Abstract
Previous work on the DNA damage checkpoint in Saccharomyces cerevisiae has shown that two complexes independently sense DNA lesions: the kinase Mec1-Ddc2 and the PCNA-like 9-1-1 complex. To test whether colocalization of these components is sufficient for checkpoint activation, we fused these checkpoint proteins to the LacI repressor and artificially colocalized these fusions by expressing them in cells harboring Lac operator arrays. We observed Rad53 and Rad9 phosphorylation, Sml1 degradation, and metaphase delay, demonstrating that colocalization of these sensors is sufficient to activate the checkpoint in the absence of DNA damage. Our tethering system allowed us to establish that CDK functions in the checkpoint pathway downstream of damage processing and checkpoint protein recruitment. This CDK dependence is likely, at least in part, through Rad9, since mutation of CDK consensus sites compromised its checkpoint function.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Adaptor Proteins, Signal Transducing
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Animals
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Checkpoint Kinase 2
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Chromatin / genetics
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Chromatin / metabolism
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Cyclin-Dependent Kinases / genetics
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Cyclin-Dependent Kinases / metabolism
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DNA Damage*
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Enzyme Activation
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Genes, cdc*
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Histones / genetics
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Histones / metabolism
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism
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Macromolecular Substances / metabolism*
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Phosphoproteins / genetics
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Phosphoproteins / metabolism
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism
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Protein Subunits / genetics
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Protein Subunits / metabolism
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Repressor Proteins / genetics
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Repressor Proteins / metabolism
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Saccharomyces cerevisiae / cytology
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
Substances
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Adaptor Proteins, Signal Transducing
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Cell Cycle Proteins
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Chromatin
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Histones
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Intracellular Signaling Peptides and Proteins
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LCD1 protein, S cerevisiae
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Macromolecular Substances
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Phosphoproteins
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Protein Subunits
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RAD24 protein, S cerevisiae
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Recombinant Fusion Proteins
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Repressor Proteins
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Saccharomyces cerevisiae Proteins
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rad9 protein
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Checkpoint Kinase 2
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MEC1 protein, S cerevisiae
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Protein Serine-Threonine Kinases
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Cyclin-Dependent Kinases
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RAD53 protein, S cerevisiae