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 gamma-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.
Alt
Jodi R
JR
Eppley Institute for Research in Cancer and Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198, USA.
Bouska
Alyssa
A
Fernandez
Mario R
MR
Cerny
Ronald L
RL
Xiao
Hua
H
Eischen
Christine M
CM
D017766:Acid Anhydride Hydrolases; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005720:Gamma Rays; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D020014:K562 Cells; D000076228:MRE11 Homologue Protein; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D017386:Sequence Homology, Amino Acid; D016622:Silver Staining; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; C512689:Mre11a protein, mouse; C112156:NBN protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D005982:Glutathione Transferase; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes