Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes

Science. 2003 Jun 6;300(5625):1542-8. doi: 10.1126/science.1083430.

Abstract

The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins / metabolism*
  • Cell Nucleus / metabolism
  • Checkpoint Kinase 1
  • DNA Damage*
  • DNA Repair
  • DNA Replication
  • DNA, Fungal / metabolism
  • DNA, Single-Stranded / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Enzyme Activation
  • Exodeoxyribonucleases*
  • HeLa Cells
  • Humans
  • Mutation
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Binding
  • Protein Kinases / metabolism
  • Protein-Serine-Threonine Kinases*
  • RNA, Small Interfering
  • Radiation, Ionizing
  • Recombination, Genetic
  • Replication Protein A
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Signal Transduction
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transfection
  • Tumor Cells, Cultured
  • Ultraviolet Rays

Substances

  • ATRIP protein, human
  • Adaptor Proteins, Signal Transducing
  • Cell Cycle Proteins
  • DNA, Fungal
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • LCD1 protein, S cerevisiae
  • Phosphoproteins
  • RFA1 protein, S cerevisiae
  • RNA, Small Interfering
  • RPA1 protein, human
  • Rad17 protein, human
  • Replication Protein A
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Protein Kinases
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • CHEK1 protein, human
  • Checkpoint Kinase 1
  • Protein-Serine-Threonine Kinases
  • Exodeoxyribonucleases
  • three prime repair exonuclease 1