A model for the state of pre-replication chromatin and cell cycle regulation in human cells. (A) During late M to G1 phase when Cdk and geminin activities are suppressed by APC/C ubiquitin ligase, ORC, CDC6 and Cdt1 form the machinery on the nuclear matrix necessary to load MCM2-7 complexes. Multiple MCM complexes are loaded onto chromatin beyond ORC binding sites. (B) When cells enter S phase, CDC45 and some other proteins are recruited around MCM dependent on Cdk and CDC7 kinase activity, unwinding DNA. Then, DNA synthetic proteins are assembled on single-stranded DNA. Activated MCM plays an essential role in DNA replication, probably as a replicative DNA helicase, and is simultaneously displaced from chromatin through an unknown mechanism. The steps after DNA unwinding are omitted in the model shown. After S phase, reloading of dissociated MCM (re-licensing) is suppressed by multiple mechanisms. One is by Cdks, which phosphorylates ORC1 and Cdt1 so that they undergo SCFSkp2-mediated proteolysis. Phosphorylation-dependent nuclear export of chromatin-unbound CDC6 could also contribute to inhibition of re-licensing. Cdt1 is further subjected to replication-coupled proteolytic regulation mediated by Cul4-DDB1Cdt2 ubiquitin ligase and PCNA. Geminin also prevents the MCM rebinding by sequestering Cdt1.