A DNA damage-induced phosphorylation circuit enhances Mec1ATR-Ddc2ATRIP recruitment to Replication Protein A.

Abstract

The cell cycle checkpoint kinase Mec1ATR and its integral partner Ddc2ATRIP are 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 Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/521831v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@139e23corg.highwire.dtl.DTLVardef@133d6e6org.highwire.dtl.DTLVardef@6198b5org.highwire.dtl.DTLVardef@1709a73_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIRfa1-S178 phosphorylation promotes Ddc2 recruitment and Ddc2-RPA complexes modulate RPA-ssDNA behaviour. C_LIO_LIDdc2 phosphorylation enhances Mec1-Ddc2 recruitment and is important for the DNA damage checkpoint in yeast. C_LIO_LIStructure of a Ddc2:RPA complex shows phosphorylation-dependent higher order assemblies stabilised by Zn2+. C_LIO_LIWe propose a Mec1-Ddc2 recruitment strategy that allows fast accumulation of Mec1-Ddc2 through DNA damage-induced phosphorylation and promotes autophosphorylation. C_LI