FIGURE 5.

An excess of MCM7 decelerates S phase entry. A, HeLa-6TR Tet-On inducible cell lines for overexpressing MCM7-WT or MCM7-S121A were generated. The cells were synchronized at G₁ phase by double thymidine treatment in the presence of tetracycline, released for 3 h, and subjected to flow cytometry analysis (left panel). The percentage of S phase cells was calculated (right panel). B and C, HCT116 and HCT116 p53^−/− cells were transfected with pCMV FLAG-MCM7-WT or pCMV FLAG-MCM7-S121A together with pCMV GFP-H₂B as the sorting marker. The cells were synchronized at G₁ phase, released for 3 h, and subjected to flow cytometry analysis. D, cell lysates from the indicated samples were immunoprecipitated with FLAG antibody and immunoblotted with MCM7-p-Ser-121 antibody. E, HeLa-6TR/MCM7-WT and HeLa-6TR/MCM7-S121A cells were treated with tetracycline for 24 h. The total cell lysates were harvested for immunoblotting (IB) with Chk1-p-Ser-345, Cdk2-p-Thr-14, and p53 antibodies. F, the chromatin-bound fractions (CSK-P) from HeLa-6TR/MCM7-WT, HeLa-6TR/MCM7-S121A, and control cells were immunoblotted with FLAG, MCM3, MCM5, RPA70, and Lamin B. *, p < 0.05; **, p < 0.01.