Site-specific phosphorylation of the DNA damage response mediator Rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1

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Date
2013
Authors
Abreu, Carla Manuela
Kumar, Ramesh
Hamilton, Danielle
Dawdy, Andrew William
Creavin, Kevin
Eivers, Sarah
Finn, Karen
Balsbaugh, Jeremy Lynn
O'Connor, Rosemary
Kiely, Patrick A.
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Volume Title
Publisher
Public Library of Science
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Abstract
The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR-specific protein kinases.
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Keywords
Double strand breaks , Budding yeast Rad9 , Saccharomyces cerevisiae Rad9 , Cell cycle , Homologous recombination , Replication checkpoint , Signaling pathways , Protein kinases , End resection , In vivo
Citation
Abreu CM, Kumar R, Hamilton D, Dawdy AW, Creavin K, Eivers S, et al. (2013) Site-Specific Phosphorylation of the DNA Damage Response Mediator Rad9 by Cyclin-Dependent Kinases Regulates Activation of Checkpoint Kinase 1. PLoS Genet 9(4): e1003310. doi:10.1371/journal.pgen.1003310