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.

Figure 1

Chromatin binding analysis of Cdc6p and Mcm proteins in the cell cycle. (A) FACS profile of YB511 after a dbf2–1 arrest (37°C) and release at 25°C. (B) Chromatin pellets of YB511 were probed for Orc3p, Orc6p, Cdc6p, and the six Mcm proteins by using monoclonal antibodies specific for each protein.

Figure 2

Growth characteristics of CDC6 ATP-binding site mutants. (A) A schematic of Cdc6p showing the regions of homology to RFC and the AAA⁺ superfamily of proteins (44, 45). Below is an alignment (from top to bottom) of the human (50), Xenopus (7), S. pombe (51), and S. cerevisiae (40) Cdc6p Walker A and B motifs (underlined). The amino acid changes in Cdc6p are indicated by the arrows at lysine 114 and the aspartic and glutamic acids 223 and 224. (B) Strains YB502, YB503, YB504, and YB505 were streaked onto SC plates lacking methionine (−Met), expressing pMET3-CDC6^WT, or containing 2 mM methionine (+Met), allowing expression of only the indicated CDC6 allele, and photographed after 2 days at 30°C and 37°C.

Figure 3

cdc6^K114E fails to initiate S phase but inhibits mitosis. Asynchronous cultures of K4055, YB504, and YB506 were grown to exponential phase in SC medium lacking methionine. Methionine was added to 2 mM to repress the wild-type pMET3-CDC6 present in all strains, and time points were processed for FACS analysis. From left to right, strains encode no CDC6, or either cdc6^K114E or cdc6^{ΔN, K114E} integrated at the LEU2 locus and under the control of the CDC6 promoter.

Figure 4

The cdc6^K114A mutant moves more slowly into S phase. YB507, YB508, and YB510 were grown to exponential phase in YPD, arrested in G1 with α-factor, released from the block and monitored by FACS for entry into S phase.

Figure 5

Cdc6 proteins bind to chromatin but Cdc6^K114Ep fails to promote loading of Mcm proteins. Western blots of chromatin fractions from cells (K4055, YB502, YB504, YB505) blocked in G2/M with nocodazole and released to an α-factor (G1) block for 30′, 60′, or 90′ expressing the indicated CDC6 alleles. Lanes 1, 6, 11, and 16 are the chromatin fractions at the nocodazole arrest. Lanes 2, 7, 12, and 17 are the chromatin fractions released to α-factor for 60′ in cells expressing wild-type CDC6 from the MET3 promoter (i.e., no methionine). The next three lanes of each set (e.g., 3–5) represent chromatin fractions released to an α-factor block for 30′, 60′, and 90′ expressing, from left to right, no CDC6, CDC6^WT, cdc6^K114E, or cdc6^{DE(223, 224)AA}. Blots were probed for Cdc6p, Mcm2p, Mcm3p, and Orc3p plus Orc6p.