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

Abstract

Cdc6p has an essential function in the mechanism and regulation of the initiation of DNA replication. Budding yeast Cdc6p binds to chromatin near autonomously 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 proteins near autonomously replicating sequence elements by an unknown mechanism. All Cdc6p homologues contain a bipartite Walker ATP-binding motif that suggests that ATP binding or hydrolysis may regulate Cdc6p activity. To determine whether these motifs are important for Cdc6p activity, mutations were 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 temperature-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, suggesting that ATP binding is essential for this activity. The mutant arrests with a G1 DNA content but retains the ability to restrain mitosis in the absence of DNA replication, unlike depletion of Cdc6p. In contrast, Cdc6p containing 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 results suggest that Cdc6p nucleotide binding is important for establishing the prereplicative complex at origins of DNA replication and that the amino terminus of Cdc6p is required for blocking entry into mitosis.

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…These structural motifs are required for loading the MCM proteins onto DNA. (37,38) Domain III consists of a bundle of a-helixes, including a winged-helix (WH) fold that suggests a DNA-binding motif. The dual lobe formed by domains I and II is remarkably similar to that of other AAA þ ATPases such as NSF-D2 and d 0 , one of the subunits of the b-clamp loader in E. coli.…”

“…(36) In addition, the ATP-binding domain of Cdc6 is essential for pre-RC assembly (see below; Refs. 37,38).…”

Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

“…In the yeast Saccharomyces cerevisiae, but also in many other organisms, the binding of Cdc6 to the origin recognition complex (ORC) is a critical step in the formation of pre-RCs (Liang et al, 1995;Cocker et al, 1996;Detweiler and Li, 1997) and essential for subsequent loading of Mcm proteins (Mcm2-7; Piatti et al, 1996;Santocanale and Diffley, 1996;Aparicio et al, 1997;Donovan et al, 1997;Tanaka et al, 1997). After licensing the origin by loading Mcms, endogenous Cdc6 dissociates from the replicative complex and only reassociates with chromatin late in M-phase Weinreich et al, 1999) when cyclin-dependent protein kinase (Clb-Cdc28) activities are absent. Although it is clear that loading of Cdc6 onto chromatin is inhibited by cyclindependent kinase (CDK) activity, the mechanism of inhibition is complex, and there are many modes of regulation (see Figure 1).…”

Synthesis and turn‐over of the replicative Cdc6 protein during the HeLa cell cycle

“…In yeasts, Cdc6p is expressed during the G1 phase [7,8], associates with stationary ORC [9,10] and loads Mcm initiation proteins in reactions requiring an intact nucleotide binding domain [11][12][13]. Once replication begins, yeast Cdc6p is phosphorylated and then rapidly destroyed by ubiquitin-mediated protein degradation.…”

“…Cdc6p associates with the chromatin-bound six-subunit origin recognition complex (ORC) and promotes, together with the Cdt1 protein [2,3], the subsequent loading of the Mcm protein complex. The fully assembled pre-replicative complex is induced to activate replication origins by at least two classes of protein phosphorylating enzymes, cyclindependent kinases (Cdk) and the Dbf4-Cdc7 kinase [4][5][6].In yeasts, Cdc6p is expressed during the G1 phase [7,8], associates with stationary ORC [9,10] and loads Mcm initiation proteins in reactions requiring an intact nucleotide binding domain [11][12][13]. Once replication begins, yeast Cdc6p is phosphorylated and then rapidly destroyed by ubiquitin-mediated protein degradation.…”