Skip to main content
. 2007 Aug 1;21(15):1921–1936. doi: 10.1101/gad.432107

Figure 4.

Figure 4.

Effect of Top1 and/or Top2 activity attenuation on chromosomal replication and checkpoint activation. (A) Wild-type (SY359), top1Δ (CY2279), top2-1 (SY2183), and top1 Δtop2-1 (CY7039) cells were arrested in G1 by α-factor treatment at permissive temperature and then released into fresh medium at 37°C. Samples were collected at the indicated time points for FACS analysis and TCA protein precipitation and further immunodetection of Rad53 protein using antibodies recognizing the protein backbone (EL7) or the phosphorylated epitopes only (F9). (B) Wild-type, top1Δ, top2-1, and top1Δ top2-1 cells were arrested in G1 and released into fresh medium containing 0.2 M HU at 37°C. Samples were collected after 2 h, genomic DNA was extracted, and replication intermediates (RI) were analyzed by 2D gel electrophoresis using a probe specific to ARS305 region. (C) Wild-type, top1Δ, top2-1, and top1Δ top2-1 cells were arrested in G1, released into fresh medium for 30 min at 25°C, and then transferred to 37°C prewarmed medium. Samples were collected at the indicated time points for FACS analysis, TCA protein precipitation, and immunodetection of Rad53 and S129-phosphorylated H2A proteins.