A PP4-phosphatase complex dephosphorylates gamma-H2AX generated during DNA replication

Mol Cell. 2008 Jul 11;31(1):33-46. doi: 10.1016/j.molcel.2008.05.016.

Abstract

The histone H2A variant H2AX is rapidly phosphorylated in response to DNA double-stranded breaks to produce gamma-H2AX. gamma-H2AX stabilizes cell-cycle checkpoint proteins and DNA repair factors at the break site. We previously found that the protein phosphatase PP2A is required to resolve gamma-H2AX foci and complete DNA repair after exogenous DNA damage. Here we describe a three-protein PP4 phosphatase complex in mammalian cells, containing PP4C, PP4R2, and PP4R3beta, that specifically dephosphorylates ATR-mediated gamma-H2AX generated during DNA replication. PP4 efficiently dephosphorylates gamma-H2AX within mononucleosomes in vitro and does not directly alter ATR or checkpoint kinase activity, suggesting that PP4 acts directly on gamma-H2AX in cells. When the PP4 complex is silenced, repair of DNA replication-mediated breaks is inefficient, and cells are hypersensitive to DNA replication inhibitors, but not radiomimetic drugs. Therefore, gamma-H2AX elimination at DNA damage foci is required for DNA damage repair, but accomplishing this task involves distinct phosphatases with potentially overlapping roles.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins / metabolism
  • Cell Division
  • Cell Line
  • Chromatin / metabolism
  • DNA Damage
  • DNA Replication*
  • Gene Silencing
  • Histones / metabolism*
  • Humans
  • Molecular Sequence Data
  • Multiprotein Complexes / metabolism*
  • Phosphoprotein Phosphatases / chemistry
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphorylation
  • Protein Phosphatase 2 / metabolism
  • Protein Subunits / metabolism
  • Protein Transport
  • Protein-Serine-Threonine Kinases / metabolism
  • Subcellular Fractions / metabolism

Substances

  • Cell Cycle Proteins
  • Chromatin
  • H2AX protein, human
  • Histones
  • Multiprotein Complexes
  • Protein Subunits
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Protein-Serine-Threonine Kinases
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2
  • protein phosphatase 4