Regulation of Mre11/Rad50 by Nbs1

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 ∼1.2 MDa. Models of M/R/N stoichiometry in light of this and previous data are discussed.

[1]  K. Cerosaletti,et al.  Nibrin Forkhead-associated Domain and Breast Cancer C-terminal Domain Are Both Required for Nuclear Focus Formation and Phosphorylation* , 2003, Journal of Biological Chemistry.

[2]  A. Aguilera,et al.  Equal sister chromatid exchange is a major mechanism of double-strand break repair in yeast. , 2003, Molecular cell.

[3]  E. Y. Lee,et al.  Functional analysis of FHA and BRCT domains of NBS1 in chromatin association and DNA damage responses. , 2002, Nucleic acids research.

[4]  T. Hirano,et al.  Hinge‐mediated dimerization of SMC protein is essential for its dynamic interaction with DNA , 2002, The EMBO journal.

[5]  K. Tanimoto,et al.  NBS1 Localizes to γ-H2AX Foci through Interaction with the FHA/BRCT Domain , 2002, Current Biology.

[6]  R. Kanaar,et al.  DNA end-binding specificity of human Rad50/Mre11 is influenced by ATP. , 2002, Nucleic acids research.

[7]  J. Tainer,et al.  The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair , 2002, Nature.

[8]  M. Weitzman,et al.  Adenovirus oncoproteins inactivate the Mre11–Rad50–NBS1 DNA repair complex , 2002, Nature.

[9]  S. Jackson,et al.  The MRE11 complex: at the crossroads of DNA repair and checkpoint signalling , 2002, Nature Reviews Molecular Cell Biology.

[10]  William F. Morgan,et al.  A Murine Model of Nijmegen Breakage Syndrome , 2002, Current Biology.

[11]  Kim Nasmyth,et al.  Molecular architecture of SMC proteins and the yeast cohesin complex. , 2002, Molecular cell.

[12]  D. Gordenin,et al.  The Mre11 Complex Is Required for Repair of Hairpin-Capped Double-Strand Breaks and Prevention of Chromosome Rearrangements , 2002, Cell.

[13]  A. Tomkinson,et al.  Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. , 2001, Molecular cell.

[14]  C. Dekker,et al.  Human Rad50/Mre11 is a flexible complex that can tether DNA ends. , 2001, Molecular cell.

[15]  H. Erickson,et al.  Structure of the Rad50·Mre11 DNA Repair Complex fromSaccharomyces cerevisiae by Electron Microscopy* , 2001, The Journal of Biological Chemistry.

[16]  S. Jackson,et al.  The yeast Xrs2 complex functions in S phase checkpoint regulation. , 2001, Genes & development.

[17]  C. Gilbert,et al.  Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex , 2001, Nature Cell Biology.

[18]  John A. Tainer,et al.  Structural Biochemistry and Interaction Architecture of the DNA Double-Strand Break Repair Mre11 Nuclease and Rad50-ATPase , 2001, Cell.

[19]  R. S. Maser,et al.  An alternative mode of translation permits production of a variant NBS1 protein from the common Nijmegen breakage syndrome allele , 2001, Nature Genetics.

[20]  K. Cerosaletti,et al.  Distinct Functional Domains of Nibrin Mediate Mre11 Binding, Focus Formation, and Nuclear Localization , 2001, Molecular and Cellular Biology.

[21]  D. Delia,et al.  The Forkhead-associated Domain of NBS1 Is Essential for Nuclear Foci Formation after Irradiation but Not Essential for hRAD50·hMRE11·NBS1 Complex DNA Repair Activity* , 2000, The Journal of Biological Chemistry.

[22]  J. Tainer,et al.  Mre11 and Rad50 from Pyrococcus furiosus: Cloning and Biochemical Characterization Reveal an Evolutionarily Conserved Multiprotein Machine , 2000, Journal of bacteriology.

[23]  John A. Tainer,et al.  Structural Biology of Rad50 ATPase ATP-Driven Conformational Control in DNA Double-Strand Break Repair and the ABC-ATPase Superfamily , 2000, Cell.

[24]  T. Paull,et al.  A mechanistic basis for Mre11-directed DNA joining at microhomologies. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Y. Shiloh,et al.  Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products , 2000, Nature.

[26]  D. Livingston,et al.  ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response , 2000, Nature.

[27]  M. Gatei,et al.  ATM-dependent phosphorylation of nibrin in response to radiation exposure , 2000, Nature Genetics.

[28]  M. Kastan,et al.  ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway , 2000, Nature.

[29]  T. Stankovic,et al.  The DNA Double-Strand Break Repair Gene hMRE11 Is Mutated in Individuals with an Ataxia-Telangiectasia-like Disorder , 1999, Cell.

[30]  J. Petrini,et al.  The Mre11-Rad50-Xrs2 Protein Complex Facilitates Homologous Recombination-Based Double-Strand Break Repair inSaccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[31]  T. Paull,et al.  Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex. , 1999, Genes & development.

[32]  J. Haber,et al.  The Many Interfaces of Mre11 , 1998, Cell.

[33]  T. Shibata,et al.  Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination , 1998, The EMBO journal.

[34]  P. Sung,et al.  Nuclease Activities in a Complex of Human Recombination and DNA Repair Factors Rad50, Mre11, and p95* , 1998, The Journal of Biological Chemistry.

[35]  L. Kirkham,et al.  The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[36]  T. Paull,et al.  The 3' to 5' exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks. , 1998, Molecular cell.

[37]  Matthias Platzer,et al.  Nibrin, a Novel DNA Double-Strand Break Repair Protein, Is Mutated in Nijmegen Breakage Syndrome , 1998, Cell.

[38]  John R Yates,et al.  The hMre11/hRad50 Protein Complex and Nijmegen Breakage Syndrome: Linkage of Double-Strand Break Repair to the Cellular DNA Damage Response , 1998, Cell.

[39]  S. Jackson,et al.  Components of the Ku‐dependent non‐homologous end‐joining pathway are involved in telomeric length maintenance and telomeric silencing , 1998, The EMBO journal.

[40]  K. Muniyappa,et al.  Alteration of telomeric sequences and senescence caused by mutations in RAD50 of Saccharomyces cerevisiae , 1997, Genes to cells : devoted to molecular & cellular mechanisms.

[41]  J. Haber,et al.  Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae , 1996, Molecular and cellular biology.

[42]  R. Ghirlando,et al.  Stoichiometry and thermodynamics of the interaction between the Fc fragment of human IgG1 and its low-affinity receptor Fc gamma RIII. , 1995, Biochemistry.

[43]  H. Ogawa,et al.  Interaction of Mre11 and Rad50: two proteins required for DNA repair and meiosis-specific double-strand break formation in Saccharomyces cerevisiae. , 1995, Genetics.

[44]  N. Kleckner,et al.  RAD50 protein of S.cerevisiae exhibits ATP-dependent DNA binding. , 1993, Nucleic acids research.

[45]  F. Fabre,et al.  XRS2, a DNA repair gene of Saccharomyces cerevisiae, is needed for meiotic recombination. , 1992, Genetics.

[46]  R. Padmore,et al.  Analysis of wild-type and rad50 mutants of yeast suggests an intimate relationship between meiotic chromosome synapsis and recombination , 1990, Cell.

[47]  Y. Shiloh,et al.  Ataxia-telangiectasia and the Nijmegen breakage syndrome: related disorders but genes apart. , 1997, Annual review of genetics.

[48]  M. Ajimura,et al.  Identification of new genes required for meiotic recombination in Saccharomyces cerevisiae. , 1993, Genetics.