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

2005; Elsevier BV; Volume: 280; Issue: 19 Linguagem: Inglês

10.1074/jbc.m413387200

ISSN

1083-351X

Autores

Jodi R. Alt, Alyssa Bouska, Mario R. Fernandez, Ronald L. Cerny, Hua Xiao, Christine M. Eischen,

Tópico(s)

Epigenetics and DNA Methylation

Resumo

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. 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. The Mre11, Nbs1, and Rad50 proteins form a complex (the M-N-R 1The abbreviations used are: M-N-R, Mre11-Nbs1-Rad50; TRITC, tetramethylrhodamine isothiocyanate; NBS, Nijmegen breakage syndrome; A-T, ataxia telangiectasia; ATLD, A-T-like disorder; ATM, ataxia telangiectasia mutated; MEF, mouse embryo fibroblast; MS, mass spectrometry; HA, hemagglutinin; GFP, green fluorescent protein; Gy, gray; E3, ubiquitin-protein isopeptide ligase; ARF, alternative reading frame of the Ink4alocus; GST, glutathione S-transferase; MSCV, murine stem cell virus; IRES, internal ribosome entry site. complex) that is essential in maintaining DNA integrity by functioning in double strand break repair, meiotic recombination, and telomere maintenance (1D'Amours D. Jackson S.P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 317-327Crossref PubMed Scopus (718) Google Scholar). Mre11 is the catalytic subunit of the complex with 3′–5′ exonuclease, single-stranded DNA endonuclease, and DNA unwinding activities (2Paull T.T. 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If DNA is unrepaired, p53-dependent apoptotic signaling pathways are activated. Mdm2 regulates p53 by binding to and inhibiting p53-dependent transcription (20Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2800) Google Scholar). In addition, Mdm2 ubiquitylates p53 (21Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1604) Google Scholar), targeting p53 for proteosomal degradation (22Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3750) Google Scholar, 23Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2860) Google Scholar). Mdm2 also functions to shuttle p53 out of the nucleus into the cytoplasm (24Roth J. Dobbelstein M. Freedman D.A. Shenk T. Levine A.J. EMBO J. 1998; 17: 554-564Crossref PubMed Scopus (533) Google Scholar). Genetic evidence that Mdm2 restricts p53 function was revealed when the early embryonic lethality of Mdm2–/– mice was rescued with loss of p53 (25Jones S.N. Roe A.E. Donehower L.A. Bradley A. Nature. 1995; 378: 206-208Crossref PubMed Scopus (1071) Google Scholar, 26Montes de Oca Luna R. Wagner D.S. Lozano G. Nature. 1995; 378: 203-206Crossref PubMed Scopus (1211) Google Scholar). The multifaceted regulation of p53 by Mdm2 is controlled by the tumor suppressor, p14ARF(human)/p19ARF(mouse), which binds to and inhibits Mdm2 (27Weber J.D. Taylor L.J. Roussel M.F. Sherr C.J. Bar-Sagi D. Nat. Cell Biol. 1999; 1: 20-26Crossref PubMed Scopus (807) Google Scholar). Recent evidence suggests that threshold levels of p53, Mdm2, and ARF are required to maintain a proper balance between apoptosis and cancer development (28Alt J.R. Greiner T.C. Cleveland J.L. Eischen C.M. EMBO J. 2003; 22: 1442-1450Crossref PubMed Scopus (104) Google Scholar, 29O'Leary K.A. Mendrysa S.M. Vaccaro A. Perry M.E. Mol. Cell. Biol. 2004; 24: 186-191Crossref PubMed Scopus (26) Google Scholar, 30Eischen C.M. Alt J.R. Wang P. Oncogene. 2004; 23: 8931-8940Crossref PubMed Scopus (32) Google Scholar). In addition to p53-dependent functions, mounting evidence suggests Mdm2 also acts independent of p53. Mdm2 transgenic mice lacking p53 have an increased incidence of malignancies as compared with mice deficient in p53 alone (31Jones S.N. Hancock A.R. Vogel H. Donehower L.A. Bradley A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15608-15612Crossref PubMed Scopus (318) Google Scholar). Mice overexpressing Mdm2 in breast epithelial cells had increased numbers of polyploid cells regardless of whether p53 was present or absent (32Lundgren K. Montes de Oca Luna R. McNeill Y.B. Emerick E.P. Spencer B. Barfield C.R. Lozano G. Rosenberg M.P. Finlay C.A. Genes Dev. 1997; 11: 714-725Crossref PubMed Scopus (212) Google Scholar). Another report showed that expression of an alternatively spliced variant of Mdm2 increased the proliferation of p53-null MEFs and increased cancer incidence in mice (33Steinman H.A. Burstein E. Lengner C. Gosselin J. Pihan G. Duckett C.S. Jones S.N. J. Biol. Chem. 2004; 279: 4877-4886Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Additionally, lymphomas arising in humans and mice that have inactivated p53 also frequently overexpress Mdm2 protein (28Alt J.R. Greiner T.C. Cleveland J.L. Eischen C.M. EMBO J. 2003; 22: 1442-1450Crossref PubMed Scopus (104) Google Scholar, 34Eischen C.M. Weber J.D. Roussel M.F. Sherr C.J. Cleveland J.L. Genes Dev. 1999; 13: 2658-2669Crossref PubMed Scopus (712) Google Scholar, 35Watanabe T. Ichikawa A. Saito H. Hotta T. Leuk. Lymphoma. 1996; 21: 391-397Crossref PubMed Scopus (65) Google Scholar). Finally, tumor cells lacking p53 died when treated with Mdm2 antisense (36Wang H. Yu D. Agrawal S. Zhang R. Prostate. 2003; 54: 194-205Crossref PubMed Scopus (84) Google Scholar). Combined, these reports suggest that Mdm2 has functions independent of p53 that contribute to transformation. Therefore, we sought to identify novel Mdm2-binding proteins that influenced tumor development independent of p53. We determined that Mdm2 bound to the M-N-R DNA repair complex in a p53-independent manner at sites of DNA double strand breaks and that Mdm2 inhibited efficient DNA repair, which was dependent on the Nbs1 binding domain in Mdm2. This finding suggests a novel role of Mdm2 in compromising DNA integrity. Silver Staining and Mass Spectrometry—HeLa cells were Dounce-homogenized in complete lysis buffer (20 mm Tris-HCl, pH 7.3, 300 mm KCl, 0.2 mm EDTA, 0.1% Nonidet P-40, 20% glycerol, 1 mm phenylmethylsulfonyl fluoride, 0.4 units/ml aprotinin, 1 mm NaF, 10 mm β-glycerophosphate, and 0.1 mm Na3VO4). Total cellular protein (60 mg) was rotated with anti-Mdm2-conjugated beads (SMP14; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 °C, washed in complete lysis buffer, and separated by SDS-PAGE. Following silver staining, protein bands were excised and digested as described previously with slight modifications (37Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7831) Google Scholar). After separation on a reversed phase LC column, eluted peptides were analyzed on a Q-TOF Ultima tandem mass spectrometer with electrospray ionization (Micromass/Waters, Toronto, Canada). The MS/MS data were processed using Masslynx software (Micromass), and the MASCOT (Matrix Science, London, UK) search engine was used to search the NCBI nonredundant data base. Protein identifications were based on a minimum random probability score of 25 and with a mass accuracy of 0.1 daltons. Cell Culture Conditions—HeLa, 293T, MCF7, MDA-MB-231, HT1080, HCT116, CLL, K562, NIH3T3, and IMR90 cell lines were cultured as described by the American Type Culture Collection (Manassas, VA). p53–/–ARF–/–, p53–/–Mdm2–/–, and p53–/– MEFs were isolated as described previously (38Zindy F. Eischen C.M. Randle D.H. Kamijo T. Cleveland J.L. Sherr C.J. Roussel M.F. Genes Dev. 1998; 12: 2424-2433Crossref PubMed Scopus (1063) Google Scholar) and maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum (Atlanta Biologicals, Atlanta, GA) and nonessential amino acids. Saos-2 cells (provided by Dr. Gerard Zambetti, St. Jude Children's Research Hospital, Memphis, TN) were grown as indicated by ATCC. The HCC1937 (a gift from Dr. Kenneth Cowan, Eppley Institute, Omaha, NE) and ATLD3 cell lines (generously provided by Dr. Matthew Weitzman, Salk Institute, La Jolla, CA) were maintained in Dulbecco's modified Eagle's medium with 20% fetal calf serum. GM11261 (A-T lymphoblast), GM07166 fibroblasts (NBS), and GM07078 lymphoblasts (NBS) were purchased from the Coriell Institute (Camden, NJ) and cultured as their protocols indicated. Generation, Expression, and Purification of Mdm2 and Nbs1 Proteins—Wild-type murine Mdm2 cDNA (generously provided by Dr. Martine Roussel, St. Jude Children's Research Hospital) was subcloned into the pGex2T vector (Amersham Biosciences), and wild-type human Nbs1 cDNA (kindly provided by Dr. Tanya Paull, University of Texas, Austin, TX) was subcloned into the pET28B vector (Novagen, San Diego, CA). GST, GST-Mdm2, and GST-Nbs1 were purified from DH5α or BL21 Escherichia coli cells. GST and GST-Mdm2 bacterial supernatant was incubated with glutathione-Sepharose beads (Amersham Biosciences), washed in phosphate-buffered saline containing 1 m NaCl2, and stored at –80 °C for use in in vitro binding assays. For GST-Nbs1 and GST used in the Nbs1 reconstitution experiment (see below), glutathione-Sepharose beads (Amersham Biosciences) were packed to a chromatography column (Bio-Rad), and GST-Nbs1 or GST bacterial supernatant was added. Following incubation, the column was washed with phosphate-buffered saline, and GST-Nbs1 and GST purified protein were eluted as fractions with 10 mm glutathione. Fractions were separated by SDS-PAGE, and proteins were stained with Coomassie Brilliant Blue (Fisher) to estimate protein concentration. Mammalian Vector Construction and Retroviral Infection—Murine wild-type (amino acids 1–489) and deletion mutant murine Mdm2 constructs (amino acids 198–489, 298–489, 198–400, and 349–489) (generously provided by Dr. Martine Roussel) were subcloned into pJ3H vector for expression of a hemagglutinin (HA) tag at the N terminus. Wild-type murine Mdm2 was used in PCR amplification to generate other HA-Mdm2 mutants (1–192, 1–314, and 198–314). Wild-type human Nbs1 cDNA (provided by Dr. John Petrini, Memorial Sloan Kettering, New York) and human Nbs1 mutants generated by restriction digestion were cloned into pCMVTag4 or pCMVTag2 (Stratagene, La Jolla, CA) to generate FLAG-tagged proteins. HA- and FLAG-tagged wild-type and mutant constructs were cloned into pCDNA3 vector (Invitrogen) for expression in mammalian cells. Murine cells (NIH3T3 and p53–/– MEFs) were infected with an MSCV-IRES-green fluorescence protein (GFP) retrovirus (from Dr. Robert Hawley) encoding wild-type Mdm2, mutant Mdm2-(198–489), mutant Mdm2-(298–489) (gifts from Dr. Martine Roussel), or a control empty vector, as previously reported (38Zindy F. Eischen C.M. Randle D.H. Kamijo T. Cleveland J.L. Sherr C.J. Roussel M.F. Genes Dev. 1998; 12: 2424-2433Crossref PubMed Scopus (1063) Google Scholar, 39Dang J. Kuo M.L. Eischen C.M. Stepanova L. Sherr C.J. Roussel M.F. Cancer Res. 2002; 62: 1222-1230PubMed Google Scholar). Cells were analyzed by flow cytometry immediately prior to analysis for GFP expression, which is an indicator of protein expression from the bicistronic retroviral vector, and only cell populations that were greater than 90% GFP-positive were utilized for analyses. Immunoprecipitation and Western Blotting—HA-tagged Mdm2 and/or FLAG-tagged Nbs1 constructs were transfected into 293T cells, and cells were collected for analysis 36 h later. For Western blots, all cells were Dounce-homogenized in EBC buffer (50 mm Tris-HCl, pH 7.5, 120 mm NaCl2, 1 mm EDTA, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 0.4 units/ml aprotinin, 1 mm NaF, 10 mm β-glycerophosphate, and 0.1 mm Na3VO4). Equal amounts of protein were immunoprecipitated at 4 °C with antibodies specific to Mdm2 (SMP14; Santa Cruz Biotechnology), HA (HA-probe, F-7; Santa Cruz Biotechnology), FLAG (M2; Sigma), Mre11, Rad50 (both from Novus Biologicals, Littleton, CO), or isotype control antibody (Santa Cruz Biotechnology). For wild-type Nbs1 reconstitution experiments (Fig. 4), 1 μg of purified GST or GST-Nbs1 protein was added to NBS fibroblast cell lysate, and Mdm2 was immunoprecipitated with an anti-Mdm2 antibody (SMP14). For nuclear and cytoplasmic extracts, HeLa cell lysates were prepared as previously described (40Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar) and then frozen at–80 °C. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Protran; Schleicher & Schuell). Membranes were serially Western blotted with antibodies specific for HA (F-7; Santa Cruz Biotechnology), FLAG (M2) Mdm2 (SMP14), Rad50 (Novus Biologicals), Mre11 (Novus Biologicals or Oncogene Research, Boston, MA), Nbs1 (Novus Biologicals or BD Transduction Laboratories, San Jose, CA), and β-actin (Sigma). Horseradish peroxidase-linked secondary antibodies (Amersham Biosciences) and ECL (Amersham Biosciences) or Supersignal (Pierce) were used to detect bound immunocomplexes. In Vitro Transcription/Translation and Binding Experiments—Plasmids encoding p53, Bax, Nbs1, and Mre11 (generously provided by Drs. Arnold J. Levine (Princeton University, Princeton, NJ), Xu Luo (University of Nebraska Medical Center, Omaha, NE), Tanya Paull (University of Texas, Austin, TX), and John Petrini (Cornell University, Memorial Sloan Kettering Cancer Center, New York), respectively) were used to generate [35S]Met-labeled proteins by in vitro transcription and translation using TNT-coupled reticulocyte lysate systems (Promega, Madison, WI). In vitro binding assays were performed in binding buffer (20 mm Tris-HCl, pH 7.3, 100 mm KCl, 0.2 mm EDTA, 0.1% Nonidet P-40, 20% glycerol). GST or GST-Mdm2 bacterial lysates bound to glutathione-Sepharose beads were incubated with [35S]Metlabeled proteins at 4 °C. Samples were washed five times, and bound proteins were separated by SDS-PAGE. Gels were dried, and 35S activity (decays/min/mm2) was quantified on a Storm PhosphorImager (Amersham Biosciences). Immunofluorescence—Primary human (IMR90) and NBS fibroblasts were grown on glass coverslips and fixed in methanol/acetone 6–8 h following γ-irradiation (12 Gy) with a cesium-137 source. Fixed cells were incubated overnight at 4 °C in phosphate-buffered saline containing 10% fetal calf serum (Atlanta Biologicals). Following incubation with antibodies specific for Rad50 (1:150; Novus Biologicals), Nbs1 (1:150; Novus Biologicals), Mdm2 (1:1000; SMP14), and/or p53 (1:400; DO-1; Santa Cruz Biotechnology), fixed cells were washed and incubated with fluorescein isthiocyanate-conjugated donkey anti-rabbit and TRITC-conjugated donkey anti-mouse antibodies (1:200; Jackson Laboratories, West Grove, PA). Coverslips were mounted to slides with Vectashield Mounting Media containing 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Cells were imaged on a Zeiss LSM 410 confocal laser-scanning microscope (Goettinger, Germany) with ×40 or ×63 magnification. DNA Damage Quantitation—Following infection (36 h) of NIH3T3 cells or p53–/– MEFs with wild-type Mdm2, Mdm2-(198–489), Mdm2-(298–489), or Mdm2-(198–400) encoded or empty MSCV-IRES-GFP retroviruses (see above), cells were γ-irradiated with 5 Gy from a cesium-137 source. Cells were incubated at 37 °C for specific intervals and harvested for comet assay analysis. Comet assays were performed under neutral conditions to optimize for detection of DNA double strand breaks, as directed by the manufacturer's protocol (Trevigen, Gaithersburg, MD). Samples were blinded, and DNA was stained with ethidium bromide. Multiple pictures per sample were taken by fluorescence microscopy (Nikon E600, Melville, NY) using a TRITC-HYQ filter. DNA damage quantitation was performed by computer scoring at least 50 cells per interval per cell line with TriTekCometScore™ software (TriTek Corp., Summerduck, VA). Tail moments were calculated by the software and represent the length of the comet tail multiplied by the percentage of DNA in the comet tail. Tail moment is a measurement of DNA double strand breaks (41Kobayashi H. Sugiyama C. Morikawa Y. Hayashi M. Sofuni T. MMS Commun. 1995; : 103-115Google Scholar, 42Collins A.R. Mol. Biotechnol. 2004; 26: 249-261Crossref PubMed Scopus (2196) Google Scholar). Following data collection and tabulation, codes identifying each set of pictures were broken to reveal the identity of each sample. The Mre11-Nbs1-Rad50 Complex Is Associated with Mdm2 Independent of p53—Mdm2 appears to have functions that are p53-independent (31Jones S.N. Hancock A.R. Vogel H. Donehower L.A. Bradley A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15608-15612Crossref PubMed Scopus (318) Google Scholar, 32Lundgren K. Montes de Oca Luna R. McNeill Y.B. Emerick E.P. Spencer B. Barfield C.R. Lozano G. Rosenberg M.P. Finlay C.A. Genes Dev. 1997; 11: 714-725Crossref PubMed Scopus (212) Google Scholar, 33Steinman H.A. Burstein E. Lengner C. Gosselin J. Pihan G. Duckett C.S. Jones S.N. J. Biol. Chem. 2004; 279: 4877-4886Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 34Eischen C.M. Weber J.D. Roussel M.F. Sherr C.J. Cleveland J.L. Genes Dev. 1999; 13: 2658-2669Crossref PubMed Scopus (712) Google Scholar, 36Wang H. Yu D. Agrawal S. Zhang R. Prostate. 2003; 54: 194-205Crossref PubMed Scopus (84) Google Scholar), yet the pathways that mediate these activities are unclear. To uncover potentially novel Mdm2 oncogenic pathways, we isolated and identified proteins that specifically bound to Mdm2 independent of p53. Utilizing unfractionated HeLa cell extracts that express the papillomavirus E6 protein, which inactivates p53, endogenous Mdm2 was immunoprecipitated with an Mdm2-specific antibody. In Mdm2 immunoprecipitates, polypeptides of 200, 150, 115, 95, and 80 kDa were readily evident in silver-stained gels, whereas polypeptides of this size were absent in isotype control immunoprecipitations (Fig. 1A and data not shown). The 150-, 95-, and 80-kDa protein bands were excised, trypsin-digested, and subjected to mass spectrometry analysis. Seventeen peptides from the 150-kDa band, 21 peptides from the 95-kDa band, and nine peptides from the 80-kDa band matched sequences in Rad50, Nbs1, and Mre11, respectively (Fig. 1A). These three proteins comprise the M-N-R complex that is responsible for repairing double strand DNA breaks (1D'Amours D. Jackson S.P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 317-327Crossref PubMed Scopus (718) Google Scholar). To verify that the M-N-R complex was associated with Mdm2, Mdm2 and its associated proteins were immunoprecipitated from HeLa cell extracts and Western blotted with antibodies specific for Nbs1, Mre11, and Rad50. All three proteins co-immunoprecipitated with the same Mdm2-specific antibody used in the previous immunoprecipitations but not with an isotype control antibody (Fig. 1B). To obtain further confirmation of the interaction between Mdm2 and the M-N-R complex, we overexpressed HA-tagged wild-type murine Mdm2 in 293T cells, which also lack functional p53. Nbs1, Mre11, and Rad50 were clearly visible in HA immunoprecipitations and absent in isotype control immunoprecipitations (Fig. 1C). For unclear reasons, we were unable to detect Mdm2 protein in immunoprecipitations of endogenous Nbs1, Mre11, and Rad50. However, when co-transfections of FLAG-tagged Nbs1 and HA-tagged Mdm2 into 293T cells were performed, Mdm2 was detected in anti-FLAG immunoprecipitations and absent in isotype control immunoprecipitations (Fig. 1D). Thus, four lines of evidence indicate that the M-N-R complex can specifically associate with Mdm2 and that this interaction appears to occur in cells that lack functional p53. To further test whether p53 or the Mdm2 regulator ARF is required for the association of Mdm2 with the M-N-R complex, we evaluated multiple cell lines. Nbs1, Mre11, and Rad50 co-immunoprecipitated with Mdm2 in p53–/–ARF–/– MEFs, whereas in immunoprecipitations with the control p53–/–-Mdm2–/– MEFs, none of the three proteins were detected (Fig. 2A). The slight differences in Nbs1, Mre11, and Rad50 expression observed between the two genotypes of MEFs were due to a small difference in protein loading (see β-actin; Fig. 2A) and do not represent differences of the endogenous levels of these proteins in the different MEFs analyzed. Evaluation of human (MDA-MB-231, 293T, Saos-2, CLL, and K562) and murine tumor cell lines that lack functional p53 showed that Mdm2 was associated with the M-N-R complex (Fig. 2B, lanes 2, 4, 5, 8, and 9; and data not shown). Similarly, in whole cell lysates from human and mouse cells lacking ARF expression (MCF7, HT1080, HCT116, and NIH3T3), Mdm2 co-immunoprecipitated Nbs1, Mre11, and Rad50 (Fig. 2B lanes 1, 6, and 7; data not shown). In addition, the M-N-R complex precipitated with Mdm2 in primary diploid human fibroblasts (IMR90; Fig. 2B lane 10) and in immortal murine fibroblasts (p53–/–ARF–/– MEFs; Fig. 2A), indicating that the association of Mdm2 with the M-N-R complex is not due to cellular transformation. The small variations in the levels of Nbs1, Mre11, and Rad50 co-immunoprecipitating with Mdm2 in the various cell lines (Fig. 2B) were repeatedly observed and appear to reflect differences in one or more of these proteins between the cell lines. Together, these results demonstrated that the interaction between Mdm2 and the M-N-R complex does not require

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