A DNA damage-induced phosphorylation circuit enhances Mec1^ATR-Ddc2^ATRIPrecruitment to Replication Protein A

Abstract

The 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.

Replication protein-A, RPA, plays a pivotal role in the maintenance of recombination checkpoint in yeast meiosis

“…DSB ends stimulate a DDR kinase, ATM/Tel1 (Ataxia telangiectasia mutated) while ssDNAs do an ATM homolog, ATR/Mec1 (Ataxia telangiectasia mutated related), both of which belong to the PI3 kinase family 11,12 . The activation of ATR is mediated by the interaction with an ATR partner, ATRIP/Ddc2, which directly interacts with RPA bound to ssDNAs [13][14][15] . In mammals, ATR activation is also mediated by the second RPA-binding protein, ETAA1 16,17 .…”