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

Received for publication, November 29, 2004, and in revised form, February 8, 2005Published, JBC Papers in Press, February 25, 2005, DOI 10.1074/jbc.M413387200

Jodi R. Alt‡§, Alyssa Bouska§, Mario R. Fernandez‡, Ronald L. Cerny¶, Hua Xiao‡§,and Christine M. Eischen‡§�

From the ‡Eppley Institute for Research in Cancer and the §Department of Pathology and Microbiology, University ofNebraska Medical Center, Omaha, Nebraska 68198 and the ¶Department of Chemistry, University of Nebraska,Lincoln, Nebraska 68588

Mdm2 directly regulates the p53 tumor suppressor.However, Mdm2 also has p53-independent activities,and the pathways that mediate these functions are un-resolved. Here we report the identification of a specificassociation of Mdm2 with Mre11, Nbs1, and Rad50, aDNA double strand break repair complex. Mdm2 boundto the Mre11-Nbs1-Rad50 complex in primary cells andin cells containing inactivated p53 or p14/p19ARF, a reg-ulator of Mdm2. Further analysis revealed that Mdm2directly bound to Nbs1 but not to Mre11 or Rad50. Aminoacids 198–314 of Mdm2 were required for Mdm2/Nbs1association, and neither the N terminus forkhead-asso-ciated and breast cancer C-terminal domains nor the Cterminus Mre11 binding domain of Nbs1 mediated theinteraction of Nbs1 with Mdm2. Mdm2 co-localized withNbs1 to sites of DNA damage following �-irradiation.Notably, Mdm2 overexpression inhibited DNA doublestrand break repair, and this was independent of p53and ARF, the alternative reading frame of theInk4alocus. The delay in DNA repair imposed by Mdm2required the Nbs1 binding domain of Mdm2, but theubiquitin ligase domain in Mdm2 was dispensable.Therefore, Nbs1 is a novel p53-independent Mdm2 bind-ing protein and links Mdm2 to the Mre11-Nbs1-Rad50-regulated DNA repair response.

The Mre11, Nbs1, and Rad50 proteins form a complex (theM-N-R1 complex) that is essential in maintaining DNA integ-

rity by functioning in double strand break repair, meiotic re-combination, and telomere maintenance (1). Mre11 is the cat-alytic subunit of the complex with 3�–5� exonuclease, single-stranded DNA endonuclease, and DNA unwinding activities(2). Mre11 binds both Nbs1 and Rad50 (3, 4). Rad50 is an SMC(structural maintenance of chromosome) family member and,with its ATPase motifs, provides the energy source for theM-N-R complex (2, 5). A forkhead-associated domain and abreast cancer C-terminal domain at the N terminus of Nbs1 arecritical for proper nuclear localization of the M-N-R complex tonuclear foci or sites of DNA strand breaks (6). The breastcancer tumor suppressor protein BRCA1 binds to Rad50 atnuclear foci and reportedly inhibits nuclease activity of theM-N-R complex (7, 8). Therefore, a multimeric complex, includ-ing Mre11, Nbs1, Rad50, and other proteins, is necessary torepair broken DNA. However, there is currently a paucity ofinformation on the regulation of the M-N-R complex and thefunction of the proteins at nuclear foci.

The biological significance of the M-N-R complex in main-taining DNA integrity was revealed with the identification ofhuman genetic disorders resulting from mutations in Mre11and Nbs1 (4, 9, 10). Humans with mutations in Nbs1 developNijmegen breakage syndrome (NBS) and have a very highincidence of cancer (4, 10, 11). An ataxia telangiectasia (A-T)-like disorder (ATLD) develops in individuals with Mre11 mu-tations (9). This disorder is similar to that seen in humansharboring mutations in ataxia telangiectasia mutated (ATM)(12), a protein that is required for phosphorylation of Nbs1,Mre11, the p53 tumor suppressor, and the p53 regulator Mdm2following �-irradiation (1, 13–18). Cells isolated from patientswith the NBS, ATLD, or A-T disorders exhibit radiation sensi-tivity, chromosomal instability, and defective cell cycle check-points (1).

The p53 tumor suppressor is critical for the checkpoint re-sponse to DNA damage by blocking cell cycle progression untilrepairs are made (19). If DNA is unrepaired, p53-dependentapoptotic signaling pathways are activated. Mdm2 regulatesp53 by binding to and inhibiting p53-dependent transcription(20). In addition, Mdm2 ubiquitylates p53 (21), targeting p53for proteosomal degradation (22, 23). Mdm2 also functions toshuttle p53 out of the nucleus into the cytoplasm (24). Geneticevidence that Mdm2 restricts p53 function was revealed whenthe early embryonic lethality of Mdm2�/� mice was rescuedwith loss of p53 (25, 26). The multifaceted regulation of p53 byMdm2 is controlled by the tumor suppressor, p14ARF(human)/p19ARF(mouse), which binds to and inhibits Mdm2 (27). Recentevidence suggests that threshold levels of p53, Mdm2, and ARFare required to maintain a proper balance between apoptosisand cancer development (28–30).

In addition to p53-dependent functions, mounting evidence

* The research was supported by NCI, National Institutes of Health(NIH), Grant CA09139 (to C. M. E.), NCI Training Grant CA09476 (toJ. R. A. and M. R. F), American Cancer Society Research Scholar GrantRSG0216501GMC (to H. X.), Nebraska Cancer and Smoking DiseaseResearch Program 04-11, the Eppley Institute for Research in Cancer,and the Wanda Rizzo Memorial Fund. The mass spectrometry facility issupported in part by NIH Grant P20 RR15635 from the COBRE pro-gram of the National Center for Research Resources, NCI, NIH, CancerCenter Support Grant P30 CA36727, and the Nebraska Research Ini-tiative. The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

� A Leukemia and Lymphoma Society Scholar. To whom correspond-ence should be addressed: Eppley Institute for Research in Cancer,987696 Nebraska Medical Center, Omaha, NE 68198. Tel.: 402-559-3894; Fax: 402-559-3739; E-mail: ceischen@unmc.edu.

1 The abbreviations used are: M-N-R, Mre11-Nbs1-Rad50; TRITC,tetramethylrhodamine isothiocyanate; NBS, Nijmegen breakage syn-drome; 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 pro-tein; Gy, gray; E3, ubiquitin-protein isopeptide ligase; ARF, alternativereading frame of the Ink4alocus; GST, glutathione S-transferase;MSCV, murine stem cell virus; IRES, internal ribosome entry site.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 19, Issue of May 13, pp. 18771–18781, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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suggests Mdm2 also acts independent of p53. Mdm2 transgenicmice lacking p53 have an increased incidence of malignanciesas compared with mice deficient in p53 alone (31). Mice over-expressing Mdm2 in breast epithelial cells had increased num-bers of polyploid cells regardless of whether p53 was present orabsent (32). Another report showed that expression of an alter-natively spliced variant of Mdm2 increased the proliferation ofp53-null MEFs and increased cancer incidence in mice (33).Additionally, lymphomas arising in humans and mice thathave inactivated p53 also frequently overexpress Mdm2 pro-tein (28, 34, 35). Finally, tumor cells lacking p53 died whentreated with Mdm2 antisense (36). Combined, these reportssuggest that Mdm2 has functions independent of p53 thatcontribute to transformation. Therefore, we sought to identifynovel Mdm2-binding proteins that influenced tumor develop-ment independent of p53. We determined that Mdm2 bound tothe M-N-R DNA repair complex in a p53-independent mannerat sites of DNA double strand breaks and that Mdm2 inhibitedefficient DNA repair, which was dependent on the Nbs1 bind-ing domain in Mdm2. This finding suggests a novel role ofMdm2 in compromising DNA integrity.

MATERIALS AND METHODS

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 phenyl-methylsulfonyl fluoride, 0.4 units/ml aprotinin, 1 mM NaF, 10 mM

�-glycerophosphate, and 0.1 mM Na3VO4). Total cellular protein (60 mg)was rotated with anti-Mdm2-conjugated beads (SMP14; Santa CruzBiotechnology, Inc., Santa Cruz, CA) at 4 °C, washed in complete lysisbuffer, and separated by SDS-PAGE. Following silver staining, proteinbands were excised and digested as described previously with slightmodifications (37). After separation on a reversed phase LC column,eluted peptides were analyzed on a Q-TOF Ultima tandem mass spec-trometer with electrospray ionization (Micromass/Waters, Toronto,Canada). The MS/MS data were processed using Masslynx software(Micromass), and the MASCOT (Matrix Science, London, UK) searchengine was used to search the NCBI nonredundant data base. Proteinidentifications were based on a minimum random probability score of25 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 werecultured as described by the American Type Culture Collection (Man-assas, VA). p53�/�ARF�/�, p53�/�Mdm2�/�, and p53�/� MEFs wereisolated as described previously (38) and maintained in Dulbecco’smodified Eagle’s medium with 10% fetal calf serum (Atlanta Biologi-cals, Atlanta, GA) and nonessential amino acids. Saos-2 cells (providedby Dr. Gerard Zambetti, St. Jude Children’s Research Hospital, Mem-phis, TN) were grown as indicated by ATCC. The HCC1937 (a gift fromDr. Kenneth Cowan, Eppley Institute, Omaha, NE) and ATLD3 celllines (generously provided by Dr. Matthew Weitzman, Salk Institute,La Jolla, CA) were maintained in Dulbecco’s modified Eagle’s mediumwith 20% fetal calf serum. GM11261 (A-T lymphoblast), GM07166fibroblasts (NBS), and GM07078 lymphoblasts (NBS) were purchasedfrom the Coriell Institute (Camden, NJ) and cultured as theirprotocols indicated.

Generation, Expression, and Purification of Mdm2 and Nbs1 Pro-teins—Wild-type murine Mdm2 cDNA (generously provided by Dr. Mar-tine Roussel, St. Jude Children’s Research Hospital) was subcloned intothe pGex2T vector (Amersham Biosciences), and wild-type human Nbs1cDNA (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 DH5� or BL21Escherichia coli cells. GST and GST-Mdm2 bacterial supernatant wasincubated with glutathione-Sepharose beads (Amersham Biosciences),washed in phosphate-buffered saline containing 1 M NaCl2, and storedat �80 °C for use in in vitro binding assays. For GST-Nbs1 and GSTused in the Nbs1 reconstitution experiment (see below), glutathione-Sepharose beads (Amersham Biosciences) were packed to a chromatog-raphy column (Bio-Rad), and GST-Nbs1 or GST bacterial supernatantwas added. Following incubation, the column was washed with phos-phate-buffered saline, and GST-Nbs1 and GST purified protein wereeluted as fractions with 10 mM glutathione. Fractions were separatedby 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 Mdm2constructs (amino acids 198–489, 298–489, 198–400, and 349–489)(generously provided by Dr. Martine Roussel) were subcloned into pJ3Hvector for expression of a hemagglutinin (HA) tag at the N terminus.Wild-type murine Mdm2 was used in PCR amplification to generateother HA-Mdm2 mutants (1–192, 1–314, and 198–314). Wild-type hu-man Nbs1 cDNA (provided by Dr. John Petrini, Memorial Sloan Ket-tering, New York) and human Nbs1 mutants generated by restrictiondigestion were cloned into pCMVTag4 or pCMVTag2 (Stratagene, LaJolla, CA) to generate FLAG-tagged proteins. HA- and FLAG-taggedwild-type and mutant constructs were cloned into pCDNA3 vector (In-vitrogen) for expression in mammalian cells. Murine cells (NIH3T3 andp53�/� MEFs) were infected with an MSCV-IRES-green fluorescenceprotein (GFP) retrovirus (from Dr. Robert Hawley) encoding wild-typeMdm2, mutant Mdm2-(198–489), mutant Mdm2-(298–489) (gifts fromDr. Martine Roussel), or a control empty vector, as previously reported(38, 39). Cells were analyzed by flow cytometry immediately prior toanalysis for GFP expression, which is an indicator of protein expressionfrom the bicistronic retroviral vector, and only cell populations thatwere greater than 90% GFP-positive were utilized for analyses.

Immunoprecipitation and Western Blotting—HA-tagged Mdm2and/or FLAG-tagged Nbs1 constructs were transfected into 293T cells,and cells were collected for analysis 36 h later. For Western blots, allcells 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 phenylmethyl-sulfonyl fluoride, 0.4 units/ml aprotinin, 1 mM NaF, 10 mM �-glycero-phosphate, and 0.1 mM Na3VO4). Equal amounts of protein were im-munoprecipitated at 4 °C with antibodies specific to Mdm2 (SMP14;Santa Cruz Biotechnology), HA (HA-probe, F-7; Santa Cruz Biotechnol-ogy), 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 �g of purifiedGST or GST-Nbs1 protein was added to NBS fibroblast cell lysate, andMdm2 was immunoprecipitated with an anti-Mdm2 antibody (SMP14).For nuclear and cytoplasmic extracts, HeLa cell lysates were preparedas previously described (40) and then frozen at �80 °C. Proteins wereseparated by SDS-PAGE and transferred to nitrocellulose membranes(Protran; Schleicher & Schuell). Membranes were serially Westernblotted with antibodies specific for HA (F-7; Santa Cruz Biotechnology),FLAG (M2) Mdm2 (SMP14), Rad50 (Novus Biologicals), Mre11 (NovusBiologicals or Oncogene Research, Boston, MA), Nbs1 (Novus Biologi-cals or BD Transduction Laboratories, San Jose, CA), and �-actin(Sigma). Horseradish peroxidase-linked secondary antibodies (Amer-sham Biosciences) and ECL (Amersham Biosciences) or Supersignal(Pierce) were used to detect bound immunocomplexes.

In Vitro Transcription/Translation and Binding Experiments—Plas-mids encoding p53, Bax, Nbs1, and Mre11 (generously provided by Drs.Arnold J. Levine (Princeton University, Princeton, NJ), Xu Luo (Uni-versity of Nebraska Medical Center, Omaha, NE), Tanya Paull (Uni-versity 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 transcrip-tion and translation using TNT-coupled reticulocyte lysate systems(Promega, Madison, WI). In vitro binding assays were performed inbinding 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 lysatesbound to glutathione-Sepharose beads were incubated with [35S]Met-labeled proteins at 4 °C. Samples were washed five times, and boundproteins were separated by SDS-PAGE. Gels were dried, and 35S activ-ity (decays/min/mm2) was quantified on a Storm PhosphorImager (Am-ersham Biosciences).

Immunofluorescence—Primary human (IMR90) and NBS fibroblastswere grown on glass coverslips and fixed in methanol/acetone 6–8 hfollowing �-irradiation (12 Gy) with a cesium-137 source. Fixed cellswere incubated overnight at 4 °C in phosphate-buffered saline contain-ing 10% fetal calf serum (Atlanta Biologicals). Following incubationwith 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 incu-bated with fluorescein isthiocyanate-conjugated donkey anti-rabbit andTRITC-conjugated donkey anti-mouse antibodies (1:200; Jackson Lab-oratories, West Grove, PA). Coverslips were mounted to slides withVectashield Mounting Media containing 4�,6-diamidino-2-phenylindole(Vector Laboratories, Burlingame, CA). Cells were imaged on a ZeissLSM 410 confocal laser-scanning microscope (Goettinger, Germany)with �40 or �63 magnification.

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DNA Damage Quantitation—Following infection (36 h) of NIH3T3cells or p53�/� MEFs with wild-type Mdm2, Mdm2-(198–489), Mdm2-(298–489), or Mdm2-(198–400) encoded or empty MSCV-IRES-GFPretroviruses (see above), cells were �-irradiated with 5 Gy from acesium-137 source. Cells were incubated at 37 °C for specific intervalsand harvested for comet assay analysis. Comet assays were performedunder neutral conditions to optimize for detection of DNA double strandbreaks, as directed by the manufacturer’s protocol (Trevigen, Gaithers-burg, MD). Samples were blinded, and DNA was stained with ethidiumbromide. Multiple pictures per sample were taken by fluorescence mi-croscopy (Nikon E600, Melville, NY) using a TRITC-HYQ filter. DNAdamage quantitation was performed by computer scoring at least 50cells per interval per cell line with TriTekCometScoreTM software(TriTek Corp., Summerduck, VA). Tail moments were calculated by thesoftware and represent the length of the comet tail multiplied by thepercentage of DNA in the comet tail. Tail moment is a measurement ofDNA double strand breaks (41, 42). Following data collection and tab-ulation, codes identifying each set of pictures were broken to reveal theidentity of each sample.

RESULTS

The Mre11-Nbs1-Rad50 Complex Is Associated with Mdm2Independent of p53—Mdm2 appears to have functions that arep53-independent (31–34, 36), yet the pathways that mediatethese activities are unclear. To uncover potentially novel Mdm2oncogenic pathways, we isolated and identified proteins thatspecifically bound to Mdm2 independent of p53. Utilizing un-fractionated HeLa cell extracts that express the papillomavirusE6 protein, which inactivates p53, endogenous Mdm2 was im-munoprecipitated with an Mdm2-specific antibody. In Mdm2immunoprecipitates, polypeptides of 200, 150, 115, 95, and 80kDa were readily evident in silver-stained gels, whereaspolypeptides of this size were absent in isotype control immu-noprecipitations (Fig. 1A and data not shown). The 150-, 95-,and 80-kDa protein bands were excised, trypsin-digested, andsubjected to mass spectrometry analysis. Seventeen peptidesfrom the 150-kDa band, 21 peptides from the 95-kDa band, andnine peptides from the 80-kDa band matched sequences inRad50, Nbs1, and Mre11, respectively (Fig. 1A). These threeproteins comprise the M-N-R complex that is responsible forrepairing double strand DNA breaks (1). To verify that theM-N-R complex was associated with Mdm2, Mdm2 and itsassociated proteins were immunoprecipitated from HeLa cellextracts and Western blotted with antibodies specific for Nbs1,Mre11, and Rad50. All three proteins co-immunoprecipitatedwith the same Mdm2-specific antibody used in the previousimmunoprecipitations but not with an isotype control antibody(Fig. 1B). To obtain further confirmation of the interactionbetween Mdm2 and the M-N-R complex, we overexpressedHA-tagged wild-type murine Mdm2 in 293T cells, which alsolack functional p53. Nbs1, Mre11, and Rad50 were clearlyvisible in HA immunoprecipitations and absent in isotype con-trol immunoprecipitations (Fig. 1C). For unclear reasons, wewere unable to detect Mdm2 protein in immunoprecipitationsof endogenous Nbs1, Mre11, and Rad50. However, when co-transfections of FLAG-tagged Nbs1 and HA-tagged Mdm2 into293T cells were performed, Mdm2 was detected in anti-FLAGimmunoprecipitations and absent in isotype control immuno-precipitations (Fig. 1D). Thus, four lines of evidence indicatethat the M-N-R complex can specifically associate with Mdm2and that this interaction appears to occur in cells that lackfunctional p53.

To further test whether p53 or the Mdm2 regulator ARF isrequired for the association of Mdm2 with the M-N-R complex,we evaluated multiple cell lines. Nbs1, Mre11, and Rad50co-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 expres-sion observed between the two genotypes of MEFs were due to

a small difference in protein loading (see �-actin; Fig. 2A) anddo not represent differences of the endogenous levels of theseproteins in the different MEFs analyzed. Evaluation of human(MDA-MB-231, 293T, Saos-2, CLL, and K562) and murine tu-mor cell lines that lack functional p53 showed that Mdm2 wasassociated with the M-N-R complex (Fig. 2B, lanes 2, 4, 5, 8,and 9; and data not shown). Similarly, in whole cell lysatesfrom human and mouse cells lacking ARF expression (MCF7,HT1080, HCT116, and NIH3T3), Mdm2 co-immunoprecipi-tated Nbs1, Mre11, and Rad50 (Fig. 2B, lanes 1, 6, and 7; datanot shown). In addition, the M-N-R complex precipitated withMdm2 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 withthe M-N-R complex is not due to cellular transformation. Thesmall 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 differencesin one or more of these proteins between the cell lines. To-gether, these results demonstrated that the interaction be-tween Mdm2 and the M-N-R complex does not require p53 orARF and is not specific to human cells or cells of a particulartissue type or transformation status.

Nbs1, Mre11, BRCA1, and Mdm2 are phosphorylated byATM (13–18, 43), and BRCA1 can associate with Rad50 (7, 8).Therefore, we questioned whether functional ATM and BRCA1were required for Mdm2-M-N-R binding. To determine this, weutilized an A-T lymphoblastoid cell line (GM11261) andHCC1937 ductal breast carcinoma cell line, containing muta-tions in ATM and BRCA1, respectively, that inactivate theproteins (12, 44). Nbs1, Mre11, and Rad50 co-immunoprecipi-tated with Mdm2 in both cell lines (Fig. 2C). Therefore, Mdm2-M-N-R complex association does not require functional ATMor BRCA1.

Amino Acids 198–314 of Mdm2 Bind to the M-N-R Com-plex—Specific domains in Mdm2 are required to mediate bind-ing to p53, ARF, and other proteins (Fig. 3A) (45). To identifythe region of Mdm2 required for M-N-R complex association,we expressed a series of HA-tagged deletion mutants of Mdm2in 293T cells (Fig. 3A). Expression of all HA-Mdm2 mutantsand the control wild-type HA-Mdm2 protein was verified byWestern blot analysis (Fig. 3B, top panel). Anti-HA immuno-precipitated wild-type HA-Mdm2 along with the M-N-R com-plex (Fig. 3B, lane 1). Similarly, the HA-Mdm2 mutant 198–489, lacking the p53 binding domain, still interacted with theM-N-R complex, since Nbs1, Mre11, and Rad50 were readilydetected by Western blot (Fig. 3B, lane 2). Another Mdm2mutant lacking both the p53 binding domain and the ringfinger ubiquitin ligase domain (residues 198–400) also re-tained binding to the M-N-R complex (Fig. 3B, lane 7). Incontrast, the M-N-R complex did not co-immunoprecipitatewith HA-Mdm2 mutants containing just the p53 binding do-main (residues 1–192) or the ring domain (residues 349–489),despite high levels of expression of these mutants (Fig. 3B,lanes 6 and 4). Thus, neither the N nor C terminus of Mdm2 isrequired to mediate the binding of Mdm2 to the M-N-Rcomplex.

To further narrow down the M-N-R binding domain inMdm2, we utilized two overlapping HA-Mdm2 mutants (1–314and 298–489) (Fig. 3A). The 1–314 HA-Mdm2 mutant bound tothe M-N-R complex (Fig. 3B, lane 5), whereas the HA-Mdm2mutant 298–489 was unable to precipitate the M-N-R complex(Fig. 3B, lane 3). Therefore, we postulated that since aminoacids 198–400 and 1–314 of Mdm2 did and amino acids298–489 of Mdm2 did not co-immunoprecipitate the M-N-Rcomplex, amino acids 198–314 were likely the domain that

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binds to the complex. As predicted, the HA-Mdm2 mutant198–314 (Fig. 3A) did bind to the M-N-R complex and precip-itated equivalent levels of Nbs1, Mre11, and Rad50 as com-pared with wild-type HA-Mdm2, despite apparently low levelsof expression of this Mdm2 mutant (Fig. 3B, lane 8). Interest-ingly, amino acids 198–314 overlap the domain in Mdm2 thatbinds to ARF and ribosomal proteins (45). Thus, the minimalessential domain of Mdm2 required for M-N-R association lieswithin amino acids 198–314.

Nbs1 Mediates the Interaction between Mdm2 and the M-N-RComplex—Mre11 brings Rad50 and Nbs1 together by directlybinding to both proteins (3, 6). ATLD patients express mutantforms of Mre11, resulting in improper Nbs1, Mre11, and Rad50assembly (9). To determine whether Mre11 mediates the inter-action between Mdm2 and the M-N-R complex, we obtained theATLD3 patient cell line (9, 46). The mutated Mre11 in theATLD3 immortalized cell line still bound to Rad50 but showeddrastically reduced association with Nbs1 (Fig. 4A), as previ-ously reported (9). Surprisingly, Nbs1, but not Mre11 or Rad50,co-immunoprecipitated with Mdm2 in whole cell lysates fromthe ATLD3 cell line (Fig. 4B, lane 1). Mre11 and Rad50 asso-ciation with Mdm2 was restored in the ATLD3 cell line stablyexpressing wild-type Mre11, since these proteins were detectedfollowing Mdm2 immunoprecipitation (Fig. 4B). Wild-typeMre11 expression also rescued the ability of Rad50 and Mre11to co-immunoprecipitate Nbs1 (Fig. 4A). Therefore, wild-typeMre11 is required for Mre11 and Rad50 association withMdm2, and more importantly, Nbs1 appears to mediate theinteraction of the M-N-R complex with Mdm2.

Most NBS patients (95%) express a mutant form of Nbs1 thatis severely truncated and lacks the Mre11 binding domain (10).

To determine whether Nbs1 is the protein in the M-N-R com-plex that mediates the binding of the complex to Mdm2, Mdm2was immunoprecipitated from an NBS fibroblast cell line thatcontains the common 657del5 mutation. Although endogenousMdm2 was immunoprecipitated from the NBS cell line, C-terminal truncated Nbs1 was not associated (Fig. 4C). Notably,in the absence of wild-type Nbs1, co-precipitation of Mre11 andRad50 with Mdm2 was also abolished (Fig. 4C). These resultsindicate that wild-type Nbs1 is required for Mre11 and Rad50association with Mdm2. To test this possibility, we generatedand purified from bacterial cultures GST-linked Nbs1 protein.The addition of purified GST-Nbs1 protein to NBS fibroblastcell lysate restored Mre11 and Rad50 co-immunoprecipitationwith Mdm2 (Fig. 4D, lane 3), whereas the control GST purifiedprotein did not (Fig. 4D, lane 2). Therefore, these results dem-onstrate that Nbs1 is the protein mediator between Mdm2 andMre11/Rad50.

The Central Region of Nbs1 Is Required for Interaction withMdm2—In contrast to fibroblasts, lymphoid cells from NBSpatients express a C-terminal p70 Nbs1 protein generated froma second translation initiation site (47). This C-terminal Nbs1protein (amino acids 221–754) still contains the Mre11 bindingdomain (Fig. 5B) and consequently retains the ability to bind toMre11 and co-immunoprecipitate Rad50 (data not shown; seeRef. 47). To determine whether Mdm2 was associated with thisp70 truncated Nbs1 protein, we immunoprecipitated Mdm2from the lymphoblastoid NBS cell line isolated from the samepatient as the NBS fibroblast cell line used in experimentsshown in Fig. 4, C and D. Notably, p70 Nbs1, along with Mre11and Rad50, were co-immunoprecipitated with Mdm2 (Fig. 5A).Thus, the N terminus of Nbs1 appears dispensable for the

FIG. 1. Nbs1, Mre11, and Rad50 co-immunoprecipitate with Mdm2. A, en-dogenous Mdm2 was immunoprecipitatedwith an Mdm2-specific antibody fromHeLa cell lysate. Following separation bySDS-PAGE, proteins were silver-stained.The arrows indicate the polypeptidebands excised and analyzed by mass spec-trometry. Peptide sequences identified bymass spectrometry for the 150-, 95-, and80-kDa bands were homologous to Rad50,Nbs1, and Mre11, respectively, and are inbrackets. B, whole cell HeLa cell lysate(WCL; 200 �g) was immunoprecipitated(4 mg) without antibody (None), with anisotype control antibody (Isotype), or withan Mdm2-specific antibody (Mdm2) andWestern blotted with antibodies specificfor the proteins indicated to the left ofeach panel. C, HA-tagged wild-typeMdm2 was transiently expressed in 293Tcells. Immunoprecipitations (4 mg) withisotype control antibody (Isotype) or an-ti-HA antibody (HA) were Western blot-ted as in B. A blank lane separates theimmunoprecipitations and the whole celllysate (WCL; 100 �g). D, 293T cells wereco-transfected with vectors encoding HA-Mdm2 and FLAG-Nbs1 (M/N) or controlvectors (V/V). Immunoprecipitations (IP;2.5 mg) with anti-FLAG (FLAG) or anisotype (Iso) control antibody and wholecell lysates (WCL; 200 �g) were Westernblotted with antibodies specific for theproteins indicated to the left of each panel.

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interaction of Nbs1 with Mdm2. To further characterize theMdm2 interaction domain of Nbs1, we generated and tran-siently expressed FLAG-tagged wild-type Nbs1 and mutants ofNbs1 in 293T cells (Fig. 5, B–D). Not surprisingly, wild typeNbs1 and the N terminus truncated 179–754 Nbs1 mutant,which is similar to the Nbs1 mutant expressed in the NBSlymphoblasts (Fig. 5B), co-immunoprecipitated with Mdm2(Fig. 5C). In contrast, the C terminus of Nbs1 (amino acids540–754) did not co-immunoprecipitate with Mdm2 (Fig. 5, Band D). Due to the low level of expression of the 540–754 Nbs1mutant, 4 times the amount of whole cell lysate from cells withthis Nbs1 mutant was used for immunoprecipitating Mdm2, ascompared with the amount used for immunoprecipitatingMdm2 in cells expressing wild-type Nbs1 or vector control (Fig.5D). Mdm2 did associate with an Nbs1 mutant lacking its Cterminus, since amino acids 1–592 of Nbs1 co-immunoprecipi-tated with Mdm2 (Fig. 5, B and C). Therefore, neither the N norC terminus of Nbs1 is required for binding to Mdm2, suggest-

ing that the central part of Nbs1 (amino acids 221–540) medi-ates the interaction with Mdm2.

Mdm2 Binds to Nbs1—Nbs1 and Mdm2 interact in cellularlysates. To determine whether the association between Nbs1and Mdm2 is direct or indirect, we performed a series of in vitrobinding experiments with purified protein. Purified GST orGST-Mdm2 fusion protein was incubated with in vitro tran-scribed and translated [35S]Met-labeled proteins. As a negativecontrol, 35S-Met-labeled Bax was added to all reactions and didnot specifically bind to GST or GST-Mdm2 (Fig. 6A). As apositive control for Mdm2 binding, [35S]Met-labeled p53 wasgenerated. In contrast to p53 (Fig. 6, A (lanes 1–3) and B) andconsistent with our previous data, Mre11 did not bind to GST-Mdm2 (Fig. 6, A (lanes 7–9) and B). Importantly, Nbs1 boundspecifically to GST-Mdm2 (Fig. 6, A (lanes 4–6) and B). Theamount of [35S]Met-labeled protein that bound to GST andGST-Mdm2 was quantified by phosphorimaging analysis.Quantitation revealed that p53 and Nbs1 bound to GST-Mdm2

FIG. 2. p53-, ARF-, ATM-, and BRCA1-independent association of the Mre11-Nbs1-Rad50 complex with Mdm2. A, protein from wholecell lysates (WCL; 100 �g) and Mdm2 immunoprecipitations (IP; 4 mg) of immortal p53�/�Mdm2�/� and p53�/�ARF�/� MEFs were Western blottedwith antibodies specific for the proteins indicated to the left of each panel. B, whole cell lysates from the indicated human cell lines wereimmunoprecipitated with an Mdm2-specific antibody and Western blotted with antibodies specific for the proteins indicated to the left of eachpanel. The lineage of each cell line is listed above the name of the cell line. C, whole cell protein lysates from an A-T lymphoblastoid cell line(GM11261) and a BRCA1 mutated breast carcinoma cell line (HCC1937) were immunoprecipitated with an Mdm2-specific antibody. Immunopre-cipitations (IP) were Western blotted with antibodies specific for the proteins indicated to the left of each panel.

FIG. 3. Amino acids 198–314 of Mdm2 are required for association with the Mre11-Nbs1-Rad50 complex. A, schematic diagrams ofwild-type HA-Mdm2 and HA-Mdm2 deletion mutants (amino acids indicated). The white boxed areas represent domains that bind to p53 and ARFor represent the E3 ubiquitin ligase ring domain (RD). The black boxes denote the location of the nuclear localization signal, nuclear export signal,and nucleolar localization signal from the N to C terminus, respectively. HA-Mdm2 mutants that co-immunoprecipitated the M-N-R complex (�)and mutants that did not (�) are indicated to the right of each diagram. B, wild-type (WT) HA-Mdm2 (residues 1–489) and the indicated HA-Mdm2mutants were immunoprecipitated from 293T cells with an anti-HA antibody following transient transfection. Immunoprecipitated proteins wereWestern blotted with antibodies specific for the proteins indicated to the left of each panel. The location of the immunoglobulin heavy chain (IgH)is indicated.

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21–25 and 4.3–5.7 times, respectively, the amount that boundto GST (Fig. 6B). Thus, the interaction between Mdm2 andNbs1 was specific and appears direct.

Mdm2 Co-localizes with Nbs1 at Sites of DNA Damage—Inunirradiated cells, Mdm2 is ubiquitously expressed throughoutthe cell (45), whereas Nbs1, Mre11, and Rad50 are predomi-nantly nuclear proteins (4). To determine whether nuclearMdm2 can bind to the M-N-R complex, we utilized nuclear andS-100 cytosolic extracts from unirradiated HeLa cells. Nbs1,Mre11, and Rad50 co-immunoprecipitated with Mdm2 in thewhole cell and nuclear extracts, but due to the low levels of thecomplex in the cytoplasm, none of the proteins in the M-N-Rcomplex were detected in Mdm2 immunoprecipitations fromS-100 extracts (Fig. 7A). Therefore, in the absence of DNAdamage, nuclear Mdm2 is able to associate with Nbs1.

To determine whether DNA damage would alter the bindingof Mdm2 to Nbs1, Mdm2 was immunoprecipitated from �-irra-diated HeLa cells. A similar amount of Nbs1 co-immunopre-cipitated from cells that were unirradiated and irradiated (Fig.7B). Although these results suggest that the amount of Mdm2that associates with Nbs1 appears to be unaltered followingDNA damage, whole cell lysates do not necessarily reflectchanges in protein association if only a fraction of the totalproteins is involved; nor do whole cell lysates provide informa-tion on altered cellular localization of protein complexes, aswas the case for BRCA1/Rad50 association (7). To test thesepossibilities, we evaluated by immunofluorescence the localiza-tion of Mdm2 and Nbs1 prior to and following �-irradiation. Inunirradiated IMR90 cells, diffuse nuclear Nbs1 staining andboth cytoplasmic and nuclear Mdm2 staining were detected(Fig. 7C). Merging these two images revealed faint and nondis-tinct regions of overlap, which indicate a lack of co-localization

of Mdm2 and Nbs1 in the absence of DNA damage. The M-N-Rcomplex forms Type II nuclear foci at locations of DNA doublestrand breaks and are visible within 8 h post irradiation inprimary diploid human fibroblast cells (IMR90) (48). 6–8 hfollowing �-irradiation and as previously reported (48), Nbs1and Rad50 co-localization was visible in DNA damage-depend-ent nuclear foci (Fig. 7C). Notably, Mdm2 had a more clusteredappearance following �-irradiation with Mdm2 localizing to afew distinct sites throughout the nucleus (Fig. 7C). Impor-tantly, multiple sites of specific Nbs1 and Mdm2 co-localizationat nuclear foci were also detected in single planes of �-irradi-ated IMR90 cells (Fig. 7C). Mdm2/Nbs1 co-localization was alsoevident at reduced levels at earlier times postirradiation (datanot shown). Although Mdm2 did not appear to co-localize withNbs1 at every focus, Mdm2/Nbs1 co-localization was observedin over 40% of cells where Nbs1 nuclear foci were detected.Moreover, these data are consistent with the observation thatBRCA1 binds to the M-N-R complex but does not always co-localize with the M-N-R complex following irradiation (49).Thus, these results illustrate that at a single point in time,Mdm2 co-localized with a subset of the Nbs1 DNA damage foci.

Although we previously showed that p53 was not requiredfor Nbs1/Mdm2 association, Mdm2 and p53 do bind (45), andthus, it was possible that p53 also localized with Mdm2 to sitesof DNA damage. Therefore, IMR90 cells were �-irradiated,fixed, and fluorescently stained for Nbs1 and p53. In unirradi-ated IMR90 cells, p53 is expressed at very low levels andappears diffuse throughout the cell (Fig. 7D). Following �-irra-diation, p53 is stabilized, and consequently the protein levels ofp53 were higher, particularly in the nucleus where p53 acts asa transcription factor (Fig. 7D). However, p53 staining does notappear to overlap Nbs1 staining at nuclear foci following DNAdamage (Fig. 7D). These results indicate that p53 does notco-localize with Nbs1 at sites of DNA damage and may alsosuggest that Mdm2/Nbs1 binding at double strand breaks isindependent of p53.

Mdm2 Overexpression Delays DNA Double Strand BreakRepair—Nbs1, together with Mre11 and Rad50, repairs DNAdouble strand breaks induced by �-irradiation (1). To evaluatewhether Mdm2 expression would influence Nbs1-mediatedDNA repair, we took advantage of the comet assay, whichunder neutral conditions measures double strand breaks on aper cell basis (41, 42). In these comet assays, the length anddarkness of broken DNA (spreading from the nucleus like thetail of a comet) directly correlates with the severity of DNAdamage (41, 42). DNA damage, as detected by a comet assay,can be measured visually or by computer analysis, and bothmethods are reported to be comparable (41). Computerizedscoring calculates tail moments, which are an accurate meas-urement of the amount of broken DNA per cell (41). In ourexperiments, tail moments of greater than 4 denote extensiveDNA damage, and tail moments of 0–4 represent slightly dam-aged and repaired and unbroken DNA, in comparison withvisual scoring.

We first analyzed the impact on DNA repair of Mdm2 over-expression in NIH3T3 cells, which lack ARF expression.NIH3T3 cells were infected with a control GFP-expressingMSCV retrovirus or an MSCV retrovirus encoding murineMdm2 and, in cis, GFP, as previously described (39). GFP�

NIH3T3 cells were �-irradiated, and DNA damage was ana-lyzed by comet assays. At 5 min postirradiation, severe DNAdamage in the vast majority of vector control and Mdm2-over-expressing cells was detected as tail moments of �4 (Fig. 8A).At 60 min following irradiation, an expected decrease in thepercentage of tail moments of �4 and an increase in the per-centage of tail moments of �4 were observed in the cells in-

FIG. 4. Nbs1 is required for Mdm2 association with the Mre11-Nbs1-Rad50 complex. A and B, whole cell lysates were prepared fromATLD3 cells expressing mutant (M) Mre11 and ATLD3 cells stablyexpressing wild-type (WT) Mre11. Following immunoprecipitations (IP)with Rad50 (A), Mre11 (A), and Mdm2 (B) specific antibodies, proteinswere Western blotted with antibodies specific for the proteins indicatedto the left of each panel. C, lysates from an NBS fibroblast cell lineexpressing mutated Nbs1 were immunoprecipitated with an Mdm2-specific antibody. Whole cell lysates (WCL) and immunoprecipitated(IP) proteins were Western blotted with antibodies specific for theproteins indicated to the left of each panel. D, following the addition (�)of GST or GST-Nbs1 purified protein to NBS fibroblast whole cell lysate(Lysate), Mdm2 was immunoprecipitated (Mdm2 IP �). Western blotswere performed with antibodies specific for the proteins indicated to theleft of each panel.

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fected with the vector control retrovirus. In contrast, 60 minpostirradiation, cells overexpressing Mdm2 showed littlechange in the percentage of tail moments that were �4 and noincrease in the tail moments that were �4 (Fig. 8A), suggestingan inhibition of DNA repair in these cells. Similar results were

obtained when DNA damage and repair were measured withvisual comet scoring (data not shown). Although in repeatedexperiments, �-irradiation of NIH3T3 cells overexpressingMdm2 consistently led to longer tail lengths for an extendedperiod of time when directly compared with cells infected with

FIG. 5. The N and C terminus of Nbs1 are dispensable for Mdm2 binding. A, whole cell lysates from an NBS lymphoblastoid cell line wereimmunoprecipitated with an Mdm2-specific antibody. Immunoprecipitated proteins (IP; 2 mg) and a whole cell lysate (WCL; 250 �g) were Westernblotted with antibodies specific for the proteins indicated to the left of each panel. B, schematic diagrams of the Nbs1 mutant expressed in NBSlymphoblastoid cells and wild-type FLAG-tagged Nbs1 and FLAG-tagged Nbs1 deletion mutants. The white boxed areas denote the forkhead-associated, the breast cancer C-terminal, and the Mre11 binding domains. The black boxes represent the location of the three nuclear localizationsequences. Amino acids are indicated above each diagram. FLAG-tagged Nbs1 mutants that did (�) or did not (�) co-immunoprecipitate withMdm2 are indicated. C and D, following transient transfection of FLAG-tagged wild-type Nbs1, the indicated Nbs1 mutants, or vector control,immunoprecipitations (2 mg in C) were performed with an Mdm2-specific antibody (Mdm2 IP) or an isotype control antibody (Isotype IP). Wholecell lysates (WCL; 100 �g in C) were run to show the level of expression of each FLAG-tagged protein. Proteins were Western blotted withanti-FLAG. The locations of immunoglobulin heavy chain (IgH) and light chain (IgL) are indicated. Four times (�4; 4 mg versus 1 mg in the IPlanes and 200 �g versus 50 �g in the WCL lanes) or the same (�1; 50 �g) amount of cell lysate from 540–754 mutant Nbs1-expressing cells ascompared with the amount used for vector control and wild-type expressing cells is indicated in D.

FIG. 6. Mdm2 binds to Nbs1. A, in vitro transcribed and translated [35S]Met-labeled p53, Nbs1, Mre11, and, as a negative control, Bax wereadded to GST- or GST-Mdm2-conjugated glutathione-Sepharose beads. Input, 12% of the protein added to each binding reaction. Bound proteinswere separated by SDS-PAGE and visualized by phosphorimaging analysis. B, three independent in vitro binding assays were quantified byphosphorimaging analysis and expressed as an average of the number of decays/min/mm2 (dpm/mm2). The error bars represent one S.D. Thenumbers above the bars indicate the amount of p53 and Nbs1 input that specifically bound to GST-Mdm2 protein relative to GST.

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vector control, Mdm2 overexpression did not block DNA repair.Specifically, 2 hours postirradiation, cells that overexpressedMdm2 or vector control had similar high percentages of tailmoments �4, indicating DNA repair occurred in the cells over-expressing Mdm2. In agreement with this, Mdm2 overexpres-sion did not correlate to an overall decrease in cell survival oraltered rates of proliferation, since a comparable number ofcells appeared to survive irradiation, and similar rates ofgrowth in short term assays were observed whether cells over-expressed Mdm2 or vector control (data not shown). Combined,these results indicate that Mdm2 overexpression delays DNArepair independent of ARF without appearing to alter cellgrowth or survival.

To determine whether p53 is required to mediate this effectof Mdm2 overexpression, we repeated these studies in p53�/�

MEFs. In accordance with the previous results, Mdm2 overex-

pression in cells lacking p53 also showed a delay in DNArepair. 90 min following �-irradiation, over 80% of the p53�/�

MEFs overexpressing Mdm2 had tail moments �4, whereasless than 50% of the vector control-infected p53�/� MEFs hadtail moments of �4 (Fig. 8B). Moreover, there was a large in-crease in the percentage of tail moments �4 in the vector control-expressing cells and only a slight increase in the percentage oftail moments �4 in cells overexpressing wild-type Mdm2 (Fig.8B). Similarly, when the 198–489 mutant Mdm2 lacking the p53binding domain and containing the Nbs1 interaction domain(Fig. 3A) was retrovirally expressed in NIH3T3 cells, mean tailmoments of �4 and �4 were not significantly different 60 minfollowing �-irradiation, whereas there was significant DNA re-pair in the vector control-infected cells (Fig. 8C). Thus, the delayby Mdm2 of DNA repair does not require the p53-binding domainof Mdm2 and is independent of p53 expression.

FIG. 7. Mdm2 and Nbs1 co-localize at sites of DNA damage. A, whole cell extract, cytoplasmic, and nuclear fractions of HeLa cells wereimmunoprecipitated with an Mdm2-specific antibody. Mdm2 immunoprecipitates (IP) and total protein (TP) were Western blotted with antibodiesspecific for the proteins indicated to the left of each panel. B, HeLa cells were left unirradiated or subjected to 10 Gy of �-irradiation. At theindicated intervals, cells were harvested, and Mdm2 was immunoprecipitated. Proteins were Western blotted with antibodies specific for Mdm2and Nbs1. C (top panel), unirradiated (No IR) IMR90 cells were fixed and then costained with Nbs1-specific (green) and Mdm2-specific (red)antibodies and fluorescein isthiocyanate- and TRITC-tagged secondary antibodies. Images were merged to reveal co-localization (yellow). C (middleand bottom panels), IMR90 cells were �-irradiated (�IR) with 12 Gy and fixed 8 h postirradiation. Fixed cells were costained with Nbs1-specific(green) and Rad50-specific (red) or Mdm2-specific (red) antibodies with co-localization in yellow (merge). Two different cells costained with Nbs1-and Mdm2-specific antibodies are shown in the bottom two panels. The white arrows point to a few areas of co-localization. D, IMR90 cells wereunirradiated (No IR) or �-irradiated with 12 Gy (�IR) and fixed 6 h postirradiation. Fixed cells were costained with Nbs1-specific (green) andp53-specific (red) antibodies with co-localization in yellow (merge). Each row of images (C and D) were visualized from an identical plane of thenucleus.

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We postulated that if Mdm2/Nbs1 interaction was necessaryfor the inhibition of DNA repair, then deletion of the Nbs1binding domain in Mdm2 should restore the rate of DNA re-pair. Therefore, p53�/� MEFs were infected with retrovirusesexpressing the 298–489 Mdm2 mutant, which lacks both thep53 binding domain and the Nbs1 binding domain (Fig. 3A),wild-type Mdm2, or vector control. p53�/� MEFs were chosen,since expression of the 298–489 Mdm2 mutant inhibits prolif-eration and induces apoptosis in cells that have wild-type p53(39). As predicted, GFP� MEFs expressing the 298–489 Mdm2mutant did not have a delay in DNA repair, since the disap-pearance of tail moments of �4 and the appearance of tailmoments of �4 occurred at rates similar to those of GFP�

vector control cells (Fig. 8D). Although there appeared to be aslight increase in the initial amount of DNA damage in theMEFs overexpressing the 298–489 Mdm2 mutant in compari-son with the DNA damaged sustained in MEFs with emptyvector, the percentage decrease in tail moments �4 and per-centage increase in tail moments �4 between the two wereanalogous. Specifically, between 5 and 60 min postirradiationin MEFs overexpressing the 298–489 Mdm2 mutant and inMEFs with empty vector, there was a 29.4 and 32.4% decreasein tail moments �4 and a 28.2 and 27.9% increase in tailmoments �4, respectively. In contrast, there was only a 7.8%decrease in tail moments �4 and a 5.7% increase in tail mo-ments �4 in MEFs overexpressing Mdm2. These results indi-cate that wild-type Mdm2 delayed DNA repair, whereas Mdm2lacking the Nbs1 binding domain did not. Therefore, Mdm2overexpression delays DNA double strand break repair, andthis requires the Nbs1 binding domain in Mdm2.

Mdm2 is an E3 ubiquitin ligase that regulates the expressionand activity of p53 and other proteins by ubiquitinylating them

(21–23, 45). To determine whether the ubiquitin ligase activityof Mdm2 is required for the delay in DNA repair imposed byMdm2, we retrovirally infected p53-null MEFs with the Mdm2mutant (residues 198–400) that lacks the entire ubiquitin li-gase domain as well as the p53-binding domain but that stillretains the Nbs1 binding domain (Fig. 3A). To our surprise,overexpression of the 198–400 Mdm2 mutant delayed DNArepair to a similar extent as wild-type Mdm2 (Fig. 8D). At 5 and60 min postirradiation, the percentage of tail moments �4 and�4 in GFP� MEFs overexpressing the 198–400 Mdm2 mutantparalleled those of MEFs that overexpressed wild-type Mdm2(Fig. 8D). There was a 12.5% decrease in tail moments �4 anda 9.2% increase in tail moments �4 in the MEFs that overex-pressed the 198–400 Mdm2 mutant, which were similar to thepercentages quantified for MEFs overexpressing Mdm2 (seeabove). These data indicate that the loss of the ubiquitin ligasedomain did not prevent Mdm2 from delaying DNA repair, andtherefore, the ubiquitin ligase activity of Mdm2 is not requiredfor its ability to inhibit DNA repair.

DISCUSSION

Mdm2 has p53-dependent and -independent functions (28,31–34, 36), both of which are likely to contribute separately totumorigenesis. Mdm2 regulation of p53 is firmly established(45), whereas p53-independent Mdm2 activity is not well char-acterized. For example, overexpression of Mdm2 in mice altersDNA ploidy and has resulted in transformation that is inde-pendent of p53 expression (31–33), yet the pathways that me-diate these Mdm2-initiated cellular processes are unknown.Here we describe the identification and characterization of thenovel interaction of Mdm2 with the DNA repair protein Nbs1.The binding of Mdm2 to Nbs1 was direct and independent of

FIG. 8. Mdm2 inhibits DNA double strand break repair. NIH3T3 cells (A and C) or p53�/� MEFs (B and D) were infected with an emptyMSCV-IRES-GFP (Vector), MSCV-Mdm2-IRES-GFP (Mdm2), MSCV-198–489Mdm2-IRES-GFP (198–489 Mdm2), MSCV-298–489Mdm2-IRES-GFP (298–489 Mdm2), or MSCV-198–400Mdm2-IRES-GFP (198–400 Mdm2) retrovirus. Following 5 Gy of �-irradiation, cells were harvested atintervals (5, 30, 60, and/or 90 min), and DNA double strand breaks were quantitated by a comet assay (41, 42) (see “Materials and Methods”). Afterblinding the samples, multiple photographs of each were taken, and the percentage of broken DNA (tail moment) was measured by computersoftware (CometScoreTM). As compared with visual scoring, tail moments of greater than 4 denote extensive DNA damage, whereas tail momentsof less than 4 reflect very little DNA damage and no DNA damage or repaired DNA. NBS fibroblasts (NBS) were used as a positive control for DNAdamage in A. Data in A–D are the averages of the percentages of tail moments �4 or �4 at each interval from three separate experiments witherror bars representing one S.D.

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both p53 and ARF. These results provide critical new insightson Mdm2 and the p53-independent oncogenic pathways thatMdm2 may influence. In particular, our finding that Mdm2 andNbs1, but not p53, bind at Nbs1 nuclear foci implicates Mdm2in a DNA repair response distinct from the DNA damage check-point response regulated by p53. Moreover, a previous reportshowed that overexpression of Mdm2 increased DNA instabil-ity and chromosomal abnormalities, which was attributed tothe inhibition by Mdm2 of p53 (50). However, in light of ourresults, Mdm2 overexpression also appears to negatively im-pact the DNA repair function of the M-N-R complex, whichcould lead to loss of DNA integrity or generation of chromo-somal abnormalities. Mdm2 inhibition of DNA double-strandbreak repair was independent of p53 and ARF expression andrelied on the domain in Mdm2 that binds to Nbs1. Additionally,Mdm2 was reported to bind to and alter the activity of DNApolymerase �, an enzyme that is important in DNA repair (51).Therefore, Mdm2 may regulate DNA repair through its inter-actions with the M-N-R complex, DNA polymerase �, and pos-sibly other proteins necessary for restoring broken DNA. Thus,Mdm2 would impact the DNA damage response in two comple-mentary ways: first by controlling the activity of p53, the sen-sor of DNA damage, and second by regulating Nbs1 and DNApolymerase �, proteins that repair DNA.

Formation of Nbs1 foci at sites of DNA damage involvesmany proteins, including BRCA1, ATM, the phosphorylatedhistone protein �H2AX, Mre11, and many others, whose ex-pression is critical for maintaining DNA integrity (1, 7, 12, 52).Although BRCA1 and ATM are necessary for DNA repair, adeficiency in BRCA1 or ATM does not inhibit Nbs1 focus for-mation (7, 48). In contrast, loss of �H2AX inhibits the forma-tion of Nbs1 foci and co-localization of BRCA1 to foci (52),suggesting that H2AX expression is requisite for Nbs1 andBRCA1 to localize to sites of DNA damage. Furthermore, theforkhead-associated and breast cancer C-terminal domains ofNbs1 are required for Nbs1 localization with �H2AX at sites ofDNA strand breaks (6, 53). However, these N-terminal do-mains of Nbs1 are dispensable for interaction with Mre11 andMdm2 (6). The C terminus of Nbs1 directly binds to Mre11 andcontrols the cellular localization of Mre11 (6). Mre11 is cytoso-lic in unirradiated cells from NBS patients, whereas Mre11 isprimarily nuclear in cells expressing wild-type Nbs1 (9). Incontrast, there was no detectable difference in Mdm2 localiza-tion in unirradiated NBS cells.2 However, similar to Nbs1control of Mre11/Rad50 localization, Nbs1 may be involvedeither directly or indirectly in relocating Mdm2 to foci uponDNA damage. On the other hand, it is interesting to speculatethat the shuttling functions of Mdm2 (24) may be important forNbs1 localization following DNA damage, since the ubiquitinligase activity of Mdm2 does not appear to be necessary for itsability to inhibit DNA repair. Alternatively, Nbs1 may retainMdm2 at sites of DNA damage rather than either proteinbringing the other protein to DNA breaks, although even whenNbs1 was expressed, Mdm2 localization was not observed atevery Nbs1 focus. These data are consistent with the observa-tion that BRCA1 did not always co-localize with the M-N-Rcomplex, which was attributed to the order of recruitment ofBRCA1 and the M-N-R complex to sites of DNA damage (49).Moreover, in yeast, a whole host of proteins are recruited tosites of DNA damage much later than the M-R-N complex,which has been shown to be one of the earliest protein com-plexes to arrive at DNA breaks (54). Similarly, the kinetics ofMdm2 localization to DNA strand breaks may differ from thatof Nbs1 in that Mdm2 may localize to DNA damage at a later

time than Nbs1 does. On the other hand, Mdm2 may onlytransiently associate with Nbs1 and only bind to Nbs1 at spe-cific times in the DNA repair process, which is dynamic andunsynchronized with other sites of DNA damage. In addition, ithas been recently postulated that the larger and more brightlystaining foci at later times following DNA damage representDNA breaks that are irreparable or delayed in their repair (55).In accordance with this theory, Mdm2 may associate with Nbs1at foci that are having difficulty repairing their DNA, as Mdm2/Nbs1 co-localization was more prominent at later time pointsthan in the first 2 h following DNA damage. Although sinceearly foci are fainter and more difficult to detect, Mdm2 local-ization at early foci may be underestimated. Nevertheless, in asingle cell at one point in time, Mdm2 co-localization in asubset of Nbs1 foci would be expected, as we observed. How-ever, further analysis is required to define the regulation ofMdm2 localization to nuclear foci, the release of Mdm2 fromthese sites, and the role Nbs1/Mdm2 interactions have in theseprocesses. In addition, investigations are needed to determinewhether Mdm2 overexpression delays DNA repair by inhibit-ing Nbs1 localization to or function at foci.

The association of Mdm2 with Nbs1 does not require Mre11,ATM, BRCA1, or p53, although all of these proteins are neces-sary for a normal cellular response to ionizing irradiation andto maintain DNA integrity (1, 45). ATM is required to signalthe DNA damage response by phosphorylating many targetproteins. Following �-irradiation, ATM phosphorylates Mdm2and p53 (13, 17). Phosphorylation of Mdm2 inhibits Mdm2 fromtargeting p53 for degradation (13), whereas phosphorylation ofserine 15 on p53 activates p53 (17). BRCA1 is also phosphoryl-ated by ATM in response to �-irradiation (43). Recently, acomplicated regulatory mechanism between Nbs1, Mre11, andATM activation has been elucidated. Nbs1 and Mre11 arephosphorylated by ATM following �-irradiation (14–16), andNbs1 and Mre11 are necessary to activate ATM (56–58). How-ever, Nbs1 is not required for ATM-dependent phosphorylationof p53 (57). Whether Nbs1 is required for Mdm2 phosphoryla-tion by ATM remains to be determined. Of note, phosphoryla-tion of serine 395 in Mdm2 by ATM is reported to be at leastpartially responsible for destabilizing Mdm2 following �-irra-diation (59). However, we observed similar amounts of Mdm2bound to Nbs1 prior to and following �-irradiation, suggestingthat the Mdm2 associated with Nbs1 may be stable. Thus,additional experiments are needed to determine whether phos-phorylation of Nbs1 and/or Mdm2 alters Mdm2 and Nbs1 bind-ing and the stability of this interaction. For example, prelimi-nary mass spectrometry analysis revealed that in unirradiatedcells, Nbs1 is phosphorylated at a site distinct from the ATMphosphorylation sites when bound to Mdm2.2 Therefore, post-translational modifications are likely to regulate the associa-tion of Mdm2 and Nbs1 and the localization and activity ofthis complex.

Acknowledgments—We thank Dr. Martine Roussel for generouslyproviding multiple Mdm2 constructs, Dr. Matthew D. Weitzman for thegift of the ATLD3 parental and Mre11 reconstituted cell lines,Drs. Tanya Paull and John Petrini for Nbs1 and Mre11 constructs,Drs. Ellen Brisch and Michelle Malott for assistance with the cometassays, and Drs. Robert Lahue, Tadayoshi Bessho, and TimothyMcKeithan for critical reading of the manuscript. We also thank JaniceTaylor for assistance with confocal microscopy and Valerie Piening,Jane Kennedy, and Silvia Plaza for technical expertise.

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Mdm2 Binds to Nbs1 and Inhibits DNA Repair 18781

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Christine M. EischenJodi R. Alt, Alyssa Bouska, Mario R. Fernandez, Ronald L. Cerny, Hua Xiao and

RepairMdm2 Binds to Nbs1 at Sites of DNA Damage and Regulates Double Strand Break

doi: 10.1074/jbc.M413387200 originally published online February 25, 20052005, 280:18771-18781.J. Biol. Chem. 

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