Maintenance of the DNA-Damage Checkpoint Requires DNA-Damage-Induced Mediator Protein Oligomerization  Takehiko Usui, Steven S. Foster, John H.J. Petrini  Molecular Cell  Volume 33, Issue 2, Pages 147-159 (January 2009) DOI: 10.1016/j.molcel.2008.12.022 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Rad9-BRCT Interacts with PIKK-Phosphorylated Rad9 SCD (A) Schematic illustrations of Rad9 functional domains and Rad9-TEV-HA system. Shown are Rad9 residues 1–231 for Chk1 activation (Blankley and Lydall, 2004), 390–457 for SCD, 542–620 for Rad53 binding (Schwartz et al., 2002), 754–947 for Tudor (Alpha-Bazin et al., 2005), and 998–1298 for tandem BRCT domains (Callebaut and Mornon, 1997). (B) WB analyses of HA immunoprecipitates (IPs) from RAD9-TEV-HA-expressing cells. Cell extracts were obtained from asynchronous cells (−) and cells treated with 0.03% MMS for 1.5 hr (+). WB with HA antibody (Ab) for Rad9 full-length (FL) or BRCT-HA and N-terminal-specific Rad9 Ab for Rad9 FL or N-SCD were shown. After incubation of HA IPs without (lanes 1–4) and with TEV protease (lanes 5–8), supernatants (lanes 1, 2, 5, and 6) were separated from beads (lanes 3, 4, 7, and 8). (C) WB analyses of GST-BRCT pull-down (PD) assay against BRCT-HA. Ten percent input “I,” PD by WT BRCT “B,” and S1129A BRCT “b.” Lane 1 was a control supernatant after incubation of HA IPs without TEV cleavage. (D) WB analyses of GST-BRCT PD assay against N-SCD. “I,” “B,” and “b” are denoted as in (C). Lane 1 was a control supernatant without TEV cleavage. Noncleaved Rad9-TEV-HA was shown (lanes 10 and 11). (E) Amino acid sequence alignments surrounding Rad9 S1129, Crb2 S658, and 53BP1 S1853 in the BRCT linker. (F) WB analyses of GST-BRCT PD assay against N-SCD prepared from the indicated strains. PD by S1129A BRCT (lanes 1 and 2) and by WT BRCT (lanes 3–8). Molecular Cell 2009 33, 147-159DOI: (10.1016/j.molcel.2008.12.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Direct Interaction between Rad9-BRCT and PIKK-Phosphorylated Rad9 SCD (A) WB analyses of GST-BRCT PD assay against N-SCD prepared from the indicated strains. PD by S1129A BRCT (lane 1) and Rad9-TEV-HA FL (lanes 11 and12) were shown. (B and C) FP of 5-FAM-labeled T427 (B) nonphospho- and (C) phosphopeptides incubated with GST fusions at indicated concentrations. Open circle, WT BRCT; closed circle, GST alone; triangle, S1129A BRCT. The nonlinear regression coefficient for the S1129A BRCT was 0.56, indicating that binding was insufficient to allow determination of Kd. The gray line for S1129A is for comparison only. Each experiment consisted of triplicate readings at each protein concentration. Error bars denote standard deviation obtained using at least two independent preparations of GST fusions. Molecular Cell 2009 33, 147-159DOI: (10.1016/j.molcel.2008.12.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 rad9-S1129A and rad9-6AQ Are Defective in Checkpoint Maintenance (A–C) Cell-cycle checkpoint in the indicated strains was tested in response to cdc13-induced telomere damage (A and C) and HO DSBs (B). (A and C) The indicated strains arrested at G1 by α factor were released at the nonpermissive temperature (37°C). Nuclear division was monitored by DAPI staining (>200 cells counted). The y axis represented the percentage of cells that arrested at telophase at the indicated times. Error bars denote standard deviation from three independent experiments. (B) G1-arrested cells of the indicated strains were plated on galactose-containing plates to induce HO DSBs. More than 150 microcolonies were examined to count the number of cells and buds at the indicated time. Experiments were performed three times, and the representative data were shown. Molecular Cell 2009 33, 147-159DOI: (10.1016/j.molcel.2008.12.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 BRCT-SCD-Mediated Rad9 Oligomerization Is Important to Maintain Rad53 Activation (A and B) Rad53 kinase activity was examined in the indicated genotypes in the cdc13 cdc15 background. Flag-Rad53 IPs were obtained from G1-arrested cells at 23°C (time 0) and 2 hr after transfer to 37°C, followed by in vitro kinase assay using purified GST-dun1-KD as a substrate. Experiments were performed three times, and the representative and quantified data were shown in (A) and (B). Relative auto- and transphosphorylation activity was determined by dividing 32P incorporation to Rad53 and GST-dun1KD by Flag WB signals. Error bars represent standard deviation. (C) Mobility shift of Flag-Rad53 was examined in the indicated genotypes in the cdc13 cdc15 background. Flag-Rad53 IPs were obtained from G1-arrested cells at 23°C (time 0) and the indicated time after transfer to 37°C. Time points 0–4 hr and 6–8 hr were obtained from separate membranes but from the same experiment. (D) Mobility shift of Flag-Rad53 and nuclear divisions in response to cdc13-induced damage were examined in the indicated genotypes in the cdc13 cdc15 background, respectively. Tubulin WB was shown as loading control. (E) Shown are WB analyses of Myc and HA IPs from cell extracts of the indicated tagged RAD9-expressing or rad9 mutant-expressing asynchronous cells. 3xHA-Rad9 and 6xMyc-Rad9 were expressed from the RAD9 genomic locus and the single copy plasmid, respectively. (F) A possibility that rad9-S1129A/rad9-6AQ dimers mediate intermolecular Rad9 interactions was tested as illustrated above the graph. rad9-6AQ cdc13 cdc15 cells were transformed with single-copy plasmids expressing RAD9 or rad9-S1129A and an empty plasmid, and cdc13-induced cell-cycle checkpoint was examined as in Figure 3A. Note that transformants adapt checkpoint arrest earlier than nontransformants in our condition. Error bars denote standard deviation from three independent experiments. Molecular Cell 2009 33, 147-159DOI: (10.1016/j.molcel.2008.12.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Rad9 Accumulates in Chromatin around DNA Damage in rad53-KD (A–C) Shown are representative images of Rad9-YFP foci in the indicated genotypes in the cdc13 cdc15 background at nonpermissive temperature for 4 hr. (D) Rad9-YFP focus formation was examined in the indicated genotypes in the cdc13 cdc15 background. Cells were released at nonpermissive temperature from G1 arrest. At least 100 cells were examined for each time point. The y axis represented the percentage of focus-positive cells at the indicated times. Error bars denote standard deviation from at least three independent experiments. P value was calculated using two-tailed Wilcoxon rank sum test. (E) Rad9 binding to DSB-proximal chromatin was tested by chip. Formaldehyde-fixed chromatin extracts were prepared at 0 and 3 hr after HO DSB induction in G2/M-arrested cells of the indicated strains, followed by anti-HA Chip. Anti-HA-bound DNA at 0.05 kb from the HO DSB site was quantified by real-time PCR. The data were obtained from two independent cultures with duplicated IPs and PCRs. Error bars represent standard deviation. P value was calculated using two-tailed Wilcoxon rank sum test. Molecular Cell 2009 33, 147-159DOI: (10.1016/j.molcel.2008.12.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Rad53 Phosphorylates Rad9 BRCT (A) 32P was incorporated to Rad9 coimmunoprecipitated with Flag-Rad53 after in vitro kinase assay. Flag-Rad53 IPs were obtained from the indicated strains without (−) and with 0.03% MMS treatment for 1.5 hr (+). WB with Flag and Rad9 Abs and 32P incorporation to Flag-Rad53 and Rad9 were shown. (B) In vitro kinase assay of purified His-Rad53 using GST fusion proteins expressing Rad9 fragments A–E. (C–E) Mobility shift of BRCT-HA was tested on longer separation gels. Top and bottom panels represent high and low molecular range of the same membrane. (C and E) HA IPs of Rad9-TEV-HA were obtained from indicated strains' extracts prepared from asynchronous cells (−) or 1.5 hr after 0.03% MMS treatment (+). HA IPs were split into two fractions subject to mock or TEV protease treatment. (D) Phosphatase treatment of HA IPs of BRCT-HA and Rad9-HA from MMS-treated cells. Molecular Cell 2009 33, 147-159DOI: (10.1016/j.molcel.2008.12.022) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Effect of Rad53 Phosphorylation of Rad9 BRCT (A) Effect of Rad53 phosphorylation of GST-BRCT on N-SCD PD. N-SCD was prepared from MMS-treated WT cells. Prior to the PD assay, GST-BRCT was treated in three kinase reactions: (X) His-rad53-KD and ATP, (Y) His-Rad53 and no ATP, and (Z) His-Rad53 and ATP. After kinase reaction, GST-BRCT was treated with CIP (lanes 4 and 5) and with CIP and PPase inhibitor, NaV (lane 6). (B) FP of 5-FAM-labeled T427 phosphopeptides as in Figure 2C. Prior to FP, GST-BRCT was phosphorylated by rad53-KD (open circles) or Rad53 (triangle) at molar ratio 25:1. Closed circle represents FP with GST alone. The gray lines are for comparison. Error bars denote standard deviation obtained by using two independent preparations of GST-BRCT. (C and D) Models of regulation of DNA-damage-induced oligomerization of Rad9 dimers via BRCT-SCD interaction. Domains are represented as in Figure 1A. (C) Positive regulation. (Ci) Upon DNA damage, Rad9 is recruited to chromatin around DNA damage in the HM-dependent mechanisms and phosphorylated by Mec1/Tel1 in SCD. (Cii) BRCT domain of naive Rad9 engages PIKK-phosphorylated Rad9 SCD in chromatin. (Ciii) Subsequent Mec1/Tel1 phosphorylation of Rad9 is promoted as naive Rad9 is newly recruited. (D) Negative regulation. Fully active Rad53 phosphorylates Rad9 BRCT to promote disassembly of the Rad9 oligomer (Di) and/or inhibit Rad9 recruitment (Dii) to increase free PIKK-phosphorylated (active) Rad9. Molecular Cell 2009 33, 147-159DOI: (10.1016/j.molcel.2008.12.022) Copyright © 2009 Elsevier Inc. Terms and Conditions