We investigated the dynamics of DNA binding of replication initiation proteins during formation of the pre-replicative complex (pre-RC) on plasmids in Xenopus egg extracts. The pre-RC was efficiently formed on plasmids at 23°C, with one or a few origin recognition complex (ORC) molecules and ϳ10 -20 mini-chromosome maintenance 2 (MCM2) molecules loaded onto each plasmid. Although geminin inhibited MCM loading, MCM interacted weakly but stoichiometrically with the plasmid in an ORC-dependent manner, even in the presence of geminin (with ϳ10 MCM2 molecules per plasmid). Interestingly, DNA binding of ORC, CDC6, and CDT1 was significantly stabilized in the presence of geminin, under which conditions ϳ10 -20 molecules each of ORC and CDC6 were bound. Moreover, a similarly stable ORC-CDC6-CDT1 complex rapidly formed on DNA at lower temperature (0°C) without geminin, with ϳ10 -20 molecules each of ORC and CDC6 bound to the plasmid, but almost no binding of MCM. However, upon shifting the temperature to 23°C, most ORC, CDC6, and CDT1 molecules were displaced from the DNA, leaving about one ORC molecule on the plasmid, whereas ϳ10 MCM2 molecules were loaded onto each plasmid. Furthermore, it was possible to load MCM onto DNA when the isolated ORC-CDC6-CDT1-DNA complex was mixed with purified MCM proteins. These results suggest that an ORC-CDC6-CDT1 complex pre-formed on DNA is directly involved in MCM loading and imply that each DNA-bound ORC molecule loads only one or a few MCM2-7 complexes during metazoan pre-RC formation.DNA replication in eukaryotes is initiated through the coordinated actions of replication initiation proteins. Previous studies have identified the proteins that are involved in the initiation step and an outline of the mechanism of DNA replication initiation in eukaryotic cells has been described (1-3). In brief, the origin recognition complex (ORC), 2 which consists of six subunits (ORC1-6), binds to DNA and then, in cooperation with CDC6 and CDT1, loads the mini-chromosome maintenance (MCM) 2-7 protein complex onto DNA to form a specific protein-DNA complex; the so-called pre-replicative complex (pre-RC). The pre-RC is formed in a cell cycle-regulated manner that involves geminin, a cell cycle-regulated protein that acts as an inhibitor of MCM loading (2, 4). Subsequent activation of the pre-RC is required for unwinding of the DNA around the origin and the start of DNA synthesis (1, 5-11).ORC and most other initiation proteins are well conserved in various eukaryotic species (1). Saccharomyces cerevisiae ORC binds specifically to the autonomously replicating sequence, a DNA sequence functioning as a replicator in DNA replication (12). Fission yeast Schizosaccharomyces pombe ORC contains AT-hook motifs within the ORC4 subunit and thus binds preferentially to A/T-rich sequences that are common in S. pombe origins (13). In contrast, ORC in metazoans such as humans, Drosophila, and Xenopus does not exhibit sequence specificity in its DNA binding (14 -18), but DNA topology has been shown to affect Droso...

We describe an improved model of DNA replication in Xenopus egg extracts, in which a circular plasmid immobilized on paramagnetic beads is used as a template. DNA synthesis occurred on either circular or linear plasmids coupled to the beads, but only DNA synthesis on the circular plasmid was inhibited by geminin and a CDK inhibitor, p21. DNA synthesis on the circular plasmid occurred after a time lag, during which nuclear formation was probably occurring. Although pre-replicative complexes (pre-RCs) were formed soon after mixing plasmids with egg extracts, binding of CDC45, RPA, Pol α, δ and ɛ, and PCNA to the circular plasmid was delayed, but still correlated with DNA synthesis. Moreover, p21 inhibited binding of these replication fork proteins to the circular plasmid. Therefore, the circular plasmid, but not the linear plasmid, assembles bona fide replication forks in egg extracts. We conclude that this improved replication system will be useful for studying the mechanism of formation of replication forks in eukaryotic DNA replication.