Fig. 6.

Psm3K106Ac is not detectable in the eso1-H17 mutant strain at the permissive temperature. (A) Cells were cultured at 25°C, arrested in early S phase by adding 12 mM hydroxyurea (HU), and released into the cell cycle. Samples were taken at the indicated time points (min). Psm3-GFP was immunoprecipitated from total protein extracts and probed with anti-Psm3^K106Ac antibodies. Equal amounts of Psm3-GFP were verified by probing with anti-GFP antibodies. (B) Psm3-GFP immunopurified from the wt was serially diluted with Psm3-GFP immunopurified from eso1Δ wpl1Δ cells and probed with anti-Psm3^K106Ac and anti-GFP antibodies as in panel A. (C) DNA content analysis before and after release from HU arrest. (D) eso1-H17 is still viable and thermosensitive for growth in a psm3^RR background. Serial dilutions of cells were spotted on YES medium and incubated at the indicated temperatures. (E) Analysis of the cohesin complex with anti-acetyl antibodies. The cohesin complex was immunoprecipitated from native protein extracts by anti-GFP antibodies and probed with the indicated antibodies. Budding yeast SMC3-PK was immunopurified from the indicated strains (8) and used as a control for anti-acetyl antibodies. (F to I) The stable cohesin fraction is reduced in the eso1-H17 mutant at the permissive temperature. The strains contain the cdc25-22 and mis4-367 alleles and were cultured and processed for nuclear spreading and ChIP as in Fig. 1. (F) Chromatin-bound Rad21-PK was quantified from nuclear spreads before (T0) and after (T150 and T180 min) the temperature shift. The error bars represent SD from duplicate samples. (G) Cell survival during the course of the experiment. (H) ChIP assay showing the amount of Rad21 bound to pericentromeric regions (imr and dg) just after (15 min) and 3 h after the temperature shift. Rad21 enrichment was calculated from duplicate samples. The error bars represent SD. (I) Fluorescence-activated cell sorter (FACS) and cell cycle staging of cells before and after the shift to 36.5°C.