title: Regulation of Mre11/Rad50 by Nbs1: effects on nucleotide-dependent DNA binding and association with ataxia-telangiectasia-like disorder mutant complexes.
abstract: 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.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Texas, Austin, Texas 78712, USA.
authors.forename: Ji-Hoon
authors.initials: JH
authors.lastname: Lee
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ghirlando
authors.forename: Venugopal
authors.initials: V
authors.lastname: Bhaskara
authors.forename: Michaela R
authors.initials: MR
authors.lastname: Hoffmeyer
authors.forename: Jian
authors.initials: J
authors.lastname: Gu
authors.forename: Tanya T
authors.initials: TT
authors.lastname: Paull
mesh_terms: D017766:Acid Anhydride Hydrolases; D000483:Alleles; D016367:Baculoviridae / Q000378:metabolism; D001667:Binding, Competitive; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics* / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C112156:NBN protein, human; C478035:Nbs1 protein, S pombe; D009687:Nuclear Proteins; C086930:RAD50 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C094191:MRE11 protein, S cerevisiae; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes