FIG. 4.

Eso1p is required for the establishment of sister chromatid cohesion during S phase but not for its maintenance in G₂ and M phases. (A) DNA replication takes place without any significant delay in eso1-H17 mutant cells. Wild-type (h⁻ leu1-32) and eso1-H17 (h⁻ eso1-H17 leu1-32) cells were grown to mid-log phase at 25°C and then nitrogen starved in MM-N medium for 24 h to be arrested in G₁. Cells were then released by transfer into MM medium preincubated at 36°C. Cell aliquots were taken every hour and analyzed for S-phase onset and progression by flow cytometry. (B) eso1-H17 cells lose viability during S phase. To determine cell number (open squares), eso1-H17 cell aliquots were taken every hour and counted. To determine cell viability (open circles), eso1-H17 cell aliquots taken every hour were plated on YEA at 25°C. To determine percent abnormal nuclear cells (filled triangles), cells were fixed with 70% ethanol and stained with DAPI, and those with abnormal chromosome structures (overcondensed chromosomes, cut, missegregation, etc.) were counted under the fluorescence microscope. (C and D) Sister chromosomes are prematurely separated in eso1-H17 mutant cells. Wild-type (h⁺ Cen1-GFP) and eso1-H17 (h⁺ eso1-H17 Cen1-GFP) cells were arrested in G₁ by nitrogen starvation at 26°C and then released in MM medium preincubated at 36°C. Live cells were observed under the fluorescence microscope. (C) Frequencies of cells showing two split Cen1-GFP signals. (D) Examples of eso1-H17 cells showing two split Cen1-GFP signals and wild-type control at 4 h. Bar, 5 μm. (E and F) Eso1p is not essential for the maintenance of cohesion in G₂ and M phases. Cells of eso1-H17 (h⁻ eso1-H17 leu1-32) and rad21-K1 (h⁻ ura4-D18 rad21-K1-ura4⁺ leu1-32) mutants were grown to saturation in YEL medium at 25°C and incubated for a further 24 h to ensure growth arrest. They were then incubated at 36°C for 3 h and released in fresh YEL medium preincubated at 36°C. (E) Flow cytometric analysis of arrested cells. (F) Lack of abnormal mitosis in eso1-H17 cells released from G₂ phase at the nonpermissive temperature. Cell number (open squares) was determined by counting aliquots taken every 30 min. To determine percent abnormal mitosis (filled triangles), cells were fixed with 70% ethanol and stained with DAPI, and those with abnormal mitotic chromosome structures (cut, missegregation, etc.) were counted under the fluorescence microscope.