Cyclin-dependent kinase (CDK) that plays a central role in preventing 
re-replication of DNA phosphorylates several replication proteins to inactivate 
them. MCM4 in MCM2-7 and RPA2 in RPA are phosphorylated with CDK in vivo. There 
are inversed correlations between the phosphorylation of these proteins and 
their chromatin binding. Here, we examined in vitro phosphorylation of human 
replication proteins of MCM2-7, RPA, TRESLIN, CDC45 and RECQL4 with 
CDK2/cyclinE, CDK2/cyclinA, CDK1/cyclinB, CHK1, CHK2 and CDC7/DBF4 kinases. 
MCM4, RPA2, TRESLIN and RECQL4 were phosphorylated with CDKs. Effect of the 
phosphorylation by CDK2/cyclinA on DNA-binding abilities of MCM2-7 and RPA was 
examined by gel-shift analysis. The phosphorylation of RPA did not affect its 
DNA-binding ability but that of MCM4 inhibited the ability of MCM2-7. Change of 
six amino acids of serine and threonine to alanines in the amino-terminal region 
of MCM4 rendered the mutant MCM2-7 insensitive to the inhibition with CDK. These 
biochemical data suggest that phosphorylation of MCM4 at these sites by CDK 
plays a direct role in dislodging MCM2-7 from chromatin and/or preventing 
re-loading of the complex to chromatin.