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

Luke A. Yates, Elias A. Tannous, R. Marc Morgan, Peter M. Burgers, Xiaodong Zhang

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

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 modeling 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 signaling.

Original languageEnglish
Article numbere2300150120
JournalProceedings of the National Academy of Sciences of the United States of America
Volume120
Issue number14
DOIs
StatePublished - 2023

Keywords

  • DNA damage signaling
  • checkpoint kinase
  • protein phosphorylation
  • replication stress
  • structural biology

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