Volume 16, Issue 5, Pages 687-700 (December 2004) DNA Replication Checkpoint Prevents Precocious Chromosome Segregation by Regulating Spindle Behavior  Vaidehi Krishnan, Saurabh Nirantar, Karen Crasta, Alison Yi Hui Cheng, Uttam Surana  Molecular Cell  Volume 16, Issue 5, Pages 687-700 (December 2004) DOI: 10.1016/j.molcel.2004.11.001

Figure 1 Precocious Chromosome Segregation in mec1-1 Cells Does Not Require Cleavage of Scc1, APC Activity, or Bipolar Occupancy of the Kinetochore (A) Wild-type (US3335) and mec1-1 (US3499) cells carrying native promoter-driven SCC1-myc18 (at its native locus) were synchronized in G1 by α factor treatment and then released in the presence of HU (0.26 M) at 24°C. In parallel, G1-synchronized wild-type and mec1-1 cells were released in the absence of HU, and samples collected at the indicated time points were scored for anaphase spindle elongation. A comparison of the timing of spindle elongation in the presence and absence of HU is represented in the graph. (B) G1-synchronized mec1-1 (US3138) and mec1-1 cdc23-1 (US3248) cells were released at 34°C (restrictive temperature for cdc23-1) into HU (0.26 M). As a control, cdc23-1 cells (US1389) were released at 34°C without HU. (C) G1-synchronized wild-type (US3329) and mec1-1 cells (US3458) with CENV marked with GFP were released into HU-containing medium at 24°C. Samples were scored for nuclear division index and number of GFP dots per cell. The left panel shows nuclei and CENV-GFP signals. Molecular Cell 2004 16, 687-700DOI: (10.1016/j.molcel.2004.11.001)

Figure 2 Premature Spindle Elongation in mec1-1 Cells Is Not a Consequence of Untimely Onset of Mitotic Entry (A) (Upper panel) Wild-type (US1363), mec1-1 (US3138), and mec1-1 clb1Δ clb2Δ GAL-CLB2 (US3377) cells were treated with α factor in YEP+Raff+Gal medium for 2 hr. Cells were filtered, resuspended in YEP+Glu containing α factor for 1 hr to shut off CLB2 expression, and then released into YEP+Glu+HU. As control, clb1Δ clb2Δ GAL-CLB2 cells (US243) were treated in an identical manner but were released into YEP+Glu without HU. (Lower panel) mec1-1 clb1Δ clb2Δ GAL-CLB2 cells expressing HA-tagged Scc1 (US4127) at the endogenous locus were similarly treated as described above, and samples collected at indicated time points were analyzed for Scc1 cleavage by Western blotting. (B) Wild-type (US1165) and mec1-1 (US3907) cells carrying native promoter-driven CLB2-HA3 were arrested with α factor treatment and released in the presence of HU (0.26 M) at 24°C. (Upper panel) Protein extracts (2 mg) prepared from samples collected at the indicated time points were used for immunoprecipitating Clb2/Cdc28 complexes using anti-HA agarose beads. Cdc28 phosphorylation status was analyzed by probing blots with anti-phosphotyrosine antibody. (Lower panel) Clb2/Cdc28 immunoprecipitates (240 min sample) were used for measurement of histone H1 kinase activity. Molecular Cell 2004 16, 687-700DOI: (10.1016/j.molcel.2004.11.001)

Figure 3 Proteins Involved in Spindle Elongation Accumulate in mec1-1 Cells Due to a Combination of Both Protein Stability and Transcription (A) G1-synchronized wild-type and mec1-1 cells carrying either CIN8-HA3 (CEN plasmid), CIN8-myc3 (tagged endogenous gene), STU2-HA3 (CEN plasmid), or STU2-myc6 (tagged endogenous gene), respectively, were released into medium containing HU. (B) (Left panel) Wild-type and mec1-1 cells expressing GAL1-CIN8-myc3 on a CEN plasmid were arrested in YEP+Raff containing α factor and released at 24°C into YEP+Raff+Gal medium containing HU for 120 min to induce Cin8 expression. Cells were then filtered and released into YEP+Glu+HU, and the fate of the Cin8 pulse was monitored by Western blotting. (Right panel) Wild-type and mec1-1 cells expressing GAL1-STU2-myc6 were released (as described for Cin8 above) in the presence of HU, and the fate of the Stu2 pulse was monitored. As a control, both strains were released in galactose medium in the absence of HU, and the level of GAL1-STU2-myc6 was determined after 120 min of induction. (C) G1-synchronized wild-type (US1363) and mec1-1 (US3138) cells were released into HU, and samples were analyzed for the level of STU2 RNA. Molecular Cell 2004 16, 687-700DOI: (10.1016/j.molcel.2004.11.001)

Figure 4 Overexpression of Cin8 Causes Spindle Elongation in S Phase-Arrested Cells, but Not in G2/M-Arrested Cells (A) Wild-type cells and wild-type cells with GAL1-CIN8-myc3 on a CEN plasmid and mec1-1 cells were treated with α factor in YEP+Raff for 2.5 hr, and galactose was added for 30 min. Cells were released into YEP+Raff+Gal+HU medium at 24°C. (B) Wild-type cells (US3839), wild-type cells carrying GAL1-CIN8-myc3 on a CEN plasmid (US3889), and mec1-1 (US3890) cells, all expressing DDC1-GFP, were treated as in (A). (C) cdc13-1 and cdc28-1N cells expressing GAL1-CIN8-myc3 on a CEN plasmid were arrested in G1 with α factor treatment in YEP+Raff. One-half of the culture was resuspended into YEP+Raff+Gal and the other half into YEP+Glu at 31°C and 37°C (restrictive temperature) for cdc13-1 and cdc28-1N, respectively. Molecular Cell 2004 16, 687-700DOI: (10.1016/j.molcel.2004.11.001)

Figure 5 Modulation of Spindle Dynamics and the Efficacy of the Replication Checkpoint (A) G1-synchronized mec1-1 (US3138), mec1-1 cin8Δ (US3641), mec1-1 stu2-10 (US3639), and mec1-1 cin8Δ stu2-10 (US3697) cells were released at 30°C (semipermissive temperature for stu2-10) in the presence of HU. (B) mec1-1 (US3138) and mec1-1 ADH-CIN8-myc3 ADH-STU2 (US3906) were synchronized in G1 and released at 24°C in the presence of HU, and samples were analyzed as before ([Inset] Tub4 staining reveals the presence of separated SPBs). (Lower right panel) Wild-type (US1363), mec1-1 (US3138), and mec1-1 ADH-CIN8-myc3 ADH-STU2 (US3906) cells were streaked on YEP+Glu plates with (0.13 M HU) or without HU and incubated at 24°C. Plates were photographed after 5 days. Molecular Cell 2004 16, 687-700DOI: (10.1016/j.molcel.2004.11.001)

Figure 6 Combined Deficiencies of Kip3 and Mad2 Can Abrogate the Replication Checkpoint (A) G1-synchronized wild-type (US1363), mec1-1 (US3138), mad2 (US312), kip3Δ (US3874), and kip3Δ mad2 (US3868) cells were released into HU at 24°C. (B) (Left panel) G1-synchronized wild-type (US1363), mec1-1 (US3138), and kip3Δ mad2 (US3868) cells were released into HU at 24°C, and the viability of cells was measured by plating 200 cells at indicated times into YEP+glucose plates. (Right panel) kip3Δ (US3874) and kip3Δ mad2 (US3868) cells were spotted onto YEP+Glu plates with or without HU (0.26 M) at 24°C and were photographed after 5 days. (C) kip3Δ mad2 (US3978) cells expressing native promoter-driven SCC1-myc12 were released in the presence of HU (0.26 M) at 24°C, and the status of Scc1 cleavage and Clb2 proteolysis was monitored. As a control, cells were also released in the absence of HU, and a comparison of spindle elongation in the presence and absence of HU is shown in the accompanying graph. Molecular Cell 2004 16, 687-700DOI: (10.1016/j.molcel.2004.11.001)

Figure 7 Rad53 Negatively Regulates Spindle Cycle during the Normal Cell Cycle (A) Wild-type cells (US1363) and wild-type cells expressing either one copy (US3695) or six copies (US3442) of GAL1-RAD53-HA2 were arrested in YEP+Raff for 2.5 hr in α factor, transferred to YEP+Raff+Gal with α factor for 1 hr, and then released into YEP+Raff+Gal at 24°C. (Left panel) A photomicrograph shows nuclei and spindles in 180 min and 300 min samples, and a comparison of the kinetics of spindle elongation is plotted in the accompanying graph. (Right panel) Western blot showing the levels of RAD53 expression from 1X-RAD53-HA2 and 6X-RAD53-HA2 constructs. (B) Wild-type (US4122, US4128) and wild-type expressing 6XGAL1-RAD53-HA2 (US4132, US4131) cells expressing Cin8-HA3 and Stu2-myc6, respectively, were treated as described in (A), and samples collected at indicated time points were analyzed for expression of Cin8 and Stu2 by Western blotting. (C) cdc28-as1 (US3558) and mec1-1 cdc28-as1 (US4029) cells were synchronized in G1 by α factor treatment and released into YEP+Glu+1NM-PP1 (500 nM), and samples collected at 15 min intervals were scored for spindle formation. (D) A putative scheme representing the functional relationship between replication checkpoint effectors and the regulators of spindle dynamics (See Discussion). Molecular Cell 2004 16, 687-700DOI: (10.1016/j.molcel.2004.11.001)