The initiation of DNA replication is a tightly controlled process that involves the formation of distinct complexes at origins of DNA replication at specific periods of the cell cycle. Pre-replicative complexes are formed during telophase and early G 1 . They rearrange at the start of S phase to form pre-initiation complexes, which are a prerequisite for DNA replication. The CDT1 protein is required for the formation of the pre-replicative complexes. Here we show that human CDT1 associates with the CDC7 kinase and recruits CDC45 to chromatin. Moreover, we show that the amount of CDT1 bound to chromatin is regulated by CDC7. We propose a model in which chromatin-bound CDT1 is first stabilized and subsequently displaced by CDC7 activity, thereby ensuring the timely execution of DNA replication.The initiation of DNA replication is a tightly regulated process involving the orderly recruitment of specific proteins to the origins of DNA replication. In current models for DNA replication, the origins are activated (fired) by the CDC7 kinase at different times during S phase (1, 2). To license the origins for DNA replication multiprotein complexes, called pre-replicative complexes (pre-RCs), 5 have to be formed. ORC proteins bind to the origins of DNA replication throughout the cell cycle, and the binding of CDC6 and CDT1 and the subsequent recruitment of the hexameric complex MCM2-7 lead to pre-RC formation in late mitosis (telophase) and early G 1 (3, 4). CDC6 and CDT1 are both required for pre-RC formation (5-9). By recruiting other replication factors the pre-RCs rearrange during S phase and form pre-initiation complexes (preICs). Pre-ICs form only after the activation of CDK2 and CDC7 at the G 1 /S transition (10 -12). The CDC45 protein is a replication factor recruited to chromatin in a CDC7-dependent manner and is required for pre-IC formation and activation of the replication origins (13-19). DBF4 binding to CDC7 activates the kinase, and this has been shown to regulate the firing of each single replication origin in S. cerevisiae, suggesting that the kinase is activated at different times during S phase (1, 2, 20 -24). Following CDC45 association, unwinding of doublestranded DNA precedes the recruitment of the single strand binding protein RPA and onset of DNA synthesis (25). The CDT1-interacting protein, Geminin, together with CDK activity, prevents the firing of the same origin of replication during S-G 2 phases, assuring that each portion of the DNA is replicated once and only once per cell cycle (26 -34).The function of pre-RC components during S phase has not been clearly elucidated. It is known that the pre-RCs are disassembled as cells exit S phase, and only ORC proteins remain on the site to establish a complex, the so-called post-replicative complex (post-RC), representing a landing platform for pre-RC formation at the following M-G 1 transition (3). MCM proteins have been found to have in vitro helicase activity and have been hypothesized to be necessary for unwinding of the double helix and progressi...
