The Cdc6 protein (Cdc6p) has essential roles in regulating initiation of DNA replication. Cdc6p is recruited to origins of replication by the origin recognition complex (ORC) late in mitosis; Cdc6p in turn recruits minichromosome maintenance (Mcm) proteins to form the pre-replicative complex. Cdc6p is thought to interact with one or more Mcm proteins but this point has not yet been demonstrated. In the present study we observed that Cdc6p interacted significantly only with Mcm2p out of six Mcm proteins in yeast two-hybrid cells. Our results indicate that the interaction of Cdc6p with Mcm2p is specific, although we cannot exclude the possibility that the interaction might not be direct. In attempts to identify domains of Cdc6p important for interaction with Mcm2p, we tested interactions of various deleted versions of Cdc6p with Mcm2p and also with Cdc4p, which was previously known to interact with Cdc6p. The portion of Cdc6p from amino acid residues 51 to 394 was able to interact with Mcm2p. During the course of the studies we also discovered a previously undetected Cdc4p interaction domain between residues 51 and 394. Interestingly, when all six putative Cdc28 phosphorylation sites in Cdc6p were changed to alanine, a 6-7-fold increase in binding to Mcm2p was observed. This result suggests that unphosphorylated Cdc6p has higher affinity than phosphorylated Cdc6p for Mcm2p; this might partly explain the previous observation that Cdc6p failed to load Mcm proteins on replication origins during S phase when the cyclin-dependent protein kinase was active, thus helping to prevent the reinitiation of activated replicons.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|