Regulation of Mre11/Rad50 by Nbs1: effects on nucleotide-dependent DNA binding and association with ataxia-telangiectasia-like disorder mutant complexes

J Biol Chem. 2003 Nov 14;278(46):45171-81. doi: 10.1074/jbc.M308705200. Epub 2003 Sep 8.


The Mre11/Rad50 complex is a critical component of the cellular response to DNA double-strand breaks, in organisms ranging from archaebacteria to humans. In mammalian cells, Mre11/Rad50 (M/R) associates with a third component, Nbs1, that regulates its activities and is targeted by signaling pathways that initiate DNA damage-induced checkpoint responses. Mutations in the genes that encode Nbs1 and Mre11 are responsible for the human radiation sensitivity disorders Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively, which are characterized by defective checkpoint responses and high levels of chromosomal abnormalities. Here we demonstrate nucleotide-dependent DNA binding by the human M/R complex that requires the Nbs1 protein and is specific for double-strand DNA duplexes. Efficient DNA binding is only observed with non-hydrolyzable analogs of ATP, suggesting that ATP hydrolysis normally effects DNA release. The alleles of MRE11 associated with ATLD and the C-terminal Nbs1 polypeptide associated with NBS were expressed with the other components and found to form triple complexes except in the case of ATLD 3/4, which exhibits variability in Nbs1 association. The ATLD 1/2, ATLD 3/4, and p70 M/R/N complexes exhibit nucleotide-dependent DNA binding and exonuclease activity equivalent to the wild-type enzyme, although the ATLD complexes both show reduced activity in endonuclease assays. Sedimentation equilibrium analysis of the recombinant human complexes indicates that Mre11 is a stable dimer, Mre11 and Nbs1 form a 1:1 complex, and both M/R and M/R/N form large multimeric assemblies of approximately 1.2 MDa. Models of M/R/N stoichiometry in light of this and previous data are discussed.

Publication types

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

MeSH terms

  • Acid Anhydride Hydrolases
  • Alleles
  • Baculoviridae / metabolism
  • Binding, Competitive
  • Cell Cycle Proteins / genetics*
  • Cell Cycle Proteins / metabolism*
  • Chromosomal Proteins, Non-Histone / genetics*
  • Chromosomal Proteins, Non-Histone / metabolism*
  • DNA / metabolism
  • DNA Damage
  • DNA Repair Enzymes*
  • DNA-Binding Proteins / metabolism*
  • Dimerization
  • Electrophoresis, Polyacrylamide Gel
  • Endodeoxyribonucleases / metabolism*
  • Exodeoxyribonucleases / metabolism*
  • Exonucleases / metabolism
  • Gene Expression Regulation*
  • Humans
  • Models, Biological
  • Mutation
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism*
  • Plasmids / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Schizosaccharomyces pombe Proteins / genetics*
  • Schizosaccharomyces pombe Proteins / metabolism*
  • Ultracentrifugation


  • Cell Cycle Proteins
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • NBN protein, human
  • Nbs1 protein, S pombe
  • Nuclear Proteins
  • RAD50 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Schizosaccharomyces pombe Proteins
  • DNA
  • Endodeoxyribonucleases
  • Exodeoxyribonucleases
  • Exonucleases
  • MRE11 protein, S cerevisiae
  • Acid Anhydride Hydrolases
  • Rad50 protein, human
  • DNA Repair Enzymes