In eukaryotes, the activation of the prereplicative complex and assembly of an 
active DNA unwinding complex are critical but poorly understood steps required 
for the initiation of DNA replication. In this report, we have used bimolecular 
fluorescence complementation assays in HeLa cells to examine the interactions 
between Cdc45, Mcm2-7, and the GINS complex (collectively called the CMG 
complex), which seem to play a key role in the formation and progression of 
replication forks. Interactions between the CMG components were observed only 
after the G(1)/S transition of the cell cycle and were abolished by treatment of 
cells with either a CDK inhibitor or siRNA against the Cdc7 kinase. Stable 
association of CMG required all three components of the CMG complex as well as 
RecQL4, Ctf4/And-1, and Mcm10. Surprisingly, depletion of TopBP1, a homologue of 
Dpb11 that plays an essential role in the chromatin loading of Cdc45 and GINS in 
yeast cells, did not significantly affect CMG complex formation. These results 
suggest that the proteins involved in the assembly of initiation complexes in 
human cells may differ somewhat from those in yeast systems.