In all eukaryotic organisms, inappropriate firing of replication origins during the G2 phase of the cell cycle is suppressed by cyclin-dependent kinases. Multicellular eukaryotes contain a second putative inhibitor of re-replication called geminin. Geminin is believed to block binding of the mini-chromosome maintenance (MCM) complex to origins of replication, but the mechanism of this inhibition is unclear. Here we show that geminin interacts tightly with Cdt1, a recently identified replication initiation factor necessary for MCM loading. The inhibition of DNA replication by geminin that is observed in cell-free DNA replication extracts is reversed by the addition of excess Cdt1. In the normal cell cycle, Cdt1 is present only in G1 and S, whereas geminin is present in S and G2 phases of the cell cycle. Together, these results suggest that geminin inhibits inappropriate origin firing by targeting Cdt1.

We report that a highly purified human origin recognition complex (HsORC) has intrinsic DNA-binding activity, and that this activity is modestly stimulated by ATP. HsORC binds preferentially to synthetic AT-rich polydeoxynucleotides, but does not effectively discriminate between natural DNA fragments that contain known human origins and control fragments. The complex fully restores DNA replication to ORC-depleted Xenopus egg extracts, providing strong evidence for its initiator function. Strikingly, HsORC stimulates initiation from any DNA sequence, and it does not preferentially replicate DNA containing human origin sequences. These data provide a biochemical explanation for the observation that in metazoans, initiation of DNA replication often occurs in a seemingly random pattern, and they have important implications for the nature of human origins of DNA replication.

The MCM2-7 complex is believed to function as the eukaryotic replicative DNA helicase. It is recruited to chromatin by the origin recognition complex (ORC), Cdc6, and Cdt1, and it is activated at the G 1 /S transition by Cdc45 and the protein kinases Cdc7 and Cdk2. Paradoxically, the number of chromatin-bound MCM complexes greatly exceeds the number of bound ORC complexes. To understand how the high MCM2-7:ORC ratio comes about, we examined the binding of these proteins to immobilized linear DNA fragments in Xenopus egg extracts. The minimum length of DNA required to recruit ORC and MCM2-7 was ϳ80 bp, and the MCM2-7: ORC ratio on this fragment was ϳ1:1. With longer DNA fragments, the MCM2-7:ORC ratio increased dramatically, indicating that MCM complexes normally become distributed over a large region of DNA surrounding ORC. Only a small subset of the chromatin-bound MCM2-7 complexes recruited Cdc45 at the onset of DNA replication, and unlike Cdc45, MCM2-7 was not limiting for DNA replication. However, all the chromatin-bound MCM complexes may be functional, because they were phosphorylated in a Cdc7-dependent fashion, and because they could be induced to support Cdk2-dependent Cdc45 loading. The data suggest that in Xenopus egg extracts, origins of replication contain multiple, distributed, initiation-competent MCM2-7 complexes.In eukaryotic organisms, DNA replication initiates at many sites (1). In Saccharomyces cerevisiae, DNA replication initiates every ϳ40 kb at autonomously replicating sequences that recruit the origin recognition complex (ORC), 1 the sixsubunit initiator protein. In metazoans, initiation sites are less rigidly defined. In embryonic cells of Xenopus laevis, DNA replication initiates once every ϳ10 kb without sequence specificity (2). In somatic cells, initiation events are less frequent, occurring once every ϳ150 kb, and recent evidence indicates that initiations are controlled by genetic elements (1). At some loci, replication initiates at a precise location, whereas at other loci, initiation events are distributed throughout zones spanning up to 50 kb.The mechanism of DNA replication initiation is highly conserved among eukaryotic organisms (3, 4). A representative model system is Xenopus egg extracts (2, 4) where two factors, Cdc6 and Cdt1, bind to sites on chromatin that are marked by ORC. Subsequently, the hexameric MCM2-7 complex binds to the ORC-Cdc6-Cdt1 complex to establish the pre-replication complex (pre-RC). At the onset of DNA replication, the pre-RC is activated by the sequential action of the protein kinases Cdc7/Dbf4 and Cdk2/cyclin E (Cdk) (5, 6). Genetic and biochemical experiments suggest that the function of Cdc7/Dbf4 is the phosphorylation of the MCM2-7 complex (7). After MCM phosphorylation by Cdc7/Dbf4, Cdk2/cyclin E stimulates the association of Cdc45 with the pre-RC, likely via a direct interaction with the MCM2-7 complex (8, 9). The binding of Cdc45 coincides with activation of a chromatin-bound helicase that unwinds the DNA, allowing binding of the single-stranded...

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A recent Molecular Cell paper by Randell et al. (2006) sheds light on the role of ATP hydrolysis by Cdc6 in promoting the stable loading of the Mcm2-7 complex onto origins of DNA replication.