Mre11 Complex and DNA Replication: Linkage to E2F and Sites of ...

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/01/$04.0010 DOI: 10.1128/MCB.21.17.6006–6016.2001

Sept. 2001, p. 6006–6016 Vol. 21, No. 17

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Mre11 Complex and DNA Replication: Linkage to E2F andSites of DNA Synthesis†

RICHARD S. MASER,1 OLGA K. MIRZOEVA,1 JULIE WELLS,2 HEIDI OLIVARES,1 BRET R. WILLIAMS,3

ROBERT A. ZINKEL,1 PEGGY J. FARNHAM,2 AND JOHN H. J. PETRINI1,3*

Laboratory of Genetics,1 McArdle Laboratory for Cancer Research,2 and Program in Cell and Molecular Biology,3

University of Wisconsin Medical School, Madison, Wisconsin 53706

Received 27 March 2001/Returned for modification 6 May 2001/Accepted 22 May 2001

We show that the Mre11 complex associates with E2F family members via the Nbs1 N terminus. This asso-ciation and Nbs1 phosphorylation are correlated with S-phase checkpoint proficiency, whereas neither is suf-ficient individually for checkpoint activation. The Nbs1 E2F interaction occurred near the Epstein-Barr virusorigin of replication as well as near a chromosomal replication origin in the c-myc promoter region and was re-stricted to S-phase cells. The Mre11 complex colocalized with PCNA at replication forks throughout S phase, bothprior to and coincident with the appearance of nascent DNA. These data suggest that the Mre11 complex sup-presses genomic instability through its influence on both the regulation and progression of DNA replication.

The Mre11 complex (composed of Mre11, Rad50, andNbs1) and the ataxia-telangiectasia mutated (ATM) proteinkinase are required to activate a DNA damage-induced S-phase checkpoint in mammalian cells (46). Mutations in theATM, MRE11, or NBS1 gene (from patients with ataxia-telan-giectasia [A-T], ataxia-telangiectasia-like disorder [A-TLD], orNijmegen breakage syndrome [NBS], respectively) abrogate thischeckpoint (12, 52, 58, 66). Mutant cells fail to repress thefiring of DNA replication origins in the presence of ionizingradiation (IR)-induced DNA damage, a phenomenon termedradioresistant DNA synthesis (RDS) (28, 42). Hence, theMre11 complex can act as a negative regulator of DNA repli-cation origins in response to DNA damage.

The Mre11 complex is also important for recombinationalDNA repair, as established by genetic analyses with Saccharo-myces cerevisiae (21). Both the conservation of Mre11 andRad50 and in vitro studies of the human Mre11 complexstrongly suggest that the human Mre11 complex also functionsin DNA recombination (43, 44, 63). DNA recombination andDNA replication functions are intrinsically linked; thus, Mre11complex recombination functions are implicated in S-phaseprogression in addition to its role in S-phase regulation. Invertebrates, null mutants of the Mre11 complex are inviable(33, 68, 73), and DT40 cells depleted of Mre11 die with chro-mosome damage indicative of failure to resolve double-strandbreaks arising during DNA replication (69). This suggests thatthe complex’s recombination functions are required for DNAreplication in a manner analogous to that of Rad51 (45, 69). InRad51-deficient cells, spontaneous chromosomal breakageduring DNA replication leads to cell death (32, 54, 56, 64). Itis not clear whether the Mre11 complex’s influence on the

S-phase checkpoint is related to its DNA recombination func-tions.

The Nbs1 protein is an important link between the Mre11complex and the ATM-controlled S-phase checkpoint. ATMphosphorylates Nbs1 (20, 31, 67, 72), and this event is requiredfor checkpoint activation (31, 72). Its role in cell cycle regula-tion is consistent with the fact that Nbs1 contains a forkhead-associated (FHA) domain and a BRCA1 C-terminal (BRCT)domain (66), each of which is found in a number of proteinsthat effect DNA damage-dependent checkpoint functions (4,10, 22, 57, 59).

We identified the E2F1 transcription factor in a screen forproteins that interacted with the Nbs1 N-terminal region andestablished evidence that this interaction occurs on chromatinnear a defined DNA replication origin. The interaction be-tween E2F1 and Nbs1 was abrogated or significantly reducedin NBS and A-TLD cells, respectively. Further, we found theMre11 complex undergoes dramatic relocalization duringDNA replication in a manner analogous to that seen in dam-aged cells (35, 37, 38). The data presented in this study suggestthat the Mre11 complex directly influences S-phase progres-sion both near replication origins via its interaction with E2F1and at replication forks.

MATERIALS AND METHODS

Cells. Normal lymphoblastoid cells (721) were obtained from B. Sugden. Raji525-7 cells were a gift from D. Eick and were grown in RPMI–10% calf serum–200 mg of hygromycin per ml. E14 embryonic stem cells were propagated asdescribed previously (47). All other cell lines have been described previously (12,58). Raji cells were synchronized by incubation in the presence of 2 mM thymi-dine for 14 h, released into drug-free medium for 11 h, and incubated in thepresence of 1 mg of aphidicolin/ml for 14 h. Cells were then released intodrug-free medium and harvested.

Immunological reagents. Nbs1 (#16) and Mre11 (#59) antisera were de-scribed previously (12, 58). E2F1 C20, E2F1 KH95, E2F2 C20, E2F3 C18, E2F3N20, E2F4 C20, retinoblastoma (Rb) 1F8, Ets1/2 C275, and promyelocytic leu-kemia protein PG-M3 antibodies were obtained from Santa Cruz Biotechnology.E2F1 mixed monoclonal antibody (KH201KH95) was purchased from UpstateBiotechnology. Anti-PCNA PC10 was from Oncogene Research Products. E2F1mixed monoclonal antibody (SQ411SQ71) was from Neomarkers. Secondaryantibodies for immunofluorescence were obtained from Jackson Immunore-search Laboratories. Murine Nbs1 rabbit polyclonal antiserum 93 was raised

* Corresponding author. Mailing address: University of Wisconsin—Madison, Laboratory of Genetics, 445 Henry Mall, Madison, WI53706. Phone: (608) 265-6043. Fax: (608) 262-2976. E-mail: [email protected].

† Report 3572 from the University of Wisconsin—Madison Labora-tory of Genetics.

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against amino acids (aa) 1 to 645 of murine Nbs1. E2F1 rabbit polyclonalantiserum 70 was raised against aa 282 to 416.

Immunoprecipitation and phosphatase assays. For coimmunoprecipitationassays, cells were lysed as described previously (58). Cleared lysates were immu-noprecipitated with preimmune serum, Nbs1 or Mre11 antiserum, or 5 mg ofantibodies against E2F1, E2F2, E2F3, E2F4, or Rb. Immunoprecipitates werefractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose using standard methods (2). Immuno-blotting was carried out as described previously (12, 58). To control for artifac-tual coimmunoprecipitation resulting from contaminating DNA in the extracts,immunoprecipitations were carried out in the presence of 300 mg of ethidiumbromide/ml to disrupt protein-DNA interactions, as described previously (25).

For phosphorylation assays, Nbs1 immunoprecipitations and phosphatase as-says were carried out, as described previously (31), using 50 U of l phosphatase(NEB) or an equivalent volume of storage buffer for mock-treated samples.

Immunofluorescence assays. Cells were plated on glass coverslips 24 h beforeeach experiment. Before fixation, in situ cell fractionation was performed asdescribed previously (39). The cells were then fixed in modified Streck tissuefixative for 30 min at room temperature (RT) and permeabilized for 15 min atRT as described previously (38).

Cells were blocked with 10% fetal calf serum (FCS) in phosphate-bufferedsaline (PBS) and then stained with primary antibody diluted in PBS with 5% FCSfor 1 h at RT, followed by staining with secondary antibody for 30 min. 49,69-Diamidino-2-phenylindole (DAPI) counterstain was used at a final concentrationof 0.05 mg/ml in the last wash. Controls with preimmune serum or secondaryantibody alone were negative.

For bromodeoxyuridine (BrdU) labeling, cells were labeled with 10 mM BrdUfor 4 h and then were washed and processed for immunofluorescent staining asdescribed above. After the secondary antibody incubation, samples were fixedagain in 4% paraformaldehyde for 30 min at RT and then were incubated with50 mM glycine for 10 min at RT. DNA was denatured with 4 N HCl plus 0.1%Triton X-100 for 10 min at RT, and then samples were extensively washed inPBS, followed by a 50 mM glycine wash. Cells were incubated for 1 h at RT withfluorescein isothiocyanate-conjugated monoclonal BrdU antibody (Becton Dick-inson) diluted at 0.8 mg/ml in PBS plus 5% FCS plus 0.3% Triton X-100.

Images were captured with a charge-coupled device camera (Princeton Instru-ments), and gray scale images were processed using IP Labs (Scanalytics) andPhotoshop 5.5 (Adobe) software.

Yeast two-hybrid assays. Two-hybrid interaction screening was performed asdescribed previously (12), using Nbs1 expressed as a fusion to the GAL4 DNAbinding domain and a human B-lymphoblastoid cDNA library. Derivatives ofE2F1 and Nbs1 were cloned by standard methods (2) as GAL4 DNA binding oractivation domain fusions. Interaction testing was performed on both histidine(with 3 mM 3-aminotriazole) and adenine selection, using three independentclones for each interaction.

Chromatin immunoprecipitations. Cells were formaldehyde cross-linked es-sentially as described previously for HeLa cells (7), except that cells were swelledin 5 mM piperazine-N,N9-bis(2-ethanesulfonic acid) (PIPES) (pH 8.0), 85 mMKCl, 0.5% IGEPAL CA-330, 0.5 mM phenylmethylsulfonyl fluoride (PMSF),100 ng of leupeptin/ml, and 100 ng of aprotinin/ml instead of reticulocyte stan-dard buffer. Chromatin was sheared to an average size of approximately 1,000 bp.Each immunoprecipitation reaction contained 1 mg of antibody. One mg of rabbitanti-mouse antibody (ICN) was added to E2F1 (KH201KH95) antibody reac-tions for the last hour of immunoprecipitation. Antibody-protein-DNA com-plexes were isolated by immunoprecipitation with preblocked protein A-positiveStaphylococcus aureus cells. Following extensive washes, bound DNA fragmentswere eluted and analyzed by PCR.

PCR analysis and Southern blotting. Immunoprecipitates were dissolved in 30ml of water (except for input samples, which were dissolved in 1,000 ml of water).Each reaction mixture contained 2 ml of immunoprecipitated DNA, 13 Taqreaction buffer, 1.5 mM MgCl2, 50 ng of each primer, 1.7 U of Taq polymerase(Promega), 200 mM each deoxynucleoside triphosphate, and 1 M betaine in afinal reaction volume of 20 ml. PCR mixtures were amplified for 1 cycle of 95°Cfor 5 min, annealing temperature of the primers for 5 min, and 72°C for 3 minand either 28 (episomal) or 33 (endogenous) cycles of 95°C for 1 min, annealingtemperature of the primers for 2 min, and 72°C for 1.5 min, followed by incu-bation at 72°C for 7 min. PCR products were electrophoresed through a 1.5%agarose gel and visualized with ethidium bromide. Linearity of PCR conditionswas tested in reaction mixtures containing 0.1, 1, 2, or 4 ml of immunoprecipi-tated DNA and was analyzed by Southern blotting (6). The PCR primers (Uni-versity of Wisconsin Biotechnology Center) used were the following: oriP 8099,59-CGCTCAGGCGCAAGTGTGTGTA-39; oriP 8512, 59-GGCAGGGACCAA

GACAGGTGAA-39; Myc 2411, 59-GGCTTCTCAGAGGCTTGGCGGC-39;Myc 2857, 59-0-39; MycDE2F, 59-GGCTTCTCAGAGGCTTGAATTC-39.

RESULTS

Nbs1 phosphorylation is not sufficient for activation of theS-phase checkpoint. NBS lymphoblastoid cells homozygous forthe common nbs1 657del5 allele (66) express an aberrant Nbs1protein species, Nbs1p70, that lacks the N-terminal 221 aa butremains associated with the Mre11 complex (36). NBS lympho-blastoid cells exhibited RDS, as shown previously for untrans-formed lymphocytes (36, 60, 61). Hence, the FHA and BRCTdomains missing from the N terminus in Nbs1p70 are likely toinfluence the S-phase regulatory function of the Mre11 complex.

ATM phosphorylates Nbs1 in response to IR, and this eventis necessary for activation of the S-phase checkpoint (20, 31,67, 72). We asked whether Nbs1p70, which contains the primaryIR-induced phosphorylation site, serine 343, was phosphory-lated in response to IR. Nbs1p70 was phosphorylated after IR(Fig. 1A). In addition, IR-induced Nbs1 phosphorylation wasobserved in A-TLD3 cells but was nearly absent in A-TLD2cells (Fig. 1B). Since each of these cells exhibits RDS, phos-phorylation of either Nbs1p70 or full-length Nbs1 (present inA-TLD cells) is not sufficient for activation of the DNA dam-age-induced S-phase checkpoint.

Nbs1 associates with E2F1. S-phase checkpoint failure inNbs1p70-containing cells, notwithstanding IR-induced phosphor-ylation, suggested that the Nbs1 N-terminal region is required forcheckpoint activation. We undertook a yeast two-hybrid screen toidentify protein interactions mediated by the FHA and BRCTdomains in the Nbs1 N terminus. Among 35 positive interactorsrepresenting 5 distinct genes, 2 encoded nearly full-length humanE2F1 (aa 27 to 437). The interaction with E2F1 occurred via theN terminus of Nbs1 (Fig. 2A, p95N) (data not shown).

FIG. 1. IR-induced Nbs1 phosphorylation is compromised in A-TLD2 but not in A-TLD3 or NBS cells. (A) Nbs1p70 is phosphorylatedafter IR. NBS cells were irradiated with 20 Gy (1) or were mocktreated (2) and harvested at 45 min post-IR. Nbs1 immunoprecipita-tions and lambda phosphatase treatments (l PP) were performed,resolved by SDS-PAGE, and immunoblotted with Nbs1 antiserum.The migration of a 68-kDa molecular size marker protein is indicated.(B) Lymphoblasts from two different A-TLD patients (A-TLD2 andA-TLD3), A-T lymphoblasts (AT3LA), and a normal control (721)were mock treated (2) or irradiated (1) with 20 Gy and harvested foranalysis at 45 min after treatment. Nbs1 immunoprecipitations wereperformed, and each immunoprecipitation reaction mixture was eithermock treated (2) or treated with lambda phosphatase (1) as de-scribed for panel A.

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Antibodies to E2F1 and E2F2 immunoprecipitated Nbs1and Mre11 from normal human lymphoblast cell lysates (Fig.2B and data not shown) as well as from Raji cells and murineembryonic stem cells. E2F1 and E2F2 antibodies failed tocoimmunoprecipitate Nbs1p70 from NBS lymphoblasts (Fig.

2B), and Nbs1p70 did not associate with E2F1 in yeast two-hybrid assays (Fig. 2C). Thus, the FHA and BRCT domainsmissing from Nbs1p70 are required for Nbs1’s association withE2F1 but not Mre11.

Deletion analyses indicated that the E2F1 Nbs1 interaction

FIG. 2. Nbs1 associates with E2F1. (A) The N terminus of Nbs1associates with E2F1. The N terminus of Nbs1 (p95N; aa 1 to 180) wastested for association with E2F1 (aa 284 to 416), empty vector, or anonspecific control (SNF1) by yeast two-hybrid testing. Positive (Nbs1)and negative (vector and SNF4) controls for E2F1 association areshown. Growth on Trp-Leu-Ade and the Trp-Leu control plates is shown;results on Trp-Leu-His plates were identical. (B) Coimmunoprecipi-tation of Nbs1 with E2F1 in wild-type but not NBS cells. Lysates wereprepared from wild-type (721) or NBS (DST) lymphoblasts, and im-munoprecipitations (IP) were performed with E2F1 or E2F2 antibod-ies or with Nbs1 or preimmune (PI) antiserum. The immune com-plexes were resolved by SDS-PAGE and serially immunoblotted (IB)with Nbs1 antiserum, E2F1, E2F2, and Rb antibodies. Reciprocal IPsfrom murine embyronic stem (ES) cell extracts were carried out withNbs1 antiserum 93, E2F1 antiserum 70, and Mre11 antiserum 59 (58)immunoblotted with Nbs1 93 and KH95. (C) Nbs1p70 associates withMre11 but not E2F1. GAL4 DNA binding domain fusions of full-length Nbs1 or Nbs1p70 were tested for interaction with GAL4 activa-tion domain fusions of E2F1 (aa 284 to 416), Mre11, empty vector, ora nonspecific control (SNF4). Growth on Trp-Leu-His and the Trp-Leu control plates is shown; results on Trp-Leu-Ade plates were iden-tical. (D) Deletion analysis of the E2F1-Nbs1 interaction domain.Various fragments of E2F1 were cloned as fusions to the GAL4 acti-vation domain and were tested for the ability to associate with full-length Nbs1 fused to the GAL4 DNA binding domain. The portions ofeach E2F1 fusion protein are shown as bars under the diagram depict-ing the locations of known motifs and interaction domains in E2F1(55). A positive interaction (1) was defined as growth on both His andAde test plates, and a weak interaction (1/2) was scored as growth onHis plates only. The shaded boxes represent minimally defined regionsof E2F1 necessary for Nbs1 association.

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domain (Fig. 2D) encompassed portions of the marked box (aa284 to 320) (30) and the transactivation and Rb-binding do-mains (aa 363 to 416) (55). Neither of these regions of E2F1alone was sufficient for interaction with Nbs1 (Fig. 2D). Al-though Rb immunoprecipitated with E2F1 and Nbs1 (Fig. 2B),this assay could not distinguish whether the Mre11 complex-E2F1 association is exclusive of E2F1 association with Rb.Since the Mre11 complex is much more abundant than E2F1,it is expected that most Mre11 complex members are notassociated with E2F. Nonetheless, E2F1 is present in Nbs1 andMre11 immunoprecipitates from murine embryonic stem cellextracts (Fig. 2B).

E2F1 targets the Mre11 complex to E2F sites. Recent find-ings for Drosophila melanogaster suggest that dE2F is impor-tant for the proper localization of the origin recognition com-plex (ORC) complex within the chorion gene cluster duringembryogenesis. This effect does not require dE2F transcrip-tional activity, suggesting that dE2F acts directly at or nearorigins of replication (51).

The Epstein-Barr virus (EBV) latent origin of replication,oriP, is regulated similarly to chromosomal origins during nor-mal growth as well as after DNA damage (1, 13, 26, 71).Therefore, we examined oriP as a potential site of Nbs1-E2Finteraction. Two E2F binding sites were found within 400 bp oforiP (Fig. 3A), as determined by DNA sequencing and mobilityshift assays (data not shown). Chromatin immunoprecipita-tions were prepared from formaldehyde-cross-linked, logarith-mically growing cells to test whether Nbs1 and E2F1 boundE2F sites near oriP and at chromosomal loci linked to repli-cation origin activity. Nbs1 (and Mre11) and E2F family mem-bers bound near EBV oriP in Raji cells containing an oriPepisome (Fig. 3B and data not shown). Nbs1, presumably viaE2F2, also bound at chromosomal E2F sites in the vicinity ofthe c-myc promoter, a region which has been shown to containreplication origin activity (62) (Fig. 3B).

We tested whether Nbs1-E2F1 binding to origin-proximalE2F sites was altered during S phase. Chromatin immunopre-cipitations were performed on synchronized cells harvested attime points during S-phase progression (Fig. 3D) and wereexamined for E2F site occupancy. Nbs1 and E2F1 binding atthe c-myc E2F site as well as at the episomal oriP site increasedmarkedly as cells progressed from G1/S phase to mid-S phase(Fig. 3E). In contrast, E2F3 and E2F4 occupancy did not ap-pear to be modulated during S-phase progression. These dataFIG. 3. Nbs1-E2F1 localizes to EBV oriP. (A) Location of oriP E2F

target sites. A segment of the EBV genome, nt 7000 to 9600 (70),including oriP, is depicted. The FR and DS elements (large gray boxes)of oriP are located at nt 7421 to 8043 and nt 8891 to 9131, respectively.The two E2F elements (small gray boxes with their correspondingsequences) are located at nt 7051 to 7058 and 9418 to 9425, respec-tively. Primers for chromatin immunoprecipitation are at the 39 end ofthe FR element at nt 8099 and 8412. (B) Nbs1 localization to E2Ftarget sites in log phase cells. Chromatin immunoprecipitations (IP)were performed on log phase Raji cells carrying an episomal plasmidcontaining a mutant E2F site in the c-myc promoter (mycDE2F),allowing Ets1/2 binding. Primers near E2F sites in oriP and the pro-moters of c-myc and mycDE2F were used to amplify DNA fragmentsfrom the indicated immunoprecipitations. Input, 0.1% of the totalisolated chromatin; mock, no-antibody control; Pre Imm, Nbs1 preim-mune antiserum (#16). PCRs were performed in the linear range (seeMaterials and Methods). (C) The Mre11 complex fails to bind to E2Fsites in NBS cells. Chromatin immunoprecipitations were performedas described in the legend to panel B with the indicated antibodiesfrom formaldehyde-cross-linked NBS lymphoblasts, and PCR was per-

formed with c-myc-specific primers to detect bound DNA. PI, preim-mune. (D) Cell cycle profiles of synchronized Raji cells used for theexperiment depicted in panel E. Cells were synchronized at the G1/Sborder and released into S phase. Cells were harvested for propidiumiodide staining and for chromatin immunoprecipitations (shown inpanel E). The cells were harvested at 0 h (G1/S phase), 3 h (early Sphase), 5 h (mid-S phase), and 7 h (late S phase) after release fromsynchrony. A representative log phase profile is shown for comparison.(E) Nbs1 localization to E2F sites in synchronized cells. Raji cells weresynchronized at the G1/S border, released into S phase, and harvestedat various times after release (given in the legend to panel D). Chro-matin immunoprecipitations using the indicated antibodies were per-formed, and PCR using primers specific to the indicated loci was usedto amplify those DNA fragments in the immunoprecipitates.

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suggest the E2F1-Nbs1 interaction at the origin-proximal E2Fsite is enhanced when replication origins are active.

Neither Nbs1 nor E2F bound to an episomal c-myc pro-moter in which the E2F site was mutated to allow Ets1/2binding (mycDE2F; Fig. 3B), nor were they found at a genomiclocus (Fra1) lacking E2F binding sites (data not shown). InNBS cells, neither Nbs1p70 nor Mre11 bound to E2F sites, where-as E2F1 binding was unaffected (Fig. 3C). Thus, Nbs1 bindingto E2F sites was dependent on E2F1.

A-TLD but not A-T cells are deficient in Nbs1-E2F1 associ-ation. To determine whether lack of Nbs1-E2F1 interactionwas correlated with abrogation of the S-phase checkpoint, wecarried out immunoprecipitations from A-TLD and A-T cells.The Mre11 complex-E2F1 association was reduced in bothA-TLD2 (R633X Mre11 mutant [58]) and A-TLD3 cells(N117S Mre11 mutant) but remained intact in A-T cells (Fig.4). Rb coimmunoprecipitated with E2F1 in both types of A-TLD cells (Fig. 4), demonstrating that the defective Nbs1-E2F1 association was intrinsic to the mutant Mre11 complex.Thus, disruption of the Mre11 complex-E2F1 association iscommon to Mre11 complex mutant cells that lack the S-phasecheckpoint. Reduced levels of Nbs1-E2F complexes in A-TLDcells may reflect that Nbs1 is less abundant and is primarilycytoplasmic in these mutants (58). This interaction remainsintact in A-T cells, consistent with the hypothesis that bothNbs1 phosphorylation and E2F interaction are necessary forS-phase checkpoint activation.

The Mre11 complex localizes with replication sites. Thephenotypic features of mammalian and yeast Mre11 complexmutants suggest that the complex plays an important role inthe regulation of DNA replication in response to DNA dam-age. We adapted an in situ fractionation technique to assesswhether the cytologic behavior of the Mre11 complex during

cell cycle progression reflected its S-phase-dependent associa-tion with origin-proximal E2F sites (24, 37). We found that 10to 20% of normally growing cells exhibited Mre11 and Nbs1foci similar to those observed in irradiated cells with the samein situ fractionation conditions (37) (Fig. 5 and data notshown). To determine whether the focus-positive cells were inS phase, normal human fibroblasts were pulsed with BrdU andstained for both BrdU and Mre11. Nuclei with Mre11 foci wereBrdU positive (Fig. 5a). PCNA positivity following extractionwas also used to identify S-phase cells (8). As with BrdU in-corporation, Mre11 foci were only seen in PCNA-positive cells(Fig. 5b). These data indicated that the Mre11 complex wasassociated with sites of DNA replication under normal (i.e.,nonstressed) growth conditions.

PCNA-containing DNA replication foci undergo character-istic changes as replication progresses (8, 24). We examinedthe temporal and spatial relationship between Mre11 andPCNA foci during S-phase progression. At the G1/S transition,PCNA is localized at a few discrete sites prior to the appear-ance of nascent DNA (24). These early PCNA foci colocalizedwith many Mre11 foci, consistent with chromatin immunopre-cipitation data placing the complex at sites adjacent to repli-cation origins (Fig. 6, top row). In early- to mid-S phase, Mre11and PCNA foci were distributed throughout the nucleus andwere substantially colocalized (Fig. 6, middle row). In late Sphase, PCNA staining is localized at heterochromatic regionscontaining late replicating DNA (24). The majority of theselate replication clusters contained both PCNA and Mre11 (Fig.6, bottom row). Thus, colocalization of Mre11 was observedwith normal DNA replication patterns throughout S phase.

We tested whether Mre11 complex localization to repli-cation sites during normal S phase was ATM dependent. A-Tfibroblasts were double labeled for Mre11 and PCNA. The pat-tern of Mre11 (and Nbs1; data not shown) and PCNA stainingin S-phase A-T cells was indistinguishable from that of normalcells (Fig. 7). Thus, as in cells treated with IR (37), ATM is notrequired for relocalization of the Mre11 complex in S phase.

DISCUSSION

In this study, we demonstrated that the Mre11 complexphysically associates with E2F protein family members via theN terminus of Nbs1. We established evidence that the Nbs1-E2F interaction occurs near an origin of DNA replication andthat interaction at those sites is essentially restricted to S-phasecells, suggesting that the interaction coincides with origin ac-tivity. Abrogation of the interaction is correlated with S-phasecheckpoint deficiency—the failure to suppress origin firingin response to DNA damage—irrespective of ATM-mediatedphosphorylation of Nbs1, suggesting that this interaction mayfacilitate both positive and negative influences on DNA repli-cation. Mre11 complex proteins were also associated with sitesof DNA replication removed from the replication origin. We

FIG. 4. Nbs1-E2F1 association is maintained in A-T cells but isreduced in A-TLD cells. Immunoprecipitations (IP) were performedas described in the legend to Fig. 2B from the indicated cell lines andwere immunoblotted serially with Nbs1 and Rb antibodies. WT, wildtype; PI, preimmune.

FIG. 5. The speckled Mre11 focus pattern is found in nonirradiated S-phase cells. (a) Speckled Mre11 foci are contained in BrdU-positive cellsand overlap with BrdU incorporation. Nonirradiated 37Lu human fibroblasts were doubly stained for Mre11 (top panel, red signal) and BrdUincorporation (middle panel, green signal), and the images were merged (bottom panel). Overlap between the two signals appears yellow. Thethree nuclei that do not stain with BrdU correspond to those containing promyelocytic leukemia protein-associated Mre11 foci (37). (b) SpeckledMre11 foci are contained in PCNA-positive S-phase cells. Cells were doubly labeled for Mre11 (top panel, red signal) and PCNA (middle panel,green signal), and the images were merged (bottom, yellow signal). Overlap between the two signals appears yellow.

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observed colocalization with PCNA at sites of DNA synthe-sis throughout S phase. Together, these data suggest that theMre11 complex is important in both the regulation and com-pletion of DNA replication.

Activation of the S-phase checkpoint suppresses the firing ofreplication origins, whereas the progression of established rep-lication forks is essentially unimpeded (27, 28, 42, 50). Thetargets of E2F transcriptional control include genes that en-

FIG. 6. Mre11 complex and PCNA colocalization during S-phase progression in normal cells. 37Lu fibroblasts were doubly labeled with Mre11(red) and PCNA (green) as for Fig. 5b, and the images were merged. Overlap between the two signals in merged images appears yellow. Top row,early S phase; middle row, early- to mid-S phase; bottom row, mid- to late-S phase. Stages were determined by the method of Kill et al. (24).

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code enzymes required for DNA synthesis; these enzymesare presumably required for replication fork progression (16).Thus, it is unlikely that the Nbs1-E2F1 interaction influencesthe S-phase checkpoint through altering E2F1-dependent tran-scriptional regulation. Supporting this interpretation, we found

that gamma irradiation of murine embryonic fibroblasts orhuman lymphoblastoid cells transfected with an E2F1-depen-dent luciferase reporter did not alter luciferase expression(data not shown).

Chromatin immunoprecipitation demonstrated E2F-Nbs1

FIG. 7. Mre11 colocalization with PCNA in A-T fibroblasts. Cells and images were prepared as described in the legend to Fig. 6. Top row, earlyS phase; middle row, early- to mid-S phase; bottom row, mid- to late-S phase. Stages were determined by the method of Kill et al. (24).

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occupancy of E2F sites in close proximity (within 500 nucleo-tides [nt]) to replication origins at both oriP and c-myc (62).These data suggest that a subset of E2F sites may be associatedwith replication origins in human cells. The E2F-Nbs1 in-teraction at sites adjacent to replication origins was largelyrestricted to cells in S phase (Fig. 3D and E). Therefore, wehypothesize that the influence of the Mre11 complex and E2F1on origins of DNA replication is direct. Precedent for thisinterpretation comes from D. melanogaster. During Drosophilaembyrogenesis, endoreduplication of the chorion gene clusterin follicle cells leads to amplification of the chorion genes (11,41). The switch from normal replication to endoreduplicationis associated with relocalization of the ORC complex at repli-cation origins within the chorion gene cluster (51). dE2F mu-tants impair ORC complex localization to those sites. Theeffect is seen in a dE2F mutant with reduced DNA bindingactivity that retains transactivation and Rb binding functions.Conversely, ORC localization is normal in a dE2F mutant thatretains DNA binding capacity but lacks transactivation and Rbbinding activity (51). Finally, evidence for direct physical in-teraction between dE2F and the ORC complex has been es-tablished (5), consistent with a similar role for human E2F1.These data support a model wherein the sequence-specificDNA binding of E2F1 directs the Mre11 complex to origins ofreplication where its functions are relevant to origin function.Such recruitment of Mre11 complex functions is also observedat mammalian telomeres via the complex’s interaction with thetelomere protection protein, TRF2 (74). Interestingly, the pre-sumptive TRF2 binding sequence, TTAGGG, is found at two

sites in oriP and one site in the myc promoter region. Sincechromatin immunoprecipitation of oriP with Nbs1 antiserumdepends on E2F binding (Fig. 3B), TRF2-Mre11 complex in-teraction does not occur at those sites.

Previous studies established that the complex’s functionin S-phase checkpoint activation required phosphorylationof Nbs1 by ATM (31, 72). In this study, we found that phos-phorylation of Nbs1p70 and Nbs1 occurred in gamma-irradi-ated NBS and A-TLD3 cells, respectively, indicating that thisevent is not sufficient for checkpoint activation. We demon-strated that NBS and A-TLD cells were deficient in Nbs1-E2F1association. Hence, Nbs1-E2F interaction and Nbs1 phosphor-ylation may both be required for suppression of origin firing inresponse to DNA damage. The interaction of E2F1-Nbs1 onchromatin or in protein extracts was not affected by DNAdamage (data not shown). This suggests that ATM acts on thecomplex at the sites of DNA replication and presumably altersits activity. This view is consistent with previous data thatsuggest ATM phosphorylates Nbs1 when it is bound to DNAdamage (37) (Fig. 8).

The nature of the change in activity imparted by Nbs1 phos-phorylation and the function(s) of the Mre11 complex at sitesof DNA replication remain to be established. Previous analyseshave not implicated these proteins in origin firing or in theassembly of the ORC complex (3, 17). The Mre11 complexmay mediate the same functions proximate to origins and atreplication forks, as suggested for the minichromosome main-tenance proteins (17). Like the homologous SbcCD complex(14), the Mre11 complex may process DNA secondary struc-

FIG. 8. Functional interaction of ATM and the Mre11 complex. The Mre11 complex relocalization in response to DNA damage (37) and duringDNA replication is independent of ATM. We therefore infer that the Mre11 complex is situated at spontaneously arising DNA breaks or proximalto sites of DNA replication as part of its function in normally growing cells. We propose that upon the induction of DNA damage, ATM is activatedand acts on the Mre11 complex engaged at those structures. This implies that the complex’s role in S-phase checkpoint activation is partiallydependent upon ATM effects on the complex’s function at those sites. It is conceivable that ATM modification may also enhance Mre11 complexDNA repair functions, as has been suggested for Tel1-dependent modification of the S. cerevisiae Mre11 complex (65).

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tures that arise at replication forks. Its presence at origins mayreflect the processing of secondary structures forming at sitesof localized helical distortions during the initiation of DNAsynthesis.

An alternative possibility is suggested by the behavior ofS. cerevisiae Mre11 complex mutants in the initiation of meioticrecombination. The formation of nuclease hypersensitive sitesin meiotic chromatin prior to the initiation of meiotic recom-bination is altered in Mre11 mutants (19, 40). The Mre11complex could affect DNA replication origins by influencinganalogous changes in local chromatin architecture (3), withspecificity for replication origins conferred by E2F1. Modula-tion of chromatin structure at the origin may also be governedby E2F1. E2F-Rb and related complexes have been shown torecruit histone modification proteins that alter local chromatinstructure during transcriptional activation (18, 48). A similarrole for dE2F in modulation of chromatin structure at repli-cation origins has been proposed (51).

The colocalization of Mre11 complex proteins with PCNAthroughout S phase indicates that the complex also functions atestablished replication forks. Recombination and replicationare intimately linked (49). Homologous recombination re-quires both leading and lagging strand replication proteins(23). Conversely, DNA recombination is required to reestab-lish collapsed replication forks (15, 34, 53), and DNA recom-bination intermediates are formed during DNA replication innormally growing S. cerevisiae cells (75).

The complex’s role in DNA recombination (9, 21) may re-flect that human Mre11 complex proteins colocalized withPCNA are functioning in the resolution of damaged or stalledreplication forks. This interpretation is consistent with thefunctions of the bacterial homologue of the Mre11 complex,SbcCD, which has been proposed to degrade secondary struc-tures on the lagging strand template and thereby inducesrecombination between sister chromatids. This mechanismwould stabilize DNA sequences, such as palindromes, that areprone to form aberrant DNA structures (14, 29). It is likelythat the spontaneous genomic instability observed in Mre11complex mutant cells stems from reduced efficiency in thisfunction and that this contributes to the disease phenotypesobserved in NBS and A-TLD patients. The loss of Mre11complex-E2F interaction must also be considered as a contrib-uting factor in the diverse pathology associated with thosesyndromes.

ACKNOWLEDGMENTS

We thank D. Eick for the gift of Raji 525-7 cells, T. de Lange forhelpful discussion and comments, and Allen Edmonds for critical in-sight.

This work was supported by the Milwaukee Foundation (J.H.J.P.),the National Institutes of Health (GM59413 to J.H.J.P., CA09681 toJ.W.), the Department of Energy (J.H.J.P.), and the Public HealthService (CA45240 to P.J.F.).

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