Figure 3.

Cells depleted for Dpb2 are defective in S phase progression. (A) A culture of haploid GD149 cells was grown to log phase, the culture was then diluted and one-half was treated with 10 μg/μl thiamine to repress dpb2⁺ gene expression. Samples were collected at the indicated times and DNA content analyzed by flow cytometry. (B) Cells stained with the DNA binding dye 4,6-diamidino-2-phenylindole after incubation with thiamine at 0, 23, and 40 h. Arrows indicate abnormal nuclear morphology, including missegregated chromosomes and anucleate cells. (C) DNA replication initiation is delayed in cell cycle synchronized cultures depleted for Dpb2. Wild-type (972) or GD149 cells were grown in the absence of nitrogen to arrest cells in G1. During nitrogen starvation, thiamine was added (10 μg/μl) to the media to repress transcription from the nmt81 promoter. After 20 h or when >90% of the cells showed a G1 arrest, cells were induced to reenter the cell cycle by addition of fresh media containing nitrogen, as well as thiamine to maintain transcriptional repression of dpb2⁺. Cells were collected every 30 min for 6 h and the DNA content analyzed by flow cytometry. The positions of 1C and 2C DNA content are indicated.