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

Weinreich, M., Liang, C., Stillman, B. (1999) The Cdc6p nucleotide-binding motif is required for loading mcm proteins onto chromatin. Proceedings of the National Academy of Sciences of the United States of America, 96 (2). pp. 441-6. ISSN 0027-8424

URL: http://www.ncbi.nlm.nih.gov/pubmed/9892652
DOI: 10.1073/pnas.96.2.441

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.

Item Type: Paper
Uncontrolled Keywords: Adenosine Triphosphate metabolism Binding Sites genetics Cell Cycle genetics physiology Cell Cycle Proteins genetics metabolism Chromatin metabolism DNA-Binding Proteins metabolism Flow Cytometry MCM1 Protein Mitosis physiology Mutation genetics Origin Recognition Complex Protein Binding Saccharomyces cerevisiae metabolism Saccharomyces cerevisiae Proteins Sequence Homology, Amino Acid Transcription Factors/ metabolism
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA replication
CSHL Authors:
Communities: CSHL labs > Stillman lab
Highlight: Stillman, Bruce W.
Depositing User: CSHL Librarian
Date Deposited: 21 Feb 2012 15:16
Last Modified: 20 Jun 2017 19:24
PMCID: PMC15155
Related URLs:
URI: http://repository.cshl.edu/id/eprint/25134

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