A DNA damage-induced phosphorylation circuit enhances Mec1ATR-Ddc2ATRIPrecruitment to Replication Protein A

Abstract

AbstractThe cell cycle checkpoint kinase Mec1ATRand its integral partner Ddc2ATRIPare vital for the DNA damage and replication stress response. Mec1-Ddc2 ‘senses’ single-stranded DNA (ssDNA) by being recruited to the ssDNA binding Replication Protein A (RPA) via Ddc2. In this study, we show that a DNA-damage induced phosphorylation circuit modulates checkpoint recruitment and function. We demonstrate that Ddc2-RPA interactions modulate the association between RPA and ssDNA and that Rfa1-phosphorylation aids in the further recruitment of Mec1-Ddc2. We also uncover an underappreciated role for Ddc2 phosphorylation that enhances its recruitment to RPA-ssDNA that is important for the DNA damage checkpoint in yeast. The crystal structure of a phosphorylated Ddc2 peptide in complex with its RPA interaction domain provides molecular details of how checkpoint recruitment is enhanced, which involves Zn2+. Using electron microscopy and structural modelling approaches, we propose that Mec1-Ddc2 complexes can form higher order assemblies with RPA when Ddc2 is phosphorylated. Together, our results provide insight into Mec1 recruitment and suggest that formation of supramolecular complexes of RPA and Mec1-Ddc2, modulated by phosphorylation, would allow for rapid clustering of damage foci to promote checkpoint signalling.Graphical AbstractHighlightsRfa1-S178 phosphorylation promotes Ddc2 recruitment and Ddc2-RPA complexes modulate RPA-ssDNA behaviour.Ddc2 phosphorylation enhances Mec1-Ddc2 recruitment and is important for the DNA damage checkpoint in yeast.Structure of a Ddc2:RPA complex shows phosphorylation-dependent higher order assemblies stabilised by Zn2+.We propose a Mec1-Ddc2 recruitment strategy that allows fast accumulation of Mec1-Ddc2 through DNA damage-induced phosphorylation and promotes autophosphorylation.