Figure 4.

pds1-m8 and pds1-m9 are defective in cell cycle arrest in response to DNA damage. (A) Y809 (cdc13-1), Y811 (cdc13-1 Δchk1), Y1074 (cdc13-1 pds1-m8), and Y1075 (cdc13-1 pds1-m9) cells were grown at 24°C in YPD and then plated on prewarmed YPD plates (30°C) and incubated at 30°C for 8 h. Cells were examined for microcolony formation. (dotted bars) Percentage of cells that showed a large-budded arrest; (hatched bars) percentage of cells that formed a microcolony. (B–D) Y809 (cdc13-1), Y811 (cdc13-1 Δchk1), Y817 (cdc13-1 Δpds1), Y1074 (cdc13-1 pds1-m8), and Y1075 (cdc13-1 pds1-m9) cells were synchronized in G₁ with α-factor at 24°C and released into YPD containing 200 mM HU for 2.5 h to synchronize cells in S phase. During the last 30 min of the HU block, cells were shifted to 32°C to inactivate cdc13-1. Cells then were released from HU at 32°C, and α-factor was added to prevent cell cycle reentry. Aliquots were withdrawn at timed intervals to examine DNA content by fluorescence-activated cell sorting analysis (B) and spindle morphology. Spindle morphology of various strains at 120 min is shown in C. Kinetics of anaphase entry is evaluated by the disappearance of short spindles (D).