FIGURE 1:

DDK temperature-sensitive alleles reduce spindle extension in S phase checkpoint mutants. (A) WT (Y300), mec1-21 (AY201), dbf4-1 (JBY999), and mec1-21 dbf4-1 (JBY927) strains were released from G₁ at 25°C in 200 mM HU media. At 15, 55, or 95 min after G₁ release, cultures were shifted to a nonpermissive temperature of 34°C. At times indicated on the x-axis, aliquots were processed for α-tubulin immunofluorescence and DAPI staining. Spindle extension was reduced in mec1-21 dbf4-1 cells shifted at 55 min. (B) WT (Y300), rad53-21 (Y301), cdc7-1 (DES956), and rad53-21 cdc7-1 (DES960) were released into 200 mM HU media as in A and were shifted to 35°C at 20, 50, and 80 min. After a total of 180 min following G₁ release, cells were processed for α-tubulin immunofluorescence and DAPI. As with mec1-21 dbf4-1 cells, rad53-21 cdc7-1 cells displayed reduced spindle extension when shifted at 50 min. (C) To more closely bracket the window for suppression of spindle extension, rad53-21 (Y301) and rad53-21 dbf4-1 (JBY1002) strains were released from G₁ into 200 mM HU at 25°C and then split into parallel cultures, which were then shifted to 34°C at the indicated times on the x-axis of the right-hand graph (shift time). Aliquots were maintained at 25°C to monitor cell budding (left graph). Spindle extension was evaluated in temperature-shifted samples at 150 min post-G₁ release using DAPI and α-tubulin immunofluorescence (right graph). Maximal suppression of spindle extension was observed when rad53-21 dbf4-1 cells were shifted at 40 min, corresponding with bud emergence and S phase entry. (D) SPC42-GFP cells (JBY1129) were transformed with vector (JBY1285), a low copy plasmid expressing RAD53 under control of the inducible GAL promoter (pCEN HIS3 GAL-RAD53, JBY1286), a high copy pGAL-DBF4 plasmid (p2μm URA3 GAL-DBF4; JBY1287), or cotransformed with both plasmids (JBY1288). Transformants were arrested in G₁ for 3.5 h in galactose media (YPGAL) to induce RAD53 and/or DBF4. Cells were then released into YPGAL containing 200 mM HU. After 3.5 h, spindle extension was evaluated using Spc42-GFP.