FIG. 3.

Stability of Polα-primase complex in wild-type and mutant cells. (A) Expression of Polα, Spp1, and Spp2 in wild-type and spp2 mutant cells. Wild-type 972h⁻, ST118 (spp2-8), and ST119 (spp2-9) cells grown at 25°C were incubated at 36°C for 4.5 h. Equal amounts of protein from cell extracts were analyzed by Western blot using antibodies against Polα, Spp1, and Spp2 as probes (lanes 1 to 3). To test the coimmunoprecipitation of primase and Polα, cells were grown at the indicated temperatures for 4.5 h. Polα-primase complex was immunoprecipitated from the cell extracts by using anti-Polα antibody and probed with appropriate antibodies as described in Materials and Methods (lanes 4 to 9). (B) Checkpoint-defective polαts13 mutant has a stable Polα-primase complex. (Left panel) Spp1 and Spp2 coimmunoprecipitated with Polα in DBts13 (polαts13) cells. DBts13 (polαts13) cells were grown at 25 or 36°C for 4.5 h. Polα-primase complex was immunoprecipitated from the cell extracts by using anti-Polα antibody and probed with antibodies against Polα, Spp1, and Spp2. (Right panel) Phenotype of wild type and DBts13 (polαts13) grown at 36°C for 6 h. The arrows indicate polαts13 cells that displayed an abnormal mitotic phenotype.