Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA r...

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Bibliographic Details
Published in:The Journal of biological chemistry 2009-07, Vol.284 (28), p.18593-18604
Main Authors: Chen, Sheng-hong, Zhou, Huilin
Format: Article
Language:English
Subjects:
Adaptor Proteins, Signal Transducing - chemistry
Amino Acid Sequence
Catalysis
Cell Cycle Proteins - chemistry
Cell Cycle Proteins - metabolism
Checkpoint Kinase 2
DNA: , Repair, Recombination, and Chromosome Dynamics
Dose-Response Relationship, Drug
Epitopes - chemistry
Gene Expression Regulation
Intracellular Signaling Peptides and Proteins - metabolism
Mass Spectrometry - methods
Molecular Sequence Data
Protein Structure, Tertiary
Protein-Serine-Threonine Kinases - metabolism
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - chemistry
Saccharomyces cerevisiae Proteins - metabolism
Sequence Homology, Amino Acid
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Summary:Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M109.018242