||In all eukaryotic cells, initiation of DNA replication is tightly controlled to ensure that DNA replication occurs just once in each cell cycle. The prerequisite for the initiation of DNA replication is the stepwise assembly of a pre-replication complex (Pre-RC), which consists of ORC, Noc3p, Cdc6p, Cdt1p, MCM complex and possibly other proteins. Cdt1p is crucial for the recruitment of MCM complex to pre-RC, and hCdt1p has been reported to interact with hMcm6p. Regulation of the activity of Cdt1p is a key control point in the initiation of DNA replication. In higher eukaryotes, a protein named Geminin has been reported to inhibit the activity of Cdt1p by binding to Cdt1p specifically, consequently providing additional safeguarding to the control of the initiation of DNA replication. To understand the molecular basis of the regulation mechanisms of hCdt1 and its interactions with hGeminin and hMcm6, we have used the yeast two-hybrid system and co-IP assays to map the interaction fragments of hCdt1p with hGeminin and hMcm6p. Besides the corresponding fragment of hCdt1 to the domain in the published X-ray structure of the mouse Cdt1-Geminin interaction complex, we found a new hGeminin interacting coiled-coil fragment of hCdt1 close to the N terminal part of the published domain. This is a novel interaction complex between hCdt1 and the coiled-coil part of hGeminin. And because the coiled-coil part of hGeminin is critical for its inhibition of DNA replication, the structure of this new complex is important for us to study the interaction between the two proteins and understand the mechanism of the inhibition of DNA replication induced by Geminin binding to Cdt1 in human cells. Our data also suggest a novel interaction model between hCdt1 and hMcm6. The central part of Mcm6 is conserved in the MCM proteins and important for the conformation of the MCM hexamer. The C terminal part of hMcm6 is unique compared to other human MCM proteins. We found the C terminal part of hMcm6 is the key for the interaction between Cdt1 and MCM hexamer in mammalian cells. Furthermore, BrdU incorporation and chromatin binding assays showed that different interaction domains of hCdt1 and hMcm6 found in our study had dominant negative effects when they were transfected into mammalian cells. The number of the cells in S phase was reduced in BrdU incorporation assay, and the chromatin association of MCM complex was also reduced in the cells transfected with the interaction fragments of hCdt1 and hMcm6. Our study suggest that these cells were defective in DNA replication and MCM chromatin association. These data provide evidence that the interaction domains we have identified are functional in vivo. In summary, our results suggest new models for the interactions of hCdt1p with hGeminin and hMcm6p and for the mechanism of hCdt1 in loading MCM proteins onto chromatin for pre-RC formation.