FIG. 5.

Spb70 associates with Orp2/Orc2 throughout the cell cycle. The cdc25-22 strain containing two-myc-six-His-spb70⁺ and orp2⁺-three-FLAG was grown at 25°C in EMM to mid-log phase and then shifted to 36°C to arrest at G₂ for 4 h. Cells were then released from G₂ arrest by shift-down to 25°C and harvested every 20 min after release from G₂ arrest. Chromatin fractions were prepared from each cell sample as described in Materials and Methods. Presence of Mcm6, Spb70, Orp2, and Spp2 in chromatin fraction was detected by Western blotting with anti-MCM6, anti-myc, anti-FLAG, and anti-Spp2 antibodies, respectively. (A) Cell cycle progression after the release from G₂ arrest. Percentage of cells in different stages of the cell cycle was shown as the septation index or mitotic index. (B) Proteins in chromatin-enriched fractions. Kinetics of the appearance of MCM6, Orp2, primase Spp2/p58, and Spb70 in the chromatin fraction after release from G₂ arrest were shown by Western blotting each protein with their respective antibody. CCB, the Coomassie brilliant blue staining of the Western blot membrane. (C) Coprecipitation of Spb70 and Orp2. Immunoprecipitates of Orp2 by anti-FLAG were Western blotted with anti-myc for Spb70 and anti-FLAG for Orp2.