Abstract
The protein kinase Cds1 is an effector of the replication checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required to stabilize stalled replication forks, and it helps to prevent the onset of mitosis until the genome is fully replicated. Mrc1 (mediator of the replication checkpoint-1) and Rad3-Rad26 kinase are required for Cds1 activation, but exactly how Mrc1 mediates Cds1 activation is unknown. Here we show that Mrc1 is required for the initial threonine 11 phosphorylation of Cds1 by Rad3-Rad26. Mrc1 specifically interacts with the forkhead-associated (FHA) domain of Cds1 in yeast two-hybrid assays. Mutations in the FHA domain that abolish this interaction also eliminate Thr-11 phosphorylation of Cds1. Weak Thr-11 phosphorylation of a "kinase-dead" mutant of Cds1 is rescued by co-expression of wild type Cds1. The requirement for Mrc1 in the replication checkpoint can be partially eliminated by expression of a Rad26-Cds1 fusion protein. These findings suggest that recognition of Mrc1 by the FHA domain of Cds1 serves to recruit Cds1 to Rad3-Rad26. This interaction mediates the initial Thr-11 phosphorylation of Cds1 by Rad3-Rad26 with subsequent intermolecular phosphorylation events leading to full activation of Cds1.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphatases / metabolism*
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Amino Acid Sequence
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Cell Cycle Proteins / metabolism*
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Checkpoint Kinase 2
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DNA Helicases / metabolism*
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DNA, Complementary / metabolism
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Dose-Response Relationship, Drug
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Forkhead Transcription Factors
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Hydroxyurea / pharmacology
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Immunoblotting
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Models, Genetic
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Molecular Sequence Data
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Mutation
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Nuclear Proteins / metabolism*
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Phosphorylation
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Plasmids / metabolism
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Precipitin Tests
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Protein Serine-Threonine Kinases / metabolism*
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / chemistry
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Saccharomyces cerevisiae Proteins / metabolism*
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Schizosaccharomyces pombe Proteins / metabolism*
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Sequence Homology, Amino Acid
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Threonine / chemistry
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Time Factors
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Transcription Factors / metabolism*
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Two-Hybrid System Techniques
Substances
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Cell Cycle Proteins
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DNA, Complementary
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Forkhead Transcription Factors
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MRC1 protein, S cerevisiae
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Nuclear Proteins
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Recombinant Fusion Proteins
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Saccharomyces cerevisiae Proteins
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Schizosaccharomyces pombe Proteins
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Transcription Factors
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rad26 protein, S pombe
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Threonine
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Checkpoint Kinase 2
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Cds1 protein, S pombe
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Protein Serine-Threonine Kinases
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Adenosine Triphosphatases
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Rad3 protein, S cerevisiae
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DNA Helicases
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Hydroxyurea