Mdm2 Binds to Nbs1 at Sites of DNA Damage and Regulates Double Strand Break Repair*

Mdm2 directly regulates the p53 tumor suppressor. However, Mdm2 also has p53-independent activities, and the pathways that mediate these functions are unresolved. Here we report the identification of a specific association of Mdm2 with Mre11, Nbs1, and Rad50, a DNA double strand break repair complex. Mdm2 bound to the Mre11-Nbs1-Rad50 complex in primary cells and in cells containing inactivated p53 or p14/p19ARF, a regulator of Mdm2. Further analysis revealed that Mdm2 directly bound to Nbs1 but not to Mre11 or Rad50. Amino acids 198–314 of Mdm2 were required for Mdm2/Nbs1 association, and neither the N terminus forkhead-associated and breast cancer C-terminal domains nor the C terminus Mre11 binding domain of Nbs1 mediated the interaction of Nbs1 with Mdm2. Mdm2 co-localized with Nbs1 to sites of DNA damage following γ-irradiation. Notably, Mdm2 overexpression inhibited DNA double strand break repair, and this was independent of p53 and ARF, the alternative reading frame of the Ink4alocus. The delay in DNA repair imposed by Mdm2 required the Nbs1 binding domain of Mdm2, but the ubiquitin ligase domain in Mdm2 was dispensable. Therefore, Nbs1 is a novel p53-independent Mdm2 binding protein and links Mdm2 to the Mre11-Nbs1-Rad50-regulated DNA repair response.

[1]  S. Subbiah,et al.  The yeast RAD50 gene encodes a predicted 153-kD protein containing a purine nucleotide-binding domain and two large heptad-repeat regions. , 1989, Genetics.

[2]  J. Petrini,et al.  The cellular response to DNA double-strand breaks: defining the sensors and mediators. , 2003, Trends in cell biology.

[3]  C. Lilley,et al.  The Mre11 complex is required for ATM activation and the G2/M checkpoint , 2003, The EMBO journal.

[4]  J L Cleveland,et al.  Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. , 1998, Genes & development.

[5]  Michel C. Nussenzweig,et al.  Genomic Instability in Mice Lacking Histone H2AX , 2002, Science.

[6]  Guillermina Lozano,et al.  Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53 , 1995, Nature.

[7]  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.

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

[9]  C. M. Eischen,et al.  Loss of one allele of ARF rescues Mdm2 haploinsufficiency effects on apoptosis and lymphoma development , 2004, Oncogene.

[10]  A. Levine,et al.  The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation , 1992, Cell.

[11]  Guillermina Lozano,et al.  MDM2, an introduction. , 2003, Molecular cancer research : MCR.

[12]  Y. Shiloh,et al.  Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Yair Andegeko,et al.  Requirement of the MRN complex for ATM activation by DNA damage , 2003, The EMBO journal.

[14]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[15]  M. Oren,et al.  Mdm2 promotes the rapid degradation of p53 , 1997, Nature.

[16]  S. Elledge,et al.  Direct DNA binding by Brca1 , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Bibikova,et al.  Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication. , 2001, Molecular cell.

[18]  G. Wahl,et al.  Accelerated MDM2 auto‐degradation induced by DNA‐damage kinases is required for p53 activation , 2004, The EMBO journal.

[19]  Dong Yu,et al.  Experimental therapy of human prostate cancer by inhibiting MDM2 expression with novel mixed‐backbone antisense oligonucleotides: In vitro and in vivo activities and mechanisms , 2003, The Prostate.

[20]  J. Petrini,et al.  DNA Damage-Dependent Nuclear Dynamics of the Mre11 Complex , 2001, Molecular and Cellular Biology.

[21]  Ji-Hoon Lee,et al.  Direct Activation of the ATM Protein Kinase by the Mre11/Rad50/Nbs1 Complex , 2004, Science.

[22]  Ying Li,et al.  Stimulation of human DNA polymerase ϵ by MDM2 , 2003 .

[23]  T. Greiner,et al.  Mdm2 haplo‐insufficiency profoundly inhibits Myc‐induced lymphomagenesis , 2003, The EMBO journal.

[24]  J. Minna,et al.  Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier. , 1998, Cancer research.

[25]  H. Saito,et al.  Overexpression of the MDM2 oncogene in leukemia and lymphoma. , 1996, Leukemia & lymphoma.

[26]  Charles J. Sherr,et al.  Nucleolar Arf sequesters Mdm2 and activates p53 , 1999, Nature Cell Biology.

[27]  Y Taya,et al.  Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. , 1998, Science.

[28]  S. Elledge,et al.  Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. , 1999, Science.

[29]  S. Lowe,et al.  Control of apoptosis by p53 , 2003, Oncogene.

[30]  M. Lovett,et al.  A single ataxia telangiectasia gene with a product similar to PI-3 kinase. , 1995, Science.

[31]  Stephen N. Jones,et al.  An Alternative Splice Form of Mdm2 Induces p53-independent Cell Growth and Tumorigenesis* , 2004, Journal of Biological Chemistry.

[32]  M. Roussel,et al.  Disruption of the ARF-Mdm2-p53 tumor suppressor pathway in Myc-induced lymphomagenesis. , 1999, Genes & development.

[33]  Hirofumi Tanaka,et al.  Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53 , 1997, FEBS letters.

[34]  M. Roussel,et al.  The RING domain of Mdm2 can inhibit cell proliferation. , 2002, Cancer research.

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

[36]  Stephen N. Jones,et al.  Regulation of p53 stability by Mdm2 , 1997, Nature.

[37]  V. Yamazaki,et al.  A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage , 2000, Current Biology.

[38]  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.

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

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

[41]  Lawrence A. Donehower,et al.  Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53 , 1995, Nature.

[42]  R. Rothstein,et al.  Choreography of the DNA Damage Response Spatiotemporal Relationships among Checkpoint and Repair Proteins , 2004, Cell.

[43]  M. E. Perry,et al.  Mdm2 Regulates p53 Independently of p19ARF in Homeostatic Tissues , 2004, Molecular and Cellular Biology.

[44]  A. Collins,et al.  The comet assay for DNA damage and repair , 2004, Molecular biotechnology.

[45]  G. Lozano,et al.  Targeted expression of MDM2 uncouples S phase from mitosis and inhibits mammary gland development independent of p53. , 1997, Genes & development.

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

[47]  L. Donehower,et al.  Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[48]  Patrick E Carroll,et al.  Centrosome hyperamplification in human cancer: chromosome instability induced by p53 mutation and/or Mdm2 overexpression , 1999, Oncogene.

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

[50]  Bo Xu,et al.  ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway , 2000, Nature.

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

[52]  J. Petrini,et al.  Human Rad50 is physically associated with human Mre11: identification of a conserved multiprotein complex implicated in recombinational DNA repair , 1996, Molecular and cellular biology.

[53]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

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

[55]  A. Levine,et al.  Nucleo‐cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein , 1998, The EMBO journal.

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

[57]  C. Wang,et al.  Association of BRCA1 with the hRad50-hMre11-p95 complex and the DNA damage response. , 1999, Science.