Email this page

Print this page

Report

Cell cycle-dependent phosphorylation of Rad53 kinase by Cdc5 and Cdc28 modulates checkpoint adaptation

Thomas Schleker, Kenji Shimada, Ragna Sack, Brietta L. Pike and Susan M. Gasser

In budding yeast the evolutionarily conserved checkpoint response varies in its sensitivity to DNA damaging agents through the cell cycle. Specifically, higher amounts of damage are needed to activate the downstream checkpoint kinase Rad53 in S-phase cells. We examined here whether phosphorylation of Rad53 itself by cell cycle-dedicated kinases regulates Rad53 activation. We found that during unperturbed growth Rad53 exhibits a small phosphorylation-dependent electrophoretic mobility shift in G2, M and G1 phases of the cell cycle that is lost in S phase. We show that Rad53 is phosphorylated in vitro by Cdc5, a mitotic Polo-like kinase, and by the yeast cyclin-dependent kinase, Cdc28. Consistently, the cell cycle-dependent Rad53 mobility shift requires both Cdc5 and Cdc28 activities. We mapped the in vitro targeted phosphorylation sites by mass spectrometry and confirmed with mass spectroscopy that serines 774, 789 and 791 within Rad53 are phosphorylated in vivo in M-phase arrested cells. By creating nonphosphorylatable mutations in the endogenous RAD53 gene, we confirmed that the CDK and Polo kinase target sites are responsible for the observed cell cycle-dependent shift in protein mobility. The loss of phospho-acceptor sites does not interfere with Rad53 activation but accelerates checkpoint adaptation after induction of a single irreparable double-strand break. We thus demonstrate that cell cycle-dependent phosphorylation can fine-tune the response of Rad53 to DNA damage.

Authors

Thomas Schleker

Friedrich Miescher Institute for Biomedical Research; Basel, Switzerland

Kenji Shimada

Friedrich Miescher Institute for Biomedical Research; Basel, Switzerland

Ragna Sack

Friedrich Miescher Institute for Biomedical Research; Basel, Switzerland

Brietta L. Pike

Friedrich Miescher Institute for Biomedical Research; Basel, Switzerland

Susan M. Gasser Corresponding author: susan.gasser@fmi.ch

Friedrich Miescher Institute for Biomedical Research; Basel, Switzerland