FIGURE 3:

Overexpressing Cdc18 and Cdt1 in late meiosis increases loading of Mcm2 onto chromatin. (A) Cells expressing Mcm2-CFP, Cdc18, and Cdt1 in late meiosis at levels similar to those seen in meiS were detergent extracted at the time points shown and imaged to detect chromatin-bound Mcm2. Chromatin-associated Mcm2 can be detected around the time of meiS (2.5 h) both in the absence and presence of HU (to trap Mcm2 on DNA) but not at later time points. The strain used was also deleted for mes1 (P1756), but similar results were obtained for a mes1⁺ strain (unpublished data). Bar, 10 μm. (B) Western blot analysis of Cdc18 and Cdt1 levels in pat1-synchronized meiosis when both mes1pr-cdc18 and mes1pr-cdt1 constructs are expressed from plasmids (P2466). Levels of proteins in log-phase wt cells or meiS cells are shown for comparison. (C) Chromatin-binding assay for Mcm2 as in A, except that strain P2466 was used, overexpressing Cdc18 and Cdt1 in late meiosis. Similar results were obtained using a mes1Δ strain (unpublished data). (D) Quantitation of percentage of cells showing Mcm2-CFP chromatin binding in meiosis when Cdc18 and Cdt1 were overexpressed. At least 100 cells were counted for each time point. Strains used were P2466 (mes1⁺) and 2467 (mes1Δ). (E) As D, except that fluorescence intensity of chromatin-associated Mcm2-CFP in mes1⁺ cells (P2466) was quantitated (arbitrary units). One hundred cells were counted for each sample, and SD is shown.