Activation of DSB processing requires phosphorylation of CtIP by ATR

Mol Cell. 2013 Feb 21;49(4):657-67. doi: 10.1016/j.molcel.2012.11.020. Epub 2012 Dec 27.

Abstract

DNA double-strand breaks (DSBs) activate a DNA damage response (DDR) that coordinates checkpoint pathways with DNA repair. ATM and ATR kinases are activated sequentially. Homology-directed repair (HDR) is initiated by resection of DSBs to generate 3' single-stranded DNA overhangs. How resection and HDR are activated during DDR is not known, nor are the roles of ATM and ATR in HDR. Here, we show that CtIP undergoes ATR-dependent hyperphosphorylation in response to DSBs. ATR phosphorylates an invariant threonine, T818 of Xenopus CtIP (T859 in human). Nonphosphorylatable CtIP (T818A) does not bind to chromatin or initiate resection. Our data support a model in which ATM activity is required for an early step in resection, leading to ATR activation, CtIP-T818 phosphorylation, and accumulation of CtIP on chromatin. Chromatin binding by modified CtIP precedes extensive resection and full checkpoint activation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins / antagonists & inhibitors
  • Cell Cycle Proteins / metabolism*
  • Cell Cycle Proteins / physiology
  • Cell Extracts / isolation & purification
  • Chromatin / metabolism
  • Conserved Sequence
  • DNA Breaks, Double-Stranded*
  • DNA Cleavage
  • DNA Repair
  • DNA-Binding Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Molecular Sequence Annotation
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Phosphorylation
  • Protein Kinase Inhibitors / pharmacology
  • Protein Processing, Post-Translational*
  • Protein Serine-Threonine Kinases / antagonists & inhibitors
  • Protein Serine-Threonine Kinases / metabolism*
  • Protein Serine-Threonine Kinases / physiology
  • Rabbits
  • Tumor Suppressor Proteins / chemistry
  • Tumor Suppressor Proteins / metabolism*
  • Xenopus Proteins / antagonists & inhibitors
  • Xenopus Proteins / chemistry
  • Xenopus Proteins / metabolism*
  • Xenopus Proteins / physiology
  • Xenopus laevis

Substances

  • Cell Cycle Proteins
  • Cell Extracts
  • Chromatin
  • DNA-Binding Proteins
  • Peptide Fragments
  • Protein Kinase Inhibitors
  • RBBP8 protein, Xenopus
  • Tumor Suppressor Proteins
  • Xenopus Proteins
  • Atr protein, Xenopus
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Protein Serine-Threonine Kinases