MCM7 is among the subunits of the MCM2-7 complex that plays a critical role in DNA replication initiation and cell proliferation of eukaryotic cells. that the distribution of MCM7-S121A is different from wild-type MCM7 and that the MCM7-S121A mutant is much less efficient to form a pre-RC complex with MCM3/MCM5/cdc45 compared with wild-type MCM7. By using the Tet-On inducible HeLa cell line we revealed that overexpression of wild-type MCM7 but not MCM7-S121A can block S phase entry suggesting that an excess of the pre-RC complex may activate the cell cycle checkpoint. WHI-P97 Further analysis indicates that the Chk1 pathway is activated in MCM7-overexpressed cells in a p53-dependent manner. We performed experiments WHI-P97 with the human normal cell line HL-7702 and also observed that overexpression of MCM7 can cause S phase block through checkpoint activation. In addition we found that WHI-P97 MCM7 could also be phosphorylated by cyclin B/Cdk1 on Ser-121 both and for 5 min. The supernatant was collected as a CSK-soluble fraction. The pellet was washed once with CSK buffer and then dissolved in SDS loading buffer as a CSK-insoluble fraction. In Vitro Kinase Assay WHI-P97 GST-fused full-length MCM7 MCM7-S121A MCM7-S197A MCM7-S365A and MCM7-T690A and truncated forms of MCM7 GST-cyclin E/cyclin A and GST-Cdk2 proteins were expressed in the BL21 strain of WHI-P97 and then purified by standard procedures. Cyclin B/Cdk1-activated complex was purchased from Millipore. For the kinase assay 1 μg of GST-MCM7 protein with 1 μg of GST-cyclin E and Cdk2 GST-cyclin A and Cdk2 or cyclinB1/Cdk1 was incubated in kinase buffer (50 mm Tris (pH 7.5) 10 mm MgCl2 0.02% BSA 0.04 mm ATP) in the presence of 0.5 μCi of [γ32P]ATP for 30 min at 30 °C. Samples were solved by 10% SDS-PAGE and autoradiographed to x-ray film. Era of Tet-On Steady Cell Lines FLAG-tagged MCM7 MCM7-S121A and MCM7-S121D had been cloned in to the HindIII-NotI sites from the pcDNATM/TO vector (Invitrogen) and transfected into T-RExTM-HeLa cells (Invitrogen). 48 h after transfection cells had been chosen with 100 μg/ml zeocin and 5 μg/ml blasticidin for 3 weeks. Monoclones had been picked and manifestation of MCM7 was Slco2a1 examined by immunoblotting in the current presence of tetracycline for 24 h. RNAi Treatment The knockdown of MCM7 was attained by transfection of HeLa cells with 50 nm siRNA for 72 h. Human being WHI-P97 MCM7 siRNA focus on sequences had been the following:.