Figure 4.

drc1–8 mutants are defective for the S-phase checkpoint. (A–D) Wild-type (Y796) and drc1–8 (Y798) cells were synchronized by α-factor at 24°C. After G₁ arrest, cultures were divided into two and released from the block into either YPD or YPD containing 0.2 M HU. At regular intervals after release, aliquots were withdrawn to examine DNA content, nuclear and spindle morphology, and cell viability. (A) FACS profile showing the DNA content of wild-type and drc1–8 cells after release from G₁ into medium with or without 0.2 M HU. Time indicates minutes after release. (Lower) Untreated asynchronous cells at 24°C and are included as a reference. (B) Photomicrographs of wild-type and drc1–8 cells at 150 min after release from α-factor into YPD with 0.2 M HU. Nuclear morphology was visualized with 4′,6-diamidino-2-phenylindole, and microtubule morphology was visualized by indirect immunofluorescence using anti-α-tubulin antibodies. (C) Kinetics of spindle elongation in the presence or absence of HU. (D) Cell viability was analyzed by scoring colony-forming units on YPD plates at 24°C. (E) Wild-type (Y300), drc1–1 (Y799), and drc1–8 (Y798) cells were synchronized in G₁ by α-factor at 24°C. After release for 30 min, HU or MMS was added to block DNA replication block or damage DNA for 1 hr. Protein extracts were prepared, separated by SDS/PAGE, and immunoblotted with antibodies to Rad53.