ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1

Mol Cell Biol. 2005 Jul;25(13):5363-79. doi: 10.1128/MCB.25.13.5363-5379.2005.

Abstract

ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Here we show that two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. We conclude that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. We propose that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage.

Publication types

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

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / metabolism*
  • Cell Extracts
  • Chromosomal Proteins, Non-Histone / chemistry*
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism*
  • Conserved Sequence
  • DNA Damage / radiation effects*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • Female
  • Fungal Proteins / chemistry*
  • Fungal Proteins / metabolism*
  • Humans
  • Models, Biological
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Oocytes
  • Phosphorylation
  • Protein Conformation
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / metabolism*
  • Schizosaccharomyces pombe Proteins / chemistry*
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism*
  • Tumor Suppressor Proteins / chemistry
  • Tumor Suppressor Proteins / metabolism*
  • Two-Hybrid System Techniques
  • Xenopus
  • Xenopus Proteins

Substances

  • Cell Cycle Proteins
  • Cell Extracts
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • Fungal Proteins
  • NBN protein, Xenopus
  • Nbs1 protein, S pombe
  • Schizosaccharomyces pombe Proteins
  • Tumor Suppressor Proteins
  • Xenopus Proteins
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Protein-Serine-Threonine Kinases