DNA replication is controlled by the stepwise assembly of a pre-replicative complex and the replication apparatus. Cdt1 is a novel component of the pre-replicative complex and plays a role in loading the minichromosome maintenance (MCM) 2-7 complex onto chromatin. Cdt1 activity is inhibited by geminin, which is essential for the G 2 /M transition in metazoan cells. To understand the molecular basis of the Cdt1-geminin regulatory mechanism in mammalian cells, we cloned and expressed the mouse Cdt1 homologue cDNA in bacterial cells and purified mouse Cdt1 to near homogeneity. We found by yeast two-hybrid analysis that mouse Cdt1 associates with geminin, MCM6, and origin recognition complex 2. MCM6 interacts with the Cdt1 carboxyl-terminal region (amino acids 407-477), which is conserved among eukaryotes, whereas geminin associates with the Cdt1 central region (amino acids 177-380), which is conserved only in metazoans. In addition, we found that Cdt1 can bind DNA in a sequence-, strand-, and conformation-independent manner. The Cdt1 DNA binding domain overlaps with the geminin binding domain, and the binding of Cdt1 to DNA is inhibited by geminin. Taken together, we have defined structural domains and novel biochemical properties for mouse Cdt1 that suggest that Cdt1 behaves as an intrinsic DNA binding factor in the pre-replicative complex.Chromosomal DNA replication is subject to strict cell cycle control, which ensures that cells enter S phase once and only once per cell cycle. A considerable body of evidence from both genetic analyses of yeast mutants and biochemical studies using Xenopus egg extracts has shown that the initiation of replication requires the stepwise assembly of protein complexes on chromatin to form a pre-replicative complex (pre-RC) 1 (1-8).The pre-RC includes the origin recognition complex (ORC), the minichromosome maintenance protein complex (MCM), and the Cdc6 and Cdt1 proteins. After the activation of S phasepromoting kinases, CDKs, and the Dbf4-dependent kinase, DNA helicase unwinds the two DNA strands, and replication protein A stabilizes single-stranded DNA, thereby allowing an initiation complex to be formed by the loading of DNA polymerases onto the pre-RC. Because most of the components of the pre-RC identified in Saccharomyces cerevisiae and Xenopus have been found in other eukaryotes including humans, it is believed that the mechanisms controlling the initiation of replication are conserved in all eukaryotes. However, the DNA helicase that is associated with the replication fork has not yet been identified, even in S. cerevisiae (1,6,8,9). The best candidate for the replicative DNA helicase is the MCM2-7 complex. The MCM2-7 complex was first identified as a set of genes required for minichromosome maintenance in S. cerevisiae, and it was subsequently identified as a critical component of the replication licensing system in Xenopus egg extracts (3, 9, 10). MCM2-7 proteins are loaded onto chromatin at late telophase and gradually released as replication forks proceed, and concomitan...

Cdt1 is an essential component for the assembly of a pre-replicative complex. Cdt1 activity is inhibited by geminin, which also participates in neural development and embryonic differentiation in many eukaryotes. Although Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi. Here, we identify the C. elegans geminin, GMN-1. Biochemical analysis reveals that GMN-1 associates with C. elegans CDT-1, the Hox protein NOB-1, and the Six protein CEH-32. GMN-1 inhibits not only the interaction between mouse Cdt1 and Mcm6 but also licensing activity in Xenopus egg extracts. RNA interference-mediated reduction of GMN-1 is associated with enlarged germ nuclei with aberrant nucleolar morphology, severely impaired gametogenesis, and chromosome bridging in intestinal cells. We conclude that the Cdt1-geminin system is conserved throughout metazoans and that geminin has evolved in these taxa to regulate proliferation and differentiation by directly interacting with Cdt1 and homeobox proteins.Eukaryotic DNA replication is controlled by the stepwise assembly of a prereplicative complex (pre-RC) 1 and the replication apparatus. The pre-RC includes the origin recognition complex (ORC), the minichromosome maintenance protein complex (MCM), as well as the Cdc6 and Cdt1 proteins. After activation of S phase-promoting kinases, pre-RC converts to the pre-initiation complex (pre-IC). This step includes the loading of Cdc45 and Gins complex, which allows DNA polymerases to assemble with the replication apparatus. Each step in the assembly of pre-RC, pre-IC, and replication apparatus is regulated by checkpoint mechanisms thus maintaining genome integrity and ploidy and avoiding disposition to carcinogensesis (1-3).Cdt1 is an essential component of licensing reaction conserved in eukaryotes including human, Xenopus, Drosophila, Caenorhabditis elegans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae (4 -11). Cdt1 directly associates with MCM2-7 complex to form a pre-RC in late mitosis and early G 1 phase. Cdt1-dependent loading of MCM2-7 onto chromatin is the limiting step for preventing reinititation, because overexpression of Cdt1 causes re-replication in Xenopus egg extract (5, 12-16). Cdt1 activity is inhibited by geminin. Geminin was first identified in Xenopus egg extract as degradable protein by anaphase-promoting complex and later characterized as the licensing inhibitor through direct interaction with Cdt1 (7,17,18). Furthermore, geminin has been found in various multicellular eukaryotes including human, Xenopus, and Drosophila, to participate in neural development and embryonic differentiation (19 -22). In contrast, geminin homologue has not been found in C. elegans as well as monocellular eukaryotes including S. cerevisiae and S. pombe yet.To better understand the relevance of the geminin-Cdt1 system in higher eukaryotes, we described the domain organization of mouse Cdt1 (8). The less conserved amino-terminal region i...

In eukaryotes, DNA replication is fired once in a single cell cycle before cell division starts to maintain stability of the genome. This event is tightly controlled by a series of proteins. Cdt1 is one of the licensing factors and is involved in recruiting replicative DNA helicase Mcm2-7 proteins into the pre-replicative complex together with Cdc6. In Cdt1, the C-terminal region serves as a binding site for Mcm2-7 proteins, although the details of these interactions remain largely unknown. Here, we report the structure of the region and the key residues for binding to Mcm proteins. We determined the solution structure of the C-terminal fragment, residues 450 -557, of mouse Cdt1 by NMR. The structure consists of a winged-helix domain and shows unexpected similarity to those of the C-terminal domain of Cdc6 and the central fragment of Cdt1, thereby implying functionalandevolutionaryrelationships.Structure-basedmutagenesis and an in vitro binding assay enabled us to pinpoint the region that interacts with Mcm proteins. Moreover, by performing in vitro binding and budding yeast viability experiments, we showed that ϳ45 residues located in the N-terminal direction of the structural region are equally crucial for recognizing Mcm proteins. Our data suggest the possibility that winged-helix domain plays a role as a common module to interact with replicative helicase in the DNA replication-licensing process.In eukaryotes, DNA replication is highly coordinated to retain the integrity of the genome. Whereas DNA replication in prokaryotes begins at a single site and stops at the end of the genome, eukaryotic genomes consist of multiple replication origins where DNA replication starts. These origins are synchronized so that they are activated only once in a single division cycle. A series of proteins, the origin recognition complex (ORC), 6 cell division cycle 6 homolog (Cdc6), chromatin licensing and DNA replication factor 1 (Cdt1), and minichromosome maintenance 2-7 (Mcm2-7) are known to play correlated roles in licensing (1-5). Oncogenic proliferation often causes abnormal expression of the proteins involved in the DNA-licensing process, thus emphasizing the importance of harmonious adjustments between these proteins (6). A complicated interaction network between these proteins has been reported (7), although the details at residue and atom levels remain largely unknown.Formation of a pre-replication complex at each origin is the first event in the replication process. ORC proteins bind initially to each replication origin of DNA. The DNA sequences of the origins where ORC binds have not been identified, except for those in Saccharomyces cerevisiae (8), suggesting that there may be other factors involved in addition to the sequences (9). The DNA strand at the origin needs to be unpaired to begin replication; therefore, the existence of replicative helicase is essential. Mcm2-7 proteins are believed to function as the replicative helicase in eukaryotes. Each Mcm2-7 protein consists of a conserved AAAϩ ATPase type C-termin...