The Cdc6p nucleotide-binding motif is required for loading Mcm proteins onto chromatin

Cdc6p has an essential function in the mechanism and regulation of the init iation of DNA replication. Budding yeast Cdc6p binds to chromatin near auto nomously replicating sequence elements in late M to early G1 phase through an interaction with Origin Recognition Complex or another origin-associated factor. It then facilitates the subsequent loading of the Mcm family of pr oteins near autonomously replicating sequence elements by an unknown mechan ism. All Cdc6p homologues contain a bipartite Walker ATP-binding motif that suggests that ATP binding or hydrolysis may regulate Cdc6p activity. To de termine whether these motifs are important for Cdc6p activity, mutations we re made in conserved residues of the Walker A and B motifs. Substitution of lysine 114 to alanine (K114A) in the Walker A motif results in a temperatu re-sensitive phenotype in yeast and slower progression into S phase at the permissive temperature. A K114E mutation is lethal. The CdC6(K114E) protein binds to chromatin but fails to promote loading of the Mcm proteins, sugge sting that ATP binding is essential for this activity. The mutant arrests w ith a G1 DNA content but retains the ability to restrain mitosis in the abs ence of DNA replication, unlike depletion of Cdc6p, In contrast, Cdc6p cont aining a double alanine mutation in the Walker B motif, DE(223, 224)AA, is functional, and the mutant exhibits an apparently normal S phase. These res ults suggest that Cdc6p nucleotide binding is important for establishing th e prereplicative complex at origins of DNA replication and that the amino t erminus of Cdc6p is required for blocking entry into mitosis.