Figure 5

Analysis of Spp1-GFP nuclear distribution after TEV protease cleavage of Cdc23 _S424 ::Tcs. A. Localization of Spp1-GFP after direct fixation (methanol/acetone) of an asynchronous culture (strain P903). B. Localization of Spp1-GFP (strain P903) after detergent extraction of cells; >50% binuclear cells show nuclear Spp1 (arrow) C. As in (B) except that cells were grown in 12 mM HU for 2 h before analysis. Bar = 10 μm. D. Quantitation of data in (B); 100 cells were counted for each data point and 100% represents the total number of binucleate or uninucleate cells counted. E. Quantitation of data in (C); data for other time points (with and without detergent extraction) are also shown. F. Chromatin binding analysis of Spp1-GFP after inactivation of Cdc23_S424::Tcs by TEV protease cleavage. Strain P1460 was grown either in the absence or presence of thiamine for 24 h at 25°C, then shifted to 37°C for 2, 4 h before analysis using the chromatin binding assay. Grey bars show the percentage of total cells showing Spp1-GFP fluorescence coincident with chromatin (DAPI-staining region) after detergent extraction. Black bars show the percentage of cells showing foci of Spp1-GFP fluorescence in the absence of general nuclear fluorescence (example shown in right-hand panels). At least 300 cells were counted for each data point. G. Chromatin association of Cdc45-YFP is not prevented by cleavage of Cdc23_S424::Tcs by TEV protease. Strain P1409 was grown either in the absence or presence of thiamine for 24 h at 25°C, then shifted to 37°C for 2, 4 h before analysis using the chromatin binding assay. Data points show the percentage of total cells showing nuclear retention of Cdc45-YFP after detergent extraction. At all time points, >95% of cells show nuclear Cdc45-YFP before detergent extraction.