the Rad9-depleted sample. Mean ± one SD, n = 3. (G) Bar graphs representing 40-min time points in (F). Filled circles represent individual data. Statistical significance was evaluated using the one-way analysis of variance with Dunnett's test.
	Figure 7. Requirement of Topbp1 and ATR in the assembly of checkpoint complexes on tailed DNA. (A) The tailed substrate shown in Figure 5A was immobilized on beads, incubated with GST-S-LacI in NPE depleted of mock (lanes 1 and 4–7) or Topbp1 (lanes 2, 3, and 8–15) supplemented with either buffer (lanes 1, 2 and 4–11) or 300 nM recombinant Topbp1 (lanes 3 and 12–15), and recovered. Input (lanes 1–3) and DNA-bound samples (lanes 4–15) were analyzed by immunoblotting with indicated antibodies. LacI was used as a loading control. (B) Relative intensities of DNA-bound Rad9 (top), ATM (middle), or ATR signals (bottom) were quantified and normalized to that of the 20-min (for Rad9 and ATR) or the 2-min (for ATM) time-point of the mock-treated sample. Mean ± one SD, n = 3. Bar graphs on the right represent the mean values at the indicated time points with individual data shown by filled circles and error bars representing the ± one SD range. Statistical significance was evaluated using paired t-test. (C) Percentages of phosphorylated Mre11 relative to total Mre11 were quantified as indicated in Supplementary Figure 10B and presented as described in (B). (D) DNA pull-down assay with tailed DNA from mock- (lanes 1 and 3–6) or ATR-depleted NPE (lanes 2 and 7–10). (E) Quantification of DNA-bound Rad9 (Top), ATM (middle), and Topbp1 signals (bottom) in (D), normalized to the 20-min (for Rad9 and Topbp1) or 2-min (for ATM) time-point of the mock-treated sample and presented as described in (B). n = 3. (F) Percentages of phosphorylated Mre11 relative to total Mre11 in (D), presented as described in (C). n = 3.
	Figure 8. A model for the 9–1–1- and MRN-dependent ATR activation and DSB end resection pathways. DSBs activate the MRN-ATM-Topbp1-dependent ATR activation pathway (47,53). After the initial DSB end resection, both the MRN and 9–1–1 pathways redundantly detect the DNA structure and stimulate Dna2-mediated DSB end resection. MRN recruits and activates ATM to induce ATR signaling. ATM facilitates the loading of Topbp1 and Dna2 and activates ATR through phosphorylation on multiple substrates including S1131 on Topbp1. 9–1–1 promotes the recruitment of Topbp1, which, in turn, stabilizes 9–1–1 and ATR. Once activated, ATR facilitates the unloading of ATM and phosphorylation of Mre11, possibly turning off the ATM pathway. See text for details.

References [+] :

Blackford, ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response. 2017, Pubmed