Cdc18 Enforces Long-Term Maintenance of the S Phase Checkpoint by Anchoring the Rad3-Rad26 Complex to Chromatin  Damien Hermand, Paul Nurse  Molecular Cell  Volume 26, Issue 4, Pages 553-563 (May 2007) DOI: 10.1016/j.molcel.2007.04.014 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Cdc18, but Not Cdt1, Is Required to Maintain the S-M Checkpoint When S Phase Is Blocked (A) A cdc25-22 cdc18-TAP cdt1-TAP strain was synchronized in late G2 and released in the presence or absence of HU as indicated. Cells from each sample were analyzed by FACS, septation index was determined, and cell extracts were processed for western blot as indicated. Note that in fission yeast, DNA replication occurs concomitantly to septation; thus, in a synchronous population, cell separation directly follows the end of S phase. (B) Thiamine was added to nmt81-cdc18 (left) and nmt81-cdt1 (right) strains, and samples were taken every hour and processed for FACS or western blot as indicated. (C) nmt81-cdc18 and nmt81-cdt1 strains were grown in the presence of HU. At time 0, thiamine was added to half of the cultures, and growth continued for an additional 6 hr. At indicated times, the proportion of aberrant mitotic cells was determined by DAPI. FACS profiles of nmt81-cdc18 and typical DAPI staining of nmt81-cdc18 cells at 6 hr are shown in the right panel. Asterisks indicate aneuploid cells, cut cells, or unequal distribution of DNA. Molecular Cell 2007 26, 553-563DOI: (10.1016/j.molcel.2007.04.014) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Cdc18 Is Required to Anchor Rad3 to Chromatin When S Phase Is Arrested (A) A cdc25-22 cdc18-TAP rad3-HA strain was synchronized in late G2 and released in the presence or absence of HU. After 70 or 120 min, cell extracts were fractionated between cytosol (left) and chromatin (middle) and were analyzed by western blot as indicated. In the right panel, Cdc18 was immunoprecipitated from the chromatin fraction, and the beads were probed by western blot for the presence of Cdc18 or Rad3 as indicated. A control strain with untagged Cdc18, and processed in the same manner, is shown in the right lane, which shows the absence of aspecific binding of Rad3. (B) A nmt81-cdc18 strain was cultured as in Figure 1C, and cell extracts from indicated time points were fractionated into cytosol and chromatin and processed by western blot as indicated. (C) Same as in (B), except a cds1-TAP strain was used, and total extracts from indicated time points were analyzed by western blot as indicated. (D) Cdc2 was immunoprecipitated from extracts, and pellets were divided for western blot and for a kinase reaction with Histone H1 as substrate as indicated. (E) The same experiment as in (A), except the temperature was raised to 36°C to inactivate Cdc25 at the time thiamine was added. Note that cells had to be blocked longer in HU because of the lower temperature (25°C). Molecular Cell 2007 26, 553-563DOI: (10.1016/j.molcel.2007.04.014) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Replication Structures Are Not Lost When Cdc18 Is Depleted Panels 1 and 2: a nmt81-cdc18 strain was cultured as in Figure 1C. Genomic DNA, prepared from the 2 hr, 4 hr, and 6 hr time points in the presence or absence of thiamine as indicated, was processed for 2D gel analysis and probed with a ars 3001 probe. Panels 3 and 4: orp1-ts and orp1-ts cdc18-ts strains were grown and treated with HU for 3 hr and 5 hr at 25°C. Then (time zero), cells were transferred to 36°C and cultured for 6 hr. Genomic DNA prepared from the 2, 4, and 6 hr time points was processed for 2D gel analysis and probed with a ars 3001 probe. An identical exposure time is presented. In all cases, undigested genomic DNA is shown in the top insets and serves as a loading control. A schematic of the replication structures is presented in Figure S3. Molecular Cell 2007 26, 553-563DOI: (10.1016/j.molcel.2007.04.014) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Rad26 Interacts with Cdc18 and Is Required for Cdc18-Rad3 Complex Formation (A) Two-hybrid assay between a Gal4-binding domain-Cdc18 (or Cdc18-15 and Cdc18-57 mutants as indicated) fusion (BD-Cdc18) and a fusion of the Gal4 activation domain with either Rad3 first half (AD-Rad3 N), Rad3 second half (AD-Rad3 C), or Rad26 (AD-Rad26). BD-p53 + AD-SV40 T antigen is the positive control from the kit. The interaction is tested on synthetic medium (SD) lacking tryptophan (W), leucine (L), and histidine (H) in the presence of 25 mM 3-aminotriazole (3AT). Ten times serial dilution are presented. (B) GST or GST-Cdc18 expressed from pGEX-4T1 (Pharmacia) was purified from E. coli on glutathione Sepharose beads. The beads were incubated for 1 hr with extracts from E. coli expressing rad26 or empty vector as indicated. The beads were washed four times, boiled, and processed for western blot as indicated. (C) A cdc25-22 cdc18-TAP strain was synchronized in late G2 and released in the presence or absence of HU. After 70 or 120 min, cell extracts were fractionated into cytosol and chromatin and analyzed by anti-Rad26 western blot as indicated. In the right panel, Cdc18 was immunoprecipitated from the chromatin fraction, and the beads were probed by western blot for the presence of Cdc18 or Rad26 as indicated. A control strain with untagged Cdc18 and processed in the same manner is shown in the right lane and demonstrates the absence of aspecific binding of Rad26. (D) A cdc25-22 cdc18-TAP rad3-HA rad26::ura4 strain was synchronized in late G2 and released in the presence or absence of HU. After 70 or 120 min, total cell extracts were analyzed by western blot as indicated. In the middle panel, Cdc18 was immunoprecipitated from a chromatin fraction, and the beads were probed by western blot for the presence of Cdc18 or Rad3 as indicated. In the right panel, a control experiment similar to that shown in Figure 2A was performed in a strain containing wild-type Rad26. Molecular Cell 2007 26, 553-563DOI: (10.1016/j.molcel.2007.04.014) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Cdc2-Cig2 Controls the Cdc18 Level (A) A cdc25-22 cdc13Δ nmt81::cdc13 strain was synchronized in late G2, and thiamine (T) was added for the rest of the experiment. Flow cytometry (FACS) profiles and septation index are presented, and S phase (S) is indicated based on the FACS data. After release into mitosis, cell extracts were prepared from samples taken every 20 min and processed for western blot as indicated. Bottom: identical experiment in a cig2Δ background. (B) Similar experiment as in (A), except HU was added at the time of release into mitosis. Molecular Cell 2007 26, 553-563DOI: (10.1016/j.molcel.2007.04.014) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Cdc18 Persistence When S Phase Is Arrested Requires the S-M Checkpoint (A) A cdc25-22 cdc18-TAP strain was synchronized in late G2 and released in the presence of HU. FACS profiles, septation index, and the percentage of “cut” cells are presented. Cell extracts were prepared from each sample and processed for western blot as indicated. Note that the FACS profile and the Cdc18 western blot form part of Figure 1A and are shown for clarity. (B) Similar experiment as in (A), except a cdc25-22 cdc18-TAP rad3::ura4 strain was used. Molecular Cell 2007 26, 553-563DOI: (10.1016/j.molcel.2007.04.014) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 A Model of Cdc18's Role and Regulation in the S Phase Checkpoint See text for details. Molecular Cell 2007 26, 553-563DOI: (10.1016/j.molcel.2007.04.014) Copyright © 2007 Elsevier Inc. Terms and Conditions