A dominant-negative MEC3 mutant uncovers new functions for the Rad17 complex and Tel1.
The Rad17-Mec3-Ddc1 complex is essential for the cellular response to genotoxic agents and is thought to be important for sensing DNA lesions. Deletion of any of the RAD17, MEC3 or DDC1 genes abolishes the G(1) and G(2) and impairs the intra-S DNA-damage checkpoints. We characterize a dominant-negative mec3-dn mutation that ... has an unexpected phenotype. It inactivates the G(1) checkpoint while it leaves the G(2) response functional, thus revealing a difference in the requirements of the DNA-damage response in different phases of the cell cycle. In an attempt to identify the molecular defect imparted by the mutation, we dissected step-by-step the signaling cascade, which is triggered by DNA lesions and requires the activity of Mec1 and Rad53 kinases. The analysis of the phosphorylation state of checkpoint factors and critical protein interactions showed that, in mec3-dn cells, the signal transduction cascade is triggered normally, and the central kinase Mec1 can be activated. In G(1) cells expressing the mutation, the signaling cannot proceed any further along the pathway, indicating that the Rad17 complex acts after the activation of Mec1, possibly recruiting targets for the kinase. We also show that the function of the G(2) checkpoint in mutant cells is maintained by an uncharacterized activity of Tel1, the yeast homologue of ATM. This work thus reports a previously undiscovered role for Tel1 in checkpoint control.
Mesh Terms:
Cell Cycle, Cell Cycle Proteins, DNA Damage, DNA-Binding Proteins, Epitopes, Fungal Proteins, Gene Deletion, Genes, Dominant, Intracellular Signaling Peptides and Proteins, Models, Biological, Mutation, Nuclear Proteins, Open Reading Frames, Phenotype, Phosphorylation, Plasmids, Precipitin Tests, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Signal Transduction, Time Factors, Two-Hybrid System Techniques
Cell Cycle, Cell Cycle Proteins, DNA Damage, DNA-Binding Proteins, Epitopes, Fungal Proteins, Gene Deletion, Genes, Dominant, Intracellular Signaling Peptides and Proteins, Models, Biological, Mutation, Nuclear Proteins, Open Reading Frames, Phenotype, Phosphorylation, Plasmids, Precipitin Tests, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Signal Transduction, Time Factors, Two-Hybrid System Techniques
Proc. Natl. Acad. Sci. U.S.A.
Date: Oct. 01, 2002
PubMed ID: 12271137
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