Phosphoproteomics Reveals a Distinct Mode of Mec1/ATR Signaling in Response to DNA End Hyper-Resection

Abstract

The Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, although how it promotes context-dependent signaling and DNA repair remains elusive. Here we uncovered a specialized mode of Mec1/ATR signaling triggered by the extensive nucleolytic processing (resection) of DNA ends. Cells lacking RAD9, a checkpoint activator and an inhibitor of resection, exhibit a selective increase in Mec1-dependent phosphorylation of proteins associated with single strand DNA transactions, including the ssDNA binding protein Rfa2, the translocase/ubiquitin ligase Uls1 and the HR-regulatory Sgs1-Top3-Rmi1 (STR) complex. Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper-resection to influence recombination outcomes. Overall, the identification of a distinct mode of Mec1 signaling triggered by hyper-resection highlights the multi-faceted action of this kinase in the coordination of checkpoint signaling and HR-mediated DNA repair.