The Transcriptional Histone Acetyltransferase Cofactor TRRAP Associates with the MRN Repair Complex and Plays a Role in DNA Double-Strand Break Repair

Abstract

Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

Transactivation-transformation domain-associated protein (TRRAP; also called PAF400) is a highly conserved 434-kDa protein, which specifically interacts with c-Myc and has homology to the ataxia-telangiectasia-mutated (ATM)/phosphatidylinositol 3-kinase (PI-3 kinase) family; however, critical residues required for kinase activity are not conserved in the kinase-like domain of TRRAP (37, 49). Null mutation of TRRAP in mice indicated that TRRAP is essential for early development and required for the mitotic checkpoint and normal cell cycle progression (26). Both TRRAP and its yeast orthologue Tra1 (designated yTra1) have been identified as subunits of two distinct types of histone acetyltransferase (HAT) complexes, containing either GCN5-type HATs (i.e., TATA binding protein [TBP]-free TBP-associated factor [TAF]-containing complex [TFTC], STAGA, or GCN5/PCAF complexes in humans or SAGA in yeast) (9, 23, 36, 40), or the TIP60/Esa1 type HATs (i.e., TIP60 or NuA4 complexes) (1, 17, 29). In addition to TRRAP/Tra1, the GCN5-type HAT complexes all contain conserved subunits belonging to the ADA, SPT, and TAF family of proteins (35), and the TIP60/Esa1 type NuA4 complexes also contain subunits (i.e., p400, DMAP1, enhancer of polycomb protein 1 [EPC1], TIP48, TIP49, BAF53a, and β-actin) with conserved composition from yeast to humans (references 17, 22, and 41 and references therein). Human GCN5/PCAF and yeast Gcn5 preferentially acetylate histone H3, while human TIP60 and its yeast orthologue, Esa1, target histone H4 (reference 12 and references therein). In addition, various human TRRAP-containing complexes have been described without GCN5 or TIP60 but including several NuA4 subunits (i.e., p400, EPC1, BAF53, TIP48, and TIP49) (22, 41). Both TRRAP and yeast Tra1 proteins were shown to serve as targets for transcriptional activators in both TFTC/SAGA and NuA4 complexes (10, 18, 20, 31, 34, 51). Thus, both TRRAP and Tra1 are important for the regulation of transcription and cell cycle progression and are required for cell viability. In addition, these HAT complexes seem to be necessary for chromatin modifications involved in DNA repair (43). TFTC and STAGA HAT complexes were implicated in UV-damaged DNA recognition, chromatin modification, and nucleotide excision repair (8, 35), while the yeast Esa1-containing NuA4 HAT complex is recruited specifically to DNA double-strand breaks (DSBs) that are generated in vivo to acetylate histones (6). The human TIP60 HAT complex was suggested to play a similar role in DSB repair (29). In agreement, the Drosophila melanogaster TIP60 chromatin-remodeling complex acetylates nucleosomal DSB marker phospho-H2Av and replaces it with an unmodified H2Av (30).

The induction of DSBs activates cell cycle checkpoint responses and the DNA repair machinery. There are two major DSB repair pathways in higher eukaryotes: homologous recombination and DNA end joining (21, 24). Although many candidate sensor proteins have been identified through cytological, biochemical, and genetic studies to participate in DSB-induced checkpoint activation, including the PI-3 kinase members ATM/ATM-Rad3 related (ATR) and DNA-dependent protein kinase, the exact mechanism of DSB detection remains unclear. MRE11, RAD50, and NBS1 form a highly conserved protein complex (the MRN complex) that is involved in signaling and repair of DSBs (15, 45). The MRN complex is also a good candidate for primary DSB detection, since it has been shown to act as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules (32). The structural role of the MRN complex in bridging DNA ends is also well characterized; however, its enzymatic role is less well understood (16, 48).

Here, we describe the identification of TRRAP as a stable component of the MRN complex. The TRRAP-containing MRN complex is not associated with detectable HAT activity but is involved in DSB repair. Thus, as TRRAP is a component of complexes playing a role in DSB repair, the NuA4/TIP60 HAT complex and the TRRAP-MRN complex, it seems that TRRAP can function as a molecular link between DSB repair, signaling, and chromatin remodeling.

MATERIALS AND METHODS

Immunoprecipitation and Western blot analysis.

Routinely, proteins from 800 μg of HeLa cell nuclear extract were immunoprecipitated with 50 μl of protein G- or protein A-Sepharose (Pharmacia) and approximately 2 to 5 μg of the different antibodies (as indicated). Immunoprecipitations (IPs) and Western blot analyses were carried out as described previously (8) with the indicated primary antibodies.

The anti-human TRRAP (hTRRAP) polyclonal antibody (PAb) (1930) and the mouse monoclonal antibody (MAb) (2TRR-2D5) were described previously (25). The 2TRR-1B3 MAb was raised against amino acids 2005 to 2024 of human TRRAP as described previously (25). The following antibodies were used in this study: anti-TAF10, anti-TBP (50), anti-GCN5 (8), anti-MRE11, anti-NBS1, anti-RAD50 (Abcam), anti-BRG1 (39), anti-TIP48, anti-BAF53a (a gift from M. Cole), anti-TIP60 (a gift from D. Trouche and B. Amati), anti-p400 (22), and anti-EPC1 (2).

In vitro HAT assay.

The in vitro HAT assay was carried out as described previously (9).

Cell-free nonhomologous end-joining inhibition assay.

The normal human AAH-1 lymphoblastoid cell line was used to make nonhomologous end-joining (NHEJ)-competent protein extracts. The cell extract and the linear DNA template were prepared as described previously (3). Prior to the end-joining reactions, AAH-1 extract (100 μg) was diluted to 20 μl with end-joining buffer (50 mM Tris-HCl [pH 7.5], 60 mM potassium acetate, 0.5 mM Mg acetate, 1 mM ATP, 1 mM dithiothreitol [DTT], and 10-μg/ml bovine serum albumin). To deplete the different components, either 1 μg of the different rabbit polyclonal antibodies (an anti-yeast TBP PAb was used as a negative control) or 5 μg of purified mouse monoclonal antibodies (a purified MAb that does not recognize nuclear proteins was used as a negative control) was added to the extracts (see Fig. 4). Reactions were incubated for 1 h on ice, and then 25 μl of end-joining buffer-equilibrated protein G-Sepharose beads were added and further incubated for 30 min at room temperature with gentle shaking. Reactions were centrifuged at 1,000 rpm, and supernatants were recovered and incubated with 40 ng of a linearized DNA substrate at 37°C for 2 h. End-joining reactions were stopped and analyzed as described previously (3). Depleted supernatants were also tested by Western blot analysis.

FIG. 4.

TRRAP plays a role in NHEJ in vitro. (A) NHEJ-competent AHH-1 cell extracts were mock depleted with protein A-Sepharose alone or depleted with the indicated antibodies; the depletion efficiency was analyzed by Western blotting. (B) These extracts were tested for their end-joining capacity and compared to a nondepleted extract (lane 2) using a linear DNA template. The addition of protein A-Sepharose to the extract had an inhibitory effect on end-joining efficiency (lane 3). As negative control for the nonpurified rabbit PAbs, an anti-yeast TBP PAb was used (lane 4); for the purified mouse MAbs, a purified MAb that does not recognize any nuclear protein was used (lane 6). TRRAP was depleted by using two different MAbs as indicated. The reaction products were separated on a 0.7% agarose gel, blotted, and hybridized with a ³²P-labeled probe. The joining products, dimer, trimer and tetramer, represent end-to-end ligation of the linear input monomer. The end-joining efficiency was calculated for the reactions as described in panel C and is shown under each lane. (C) Statistical analysis of three independent experiments. Each bar represents the end-joining products together as a percentage of the total input signal. The error bars represent the average of three independent experiments.

Glycerol gradient sedimentation.

Molecular-weight markers (HMW; Pharmacia) and supernatant of the second IP (SN2) were first dialyzed against buffer D10 (Tris-HCl [pH 7.9], 10% glycerol, 0.1 mM EDTA, 0.5 mM DTT, 50 mM KCl), layered on a 20 to 40% linear glycerol gradient (3.8 ml), and centrifuged for 8 h at 55,000 rpm with an SW60 Ti rotor as described previously (5).

MALDI MS.

TRRAP-containing complexes were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Protein bands were visualized by Coomassie G250 (Bio-Rad) staining, excised, and in gel digested with trypsin (Roche). Gel-eluted peptides (0.5 μl) were mixed with an equal volume of saturated α-cyano-4 hydroxycinnamic acid (LaserBio Labs) in 50% acetonitrile and applied onto the mass spectrometry (MS) target. Mass measurements were carried out on a Bruker Reflex IV matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometer in the positive-ion reflector mode. The acquisition mass range was 800 to 5,000 Da with the low-mass gate set at 700 Da. Internal calibration was performed with autolytic trypsin peptides (MH⁺ with m/z = 842.51, 2211.11, and 2807.47). Monoisotopic peptide masses were assigned manually using the Bruker X-TOF software. Database searches were performed on Swiss-Prot and the National Center for Biotechnology Information human protein databases using Profound software (http://prowl.rockefeller.edu/) with standard parameters.

Plasmid-based host cell end-joining assay using a GFP expression vector.

The pEGFP-N3 (Clontech) plasmid was digested by EcoRI, which has a unique cutting site in the multiple cloning site between the cytomegalovirus (CMV) promoter and the green fluorescent protein (GFP) gene. Complete digestion was verified on an agarose gel and by transformation of 10 ng of linearized or circular pEGFP-N3 plasmid in Escherichia coli. Our tests indicated that the linear plasmid preparation was 99.43% digested.

To measure the end-joining activity of small interfering RNA (siRNA)-treated cells by counting GFP-positive cells, HeLa cells seeded in six-well plates at 40 to 50% confluence were transfected with 40 ng of anti-hTRRAP short double-stranded siRNA (the sequence is available upon request) or 40 ng of negative-control luciferase siRNA (from Dharmacon Research) with 3 μl of Lipofectamine 2000 (Invitrogen). Six hours later, the medium was replaced, and 1 μg of circular or linear pEGFP-N3 plasmid was transfected with JET-PEI (Polyplus Transfection). At 48 h and 72 h after transfection, cells were harvested and washed with cold 1× phosphate-buffered saline (PBS). Half of the cells were lysed in buffer B (20 mM Tris-HCl [pH 7.5], 400 mM KCl, 2 mM DTT, 20% [vol/vol] glycerol, and 1× protease inhibitor cocktail [2.5-ng/ml each leupeptin, pepstatin, chymostatin, antipain, and aprotin) to obtain protein extracts; TRRAP expression was analyzed by Western blotting (see above). The other half of the cells were submitted directly to fluorescence-activated cell sorter (FACS) analysis, and the percentage of green cells was automatically counted (for each sample, a total of 30,000 cells were counted).

Overexpression of TRRAP, MRE11, RAD50, and NBS1 in the baculovirus system.

The TRRAP cDNA was excised from the eukaryotic expression vector pcDNA3 (51) by SpeI and ClaI and cloned into the XbaI and BamHI sites of the pVL1392 expression vector using a ClaI-BamHI linker. SF9 insect cells were infected using recombinant baculoviruses expressing TRRAP, RAD50, NBS1, and MRE11 as described previously (7, 42). Cells were harvested 48 h after infection, washed in 1× PBS, resuspended in extraction buffer (20 mM Tris-HCl [pH 7.5], 250 mM NaCl, 10% glycerol, 2 mM phenylmethylsulfonyl fluoride, 10 mM NaF; 2 mM Na₃VO₄, 10 mM β-glycerophosphate, 1 mM β-mercaptoethanol), and homogenized by Dounce homogenization with a B-type pestle. Cell debris and insoluble material were removed by spinning at 12,000 × g for 20 min, and the crude protein extract was recovered. Anti-TRRAP and control IPs were performed as described for endogenous protein purification.

NHEJ assay in ES cells.

Generation of conditional knockout of TRRAP in mouse embryonic stem (ES) cell lines (Trrap^f/Δ) containing stably integrated hypoxanthine phosphoribosyltransferase (HPRT)-GFP substrate will be described elsewhere (R. Murr et al., in press). ES cells were grown in Dulbecco's modified Eagle's medium containing 15% fetal calf serum supplemented with penicillin, streptomycin, glutamine, and leukemia inhibitory factor. For all transfections, 50 μg of the expression vectors (pCBASce for I-SceI endonuclease; pMC-Cre for Cre recombinase) was electroporated into 2 × 10⁷ ES cells suspended in PBS in a 0.4-cm cuvette at 0.26 kV and 500 μF. Following electroporation, cells were grown on feeders and harvested by trypsinization. Protein extracts for Western blot analysis were prepared 48 h after electroporation and probed for the presence of TRRAP (polyclonal antibody 1930) (25) and MDC1 (for mediator of DNA checkpoint arrest 1; a polyclonal antibody that was a kind gift from J. Chen) as a loading control. For PCR detection of NHEJ, genomic DNA was isolated from ES cells and DNA surrounding the I-SceI site was amplified by PCR using primers #F15 (5′-GGTTCGGCTTCTGGCGTG) and #R2 (5′-GACCTCGAGTTACTTGTACAGCTCGTCCA). The PCR products were digested with I-SceI endonuclease (New England BioLabs), separated on 1.2% agarose gel, and stained with ethidium bromide. The density of DNA bands was quantified using a Fluor-S MultiImager (Bio-Rad).

RESULTS

Characterization of TRRAP-associated proteins.

To characterize TRRAP-associated proteins different from those present in the TFTC-type complexes (9, 50), we have developed a double-IP scheme (Fig. 1A). First, TRRAP and TRRAP-associated proteins were immunopurified from HeLa cell nuclear extract by using an anti-TRRAP polyclonal antibody. Bound proteins were extensively washed with a buffer containing 300 mM KCl and eluted with an excess of epitope peptide (E1) (Fig. 1B). This eluate (E1) contained TFTC-type complexes and additional TRRAP-containing complexes. The TBP, a basal transcription factor lacking in TFTC and other HAT complexes, was absent from immunoprecipitated E1 fraction, confirming the specificity of the experiment and the absence of other TBP-containing complexes in this fraction (Fig. 1B). To separate the TFTC/STAGA-type complexes from other TRRAP-containing complexes, we immunodepleted the E1 fraction in TAF10 (a TFTC/STAGA/PCAF-specific subunit) by using an anti-TAF10 monoclonal antibody (50); bound TFTC/STAGA complexes were eluted with an excess of epitope peptide (E2) (Fig. 1A). The supernatant of this second IP (SN2), the E2 fraction and the E1 fraction were then tested for the presence of TRRAP, GCN5, and TAF10 by Western blot analysis (Fig. 1B). TAF10 was completely depleted from the SN2 fraction, indicating that all the TFTC/STAGA/PCAF-type complexes were depleted from the SN2 fraction (Fig. 1B). Since TRRAP was also described as a subunit of the NuA4/TIP60 complexes (17, 22, 29), we verified the presence of some NuA4/TIP60 complex subunits in the SN2 fraction. TIP60, TIP48, and BAF53a proteins were detected in the SN2 fraction by immunoblotting, suggesting that this fraction contained the NuA4/TIP60 complex (Fig. 1C and data not shown). Interestingly, GCN5 was present not only in the TFTC-type complexes (E2 fraction) but also in the SN2 TRRAP-containing fraction free of TFTC (Fig. 1B), suggesting that TRRAP and GCN5 exist together in an as-yet-uncharacterized complex (without TAF10), since no GCN5 has been identified in the NuA4/TIP60 complexes (17, 29). The SN2 fraction was then resolved by SDS-PAGE, and proteins were analyzed by MALDI MS to identify TRRAP-associated proteins. As expected, we have identified several components of the NuA4/TIP60 complex in this TRRAP-containing SN2 fraction: p400, DMAP1, TIP49, TIP48, BAF53a, β-actin, and TRCp120 (Fig. 1D and data not shown). Unexpectedly, we also identified three proteins, MRE11, NSB1, and RAD50 (Fig. 1D), which have never been described in association with TRRAP and which have been described to form the MRN complex that plays a role in DSB repair. BRG1 and several other components of the SWI/SNF complex were also identified (Fig. 1D).

FIG. 1.

Purification, separation, and characterization of different hTRRAP-containing complexes. (A) Schematic representation of the purification of TRRAP-containing complexes. NE, HeLa cell nuclear extract; E, eluate; SN, supernatant. (B) TRRAP-containing complexes were separated as depicted in panel A and analyzed by Western blotting with the indicated antibodies. (C) Immunodetection of subunits of the NuA4/TIP60 complex, TIP60 and BAF53a, in the SN2 fraction. (D) Determination of the TRRAP-associated proteins in the SN2 fraction by MALDI-TOF MS analysis. Proteins associated with TRRAP in the SN2 fraction were separated on a 10% SDS-PAGE gel, visualized by Coomassie brillant blue staining, and photographed; the bands were then cut and processed for MALDI-TOF MS analysis. Migration of molecular mass standards is depicted on the left of the panel in kilodaltons. The identified proteins, their approximate position in the original gel, and their percentage of sequence coverage by the obtained masses are shown on the right of the panel. Protein species, which were also present in a control IP together with collagen and albumin, were considered as nonspecific contaminants (NS). The position of the heavy chain of the antibody is indicated (immunoglobulin GH [IgGH]).

Characterization of the specificity of interaction between TRRAP and the MRN complex.

Since TRRAP has not been reported to be associated with the MRN complex, we verified the specificity of the association between TRRAP and the components of the MRN complex. TRRAP-containing complexes were immunoprecipitated as before, antibody-bead-bound proteins were washed with IP buffers with increasing stringency, and the retained complexes analyzed by Western blotting (Fig. 2A). These experiments confirmed that the association between TRRAP and the components of the MRN complex was specific and stable, since MRE11 and RAD50 were detected in the anti-TRRAP IP even after the beads were washed with the 0.8 M KCl-containing buffer (Fig. 2A). The reciprocal experiment was also carried out with an anti-MRE11 antibody to further verify the stable association of TRRAP with the MRN complex (Fig. 2B). As expected, TRRAP could be detected in the reciprocal anti-MRE11 IP after the beads were washed with the 0.5 M KCl-containing buffer (Fig. 2B). Under higher-stringency washing conditions, TRRAP was dissociated from the MRN complex (Fig. 2B). The difference between the resistance of the TRRAP-MRN complex to the different salt concentrations during the anti-TRRAP or anti-MRE11 IPs could be explained by the epitopes of the antibodies with respect to the TRRAP-MRN interaction surface. In the corresponding control IPs, neither TRRAP nor components of the MRN complex were detected. In contrast, the association of SWI/SNF complex with TRRAP, detected in the SN2 fraction by mass spectrometry, did not resist these stringent washing conditions as monitored with an anti-BRG1 antibody (Fig. 2A). Thus, we considered the interaction between TRRAP and the SWI/SNF complex to be less specific.

FIG. 2.

Stable and specific association between TRRAP and subunits of the MRN complex. TRRAP-containing (A) or MRE11-containing (B) complexes have been immunoprecipitated with corresponding purified antibodies. In parallel, negative control IPs were carried out with polyclonal antibodies recognizing yeast or Drosophila proteins. Bound proteins were washed with IP buffer containing the indicated KCl or NP-40 (1% NP-40) concentrations. Beads were then boiled and bound proteins were separated on a SDS-PAGE gel. The indicated inputs represent 10% of the total extract used for the IP. The presence of the indicated proteins was tested by Western blotting. (C) Glycerol gradient sedimentation analysis of the TRRAP-containing SN2 fraction. A total of 200 μl of SN2 was loaded to a 20 to 40% glycerol gradient and centrifuged for 8 h at 55 krpm, using an SW60Ti rotor. Sedimentation standards were centrifuged in a parallel gradient. Fractions (each 200 μl) were collected. Aliquots (each 20 μl) from indicated fractions were resolved parallel with the unfractioned SN2 extract on a SDS-PAGE gel, transferred to a nitrocellulose filter, and probed with the indicated antibodies. The position of the 0.67-MDa molecular mass marker, as well as some extrapolated molecular masses, are indicated on the top of the panel. The numbers of the analyzed fractions are indicated at the bottom of the panel. (D) TRRAP- or MRE11-containing complexes were immunoprecipitated with the corresponding antibodies or an unrelated control antibody (as indicated) and immunoprecipitated proteins were analyzed by Western blotting with the indicated antibodies. (E) TRRAP interaction with the MRN is direct. Sf9 cells were coinfected with recombinant baculoviruses expressing TRRAP, MRE11, RAD50, and NBS1 for 48 h; cell extracts were made; and proteins were immunoprecipitated using a polyclonal anti-TRRAP antibody or a mock antibody. Antibody-bead-bound proteins were analyzed by Western blotting with the indicated antibodies.

The comparison of the amounts of TRRAP and MRN complex components in the anti-TRRAP or anti-MRE11 IPs indicates that in the cells a portion of the MRN complex can stably associate with TRRAP. In spite of the fact that the association of TRRAP with the MRN complex is specific, it is weaker than the association of the other components of the MRN complex, which cannot be dissociated by very-high-salt washing conditions (Fig. 2B), suggesting that TRRAP is recruited to the MRN complex at a later step from a free pool of TRRAP (see Discussion). The presence of ethidium bromide in the different IP buffers did not change the association of TRRAP with the MRN complex (data not shown), indicating that TRRAP association with the MRN complex is independent of DNA. Thus, TRRAP seems to be stably associated with the MRN complex. In good agreement with our finding, a nonidentified 400-kDa component in the highly purified MRN complex was already described (11).

Next, the different TRRAP-containing complexes present in SN2 fraction were separated on a glycerol gradient to test whether the TRRAP-MRN complex could be identified as an independent entity. Immunoblot analysis of the different fractions indicated that in fraction 9, corresponding to masses of around 0.8 to 1 MDa, TRRAP, MRE11, NBS1, and RAD50 cosedimented, suggesting that this fraction contained a complex that would be composed of TRRAP and MRN (Fig. 2C). We did not detect either GCN5 or TIP60 HATs in this fraction by Western blot analysis. As the E1A binding protein complex lacks the TIP60 HAT but contains TRRAP, p400, the human homologue of the Drosophila EPC1 and other subunits (22), we tested whether p400 and EPC1 would be present in an MRE11 IP together with TRRAP. Our Western blot analysis showed that in an MRE11 IP neither p400 nor EPC1 are associated with the MRN complex (Fig. 2D), while in the TRRAP IP they could be detected, further suggesting that TRRAP, MRE11, NBS1, and RAD50 can stably associate without at least the tested subunits of previously described TRRAP-containing complexes.

To further verify whether the interaction between TRRAP and the three subunits of the MRN complex is direct, we coexpressed TRRAP, MRE11, RAD50, and NBS1 by recombinant baculoviruses in Sf9 cells. Whole-cell extracts were made, and TRRAP-associated proteins were purified by an anti-TRRAP IP and analyzed by Western blotting (Fig. 2E). All the three components of the MRN complex were coimmunoprecipitated with TRRAP, while in a control IP, carried out with an unrelated antibody, none of these proteins were present (Fig. 2E). These results demonstrate that TRRAP and MRN can directly be associated in the cells, independently of other factors.

TRRAP-MRN complex is not associated with a detectable HAT activity.

Using an in vitro acetylation test, we have first demonstrated that the purified hTRRAP-containing complexes have the expected HAT activities (Fig. 3A). TFTC/STAGA/PCAF complexes acetylated mainly histone H3 (TFTC and E2) (Fig. 2A), while the SN2 fraction that contained the TIP60/NuA4 HAT complex and another TRRAP/GCN5 complex (see above) acetylated histones H3 and H4 and, to a lesser extent, H2A (Fig. 3A). These results were in good agreement with Western blot analysis (Fig. 3A, top) and confirmed an efficient purification of TRRAP complexes with the corresponding HAT activities. Since the TRRAP-containing TIP60/NuA4 complex has been involved in linking HAT activity, DSB repair, and chromatin remodeling (29), we examined the ability of the MRN/hTRRAP complex to acetylate histones. To this end, purified resin-antibody-bound complexes were used. While no GCN5 was found in the anti-MRE11 or anti-NBS1 IPs (Fig. 2B and data not shown), we were unable to verify the presence of TIP60 in the same IPs by Western blotting, as TIP60 migrated at the same place as the heavy chains of the antibodies. Anti-TRRAP antibody-bound complexes showed the same acetylation pattern as the eluted TRRAP-containing SN2 fraction (compare Fig. 3A, lane 4, with Fig. 3B, lane 1). In contrast, no HAT activity was detected when the anti-MRE11 antibody-bound complex (Fig. 3B, lane 2) or the anti-NBS1 antibody-bound complex (lane 3) was used; both these contained the MRN complex and hTRRAP (Fig. 3B, top). Note that we did not detect any HAT activity in lanes 2 and 3, even when gels were exposed for a very long time (data not shown). In contrast to the TIP60/NuA4 HAT complex, the TRRAP-containing MRN complex did not have detectable HAT activity. Thus, it seems that the role of TRRAP in this complex is other than the direct recruitment of a HAT activity to DSB sites.

FIG. 3.

The TRRAP/MRN complex is not associated with a detectable HAT activity. Histone acetylase activity was measured using free histones as substrates and the indicated purified and eluted protein fractions (A) or protein G-Sepharose resin (Res)-antibody-bound complexes (B). The amount of the different protein complexes (Fig. 1A) was verified by Western blotting (WB). Histones were separated by SDS-PAGE and stained with Coomassie blue (CBB), and the acetylated histones were visualized by autoradiography (bottom). The positions of each of the core histones are indicated.

TRRAP-depleted extracts show an inhibition of in vitro end-joining activity.

To test the functional involvement of TRRAP in DSB repair, we used an in vitro cell free end-joining system (4). In this system, MRE11 and RAD50 antibodies were already shown to specifically inhibit the NHEJ of a linearized double-stranded DNA (dsDNA) template (28, 52). Using the normal human lymphoblastoid AHH-1 cell line extract depleted with two different anti-TRRAP mouse monoclonal antibodies (MAbs) (Fig. 4A and data not shown), we show that TRRAP-depleted extracts had a significantly weaker end-joining efficiency than the appropriate control MAb-treated extract (Fig. 4B, compare lanes 7 and 8 to lane 6; Fig. 4C). In the same system, extracts depleted with a crude anti-MRE11 polyclonal sera showed a fivefold reduction in NHEJ activity compared to the nonspecific polyclonal antibody control (Fig. 4B, compare lane 4 with lane 5; Fig. 4C). The observation that in TRRAP-depleted extracts a weaker inhibition was seen than in the MRE11-depleted extracts is consistent with the fact that TRRAP is only associated with a fraction of the cellular MRN complexes. However, the inhibition of the in vitro end-joining activity in the TRRAP-depleted extract suggests that TRRAP may be involved in the end-joining reaction of naked dsDNA fragments via the MRN complex (see Discussion).

Cell-based assay indicates that the downregulation of TRRAP affects DSB end joining in vivo.

To confirm the data obtained in vitro, we adopted a cell-based in vivo assay to test the role of TRRAP in the DSB repair processes. The efficiency of in vivo DNA end joining was assessed using a plasmid-based host cell end-joining assay (19). To this end, a DSB was introduced by EcoRI restriction endonuclease, which cuts at a unique site (in the multiple cloning site) between the immediate early promoter of CMV and the GFP coding sequences in the pEGFP-N3 plasmid. This linearized plasmid (99.43% linear) was then transfected into HeLa cells. As only recircularized plasmids can express GFP, the DSB end-joining efficiency was assessed by counting GFP-positive cells by FACS analysis at different time points after transfection. To test the effect of TRRAP on DSB end joining, cells were first transfected with an siRNA directed against TRRAP (siTRRAP) to knock down TRRAP expression. In control experiments, cells were treated with an siRNA directed against firefly luciferase (siLuc). Note that the linearized GFP expression vector or the circular GFP expression vector (see below) was transfected 6 h after the siRNAs. The efficiency of siTRRAP knockdown was analyzed by Western blotting (Fig. 5A). At the 72-h time point, where TRRAP expression was inhibited (Fig. 5A), there were 20% fewer GFP-positive cells than siLuc-treated samples. These data further suggest that TRRAP is involved in DSB end joining in the cells. However, as TRRAP may be involved in the regulation of transcription from the CMV promoter, we also verified the effect of TRRAP knockdown on transcription-expression from the circular expression vector. Our results indicated that TRRAP knockdown has a significantly stronger inhibitory effect (two- to fivefold) on the end joining and consequent transcription from the linearized vector than on the transcription from the circular plasmid (Fig. 5B). A general downregulation of all repair factors' expression cannot explain this observation, since NBS1, RAD50, MRE11, Ku70, and Ku80 expression levels were not affected by siTRRAP transfection (Fig. 5A and data not shown). Thus, TRRAP is involved in the regulation of transcription efficiency from the CMV promoter (circular plasmid) and also in DSB end joining (linear plasmid) in vivo. Moreover, our cell-based assay indicates that the downregulation of TRRAP affects the DSB end joining two to five times more than transcription and consequent GFP protein expression (Fig. 5B; for raw data, see Fig. S1 in the supplemental material). The above data suggest that TRRAP plays a role in DSB repair in vivo.

FIG. 5.

TRRAP knockdown inhibits DSB end joining in vivo. (A) The inhibition of TRRAP expression by RNA interference described for panel B was analyzed by Western blotting of whole-cell extracts. Extracts were prepared from the same batches of cells that were transfected with GFP vectors and siRNAs directed against luciferase (siLuc) or TRRAP (siTRRAP) as indicated and also used in the experiments shown in panel B. Ten micrograms of total protein from each extract was loaded on a 8% SDS-PAGE gel and analyzed by Western blotting with the indicated antibodies. (B) In three independent experiments (exp1 to -3), HeLa cells were first transfected with siTRRAP or siLuc; 6 h later, the cells were further transfected with a linear or circular pEGFP-N3 plasmid. Cells were collected 72 h after the transfection of pEGFP, washed with PBS, and subjected to FACS analysis. For each sample, 30,000 cells were counted, and the number of green cells in the siLuc-treated samples was taken as 100%. Each bar represents the percentage of inhibition observed in the siTRRAP-treated samples compared to the siLuc-treated samples. Light gray bars represent the percentage of inhibition observed on GFP expression from the transfected circular pEGFP plasmid in the presence of siTRRAP, whereas black bars represent the percentage of inhibition observed on GFP expression from the transfected linear pEGFP plasmid in the presence of siTRRAP. As transfection efficiency varied significantly between independent experiments, the three experiments are displayed independently (exp1, exp2, and exp3) (see Fig. S1 in the supplemental material). (C) Inhibition ratio values represented in panel B for the three different experiments.

Cells lacking TRRAP are impaired for precise NHEJ.

To further determine the repair pathway that is affected by TRRAP inhibition, we took advantage of another repair fidelity assay, based on I-SceI endonuclease induction in vivo (46). A reporter HPRT-GFP construction bearing a unique I-SceI site in the GFP coding sequence (46) was electroporated into Trrap “floxed conditional” knockout ES (Trrap^f/Δ) cell genome (26). Following homologous recombination and selection, ES clones with correctly integrated reporter were identified by Southern blot analysis (data not shown). To knock out the second “floxed” Trrap allele, the cells were transfected with the pMC-Cre expression vector expressing Cre recombinase and grown for 24 h. To induce a DSB and to trigger the DNA repair process, ES cells were then transfected by the I-SceI expression vector. Cells were harvested 16, 24, and 48 h following this second transfection; genomic DNA was prepared. The genomic DNA regions surrounding the I-SceI site were amplified by PCR in both TRRAP-containing and TRRAP-depleted cells. Note that TRRAP expression was significantly decreased 48 h after pMC-Cre transfection (Fig. 6D). PCR products were then either kept undigested (Fig. 6A, top) or digested with I-SceI endonuclease (bottom) and separated on agarose gels. This allowed us to assess NHEJ repair efficiency and to distinguish between precisely and nonprecisely repaired sequences. Imprecise NHEJ results in I-SceI site loss that was detected by resistance to I-SceI enzyme cleavage of PCR product in vitro (46). As shown in Fig. 6A and B, the I-SceI site was lost in 43 to 57% of the amplified fragments in TRRAP-depleted cells compared to 23 to 35% of that in TRRAP-containing cells. This is comparable to what has been previously observed with cells mutant for Ku70, a protein known to be a major player in NHEJ (46). The expression levels of I-Sce I in TRRAP-containing (−pMC-Cre) and TRRAP-depleted (+pMC-Cre) cells were comparable (Fig. 6C), suggesting that impaired precise NHEJ in cells lacking TRRAP is not due to compromised expression of I-SceI. Since I-SceI cleavage generates precisely ligatable overhangs, significantly lower levels (∼2-fold decrease) of the I-SceI-sensitive fragments suggests that cells lacking TRRAP are impaired for precise NHEJ.

FIG. 6.

Impaired NHEJ fidelity in the absence of TRRAP. (A) TRRAP “conditional” knockout mouse ES cells (Trrapf/Δ) containing stably integrated HPRT-GFP substrate were mock electroporated (−pMC-Cre; TRRAP-proficient cells) or electroporated with pMC-Cre (+pMC-Cre; TRRAP-deficient cells), expressed for 24 h, and then transfected by I-SceI vector to induce DSB. At indicated time points after I-SceI transfection, genomic DNA was extracted and amplified by PCR with specific primers flanking the I-SceI site. PCR products were then either kept undigested (top) or digested with I-SceI endonuclease (bottom) and separated on an agarose gel. I-SceS and I-SceR, PCR products that are sensitive and resistant, respectively, to the endonuclease. The error bars represent the average of three independent experiments. (B) Quantification of I-SceI loss shown in panel A by densitometric analysis and normalization for fragment length. (C) Northern blot analysis of I-SceI expression. Trrapf/Δ ES cells were mock electroporated (−pMC-Cre) or electroporated with pMC-Cre vector (+pMC-Cre), cultured for 24 h, and then transfected by I-SceI expression vector. At 24 h after I-SceI transfection, total RNA was extracted, and Northern blot analysis was carried out using an I-Sce-specific probe. Equal RNA loading was checked by an actin-specific probe. (D) Western blot analysis of TRRAP protein levels in ES cells. At 48 h after mock electroporation (−pMC-Cre) or pMC-Cre electroporation (+pMC-Cre), whole-protein lysates were prepared from Trrapf/Δ ES cells and probed with a rabbit anti-TRRAP polyclonal antibody (1930) and a rabbit anti-MDC1 polyclonal antibody to verify equal loading on the Western blot.

DISCUSSION

In an attempt to purify human TRRAP-containing complexes by double immunoprecipitation from HeLa cell nuclear extract, we isolated a novel TRRAP-containing complex, involving the well-characterized MRE11-RAD50-NBS1 complex. We also show that antibodies against several components of the MRN complex copurify TRRAP (Fig. 2 and 3). The different endogenous TRRAP-containing complexes were separable by glycerol gradient sedimentation; TRRAP, MRE11, RAD50, and NBS1 cofractionated in a fraction distinct from the other TRRAP-associated proteins (Fig. 2C). Moreover, when coexpressed in Sf9 cells, TRRAP formed a complex with MRN (Fig. 2E), further suggesting that the MRN complex can also stably associate with TRRAP in human cells. Our finding is in good agreement with the fact that a nonidentified 400-kDa component was previously described in the highly purified MRN complex (11). Nevertheless, from the MRE11 IP, it is clear that not all the cellular MRN complexes contain TRRAP. At present, we cannot distinguish between the following possibilities. (i) In the analyzed human cells, both TRRAP-containing and -lacking MRN complexes exist together, and the association of TRRAP with the MRN complex could be regulated by certain cellular signals. (ii) There is a subpopulation of cells in which TRRAP is always associated with the MRN complex. Interestingly, TRRAP was shown to be recruited to chromatin in a temporally regulated manner, before the recruitment of GCN5 or TIP60 and consequent acetylation of histone H3 and H4 by TFTC- and NuA4/TIP60-type complexes (38). This finding suggested that a free pool of TRRAP exists in the cells that can associate with different HAT complexes when chromatin remodeling is needed. Similarly, it is conceivable that the MRN complex can also functionally associate with free TRRAP at sites where MRN function is needed. In agreement with the idea that free TRRAP may exist in the cells, a significant amount of TRRAP was detected in a fraction(s) in our glycerol gradient separation of TRRAP-containing complexes where, according to the molecular markers, it cannot be associated with any other protein (Fig. 2C, fraction 3).

Although yTra1 or hTRRAP has not been directly involved in DSB repair, yeast and human NuA4/TIP60 complexes have been suggested to play a role in DSB repair. Yeast Esa1-containing NuA4 HAT complex is recruited specifically to DNA double-strand breaks that are generated in vivo, and the purified complex acetylates linear nucleosomal arrays with a greater efficiency than circular arrays in vitro, indicating that it preferentially acetylates nucleosomes near DSB sites (6). Furthermore, Ikura et al. studied the involvement of human homologue of Esa1, TIP60, in DSB repair and reported that cells expressing a HAT-deficient TIP60 derivative accumulate DNA DSBs upon irradiation and fail to undergo apoptosis (29). This was confirmed recently by showing that a Drosophila TRRAP-containing TIP60 complex acetylates the DSB marker phospho-H2Av, to allow its exchange against a nonphosphorylated H2Av (30). Thus, the functional involvement of TRRAP in DSB repair described in this study could be attributed to TRRAP-MRN complex and/or concomitantly to the NuA4/TIP60 complex. Since the purified TRRAP-MRN complex (i) is devoid of any HAT activity (Fig. 3B), (ii) is not associated with any of the tested subunits of NuA4 complexes (Fig. 2D), and (iii) seems to play a role also in the end-joining of naked dsDNA templates (Fig. 4), its function will necessarily be different from that of NuA4/TIP60-type complexes. TRRAP may arrive alone to DNA DSBs and associate with MRN at these sites (see above) or together with the MRN complex. TRRAP may then participate in DNA damage signaling, probably together with other members of the ATM kinase family (see below), and mediate the recruitment of the NuA4 complex, which would then participate in the alteration of the chromatin structure around the broken DNA ends. As TRRAP has been shown to arrive to the chromatin before the NuA4 complex (38), it is conceivable that TRRAP arrives to DSB sites with the MRN complex. At these sites, TRRAP would then be released from MRN and subsequently incorporated into the NuA4 complex, thus participating in DSB signaling and the recruitment of the NuA4 complex to the DSB sites. Our in vivo results on TRRAP involvement in NHEJ suggest that the TRRAP-MRN complex may have a role at the DSB repair sites that promotes repair fidelity.

Apart from its direct role in DSB repair, the MRN complex is an important sensor of DNA damage, able to activate the G₂/M checkpoint by an ATM/Chk2 signaling pathway (13). It also contributes to the accurate replication of chromosomes and the avoidance of bridge formation between sister chromatids (14). Mutations of genes encoding either NBS1 or MRE11 cause Nijmegen breakage syndrome or ataxia-telangiectasia-like disorder, respectively, which show genomic instability, hypersensitivity to DSB-inducing agents, and a defect in triggering cell cycle checkpoints (44). Interestingly, mouse TRRAP knockout cells show a defect in G₂/M checkpoint regulation and error-prone replication of chromosomes that do not segregate properly at the end of mitosis (26, 27, 33). Thus, TRRAP and the MRN complex seem to control similar cellular processes in vivo. It is thus possible that they do so as components of the same complex.

The maintenance of genome integrity requires a rapid and specific response to many types of DNA damage. The conserved and related PI-3-like ATM and ATR kinases orchestrate signal transduction pathways in response to genomic insults, such as DSBs (45, 47). In the light of our results, it is remarkable that TRRAP is a highly related member of the PI-3 kinase/ATM family of proteins (37, 49). It is thus possible that TRRAP is also an essential factor in DNA damage signaling, even though its kinase domain seems to be inactive. Moreover, TRRAP could play a DSB signaling role (direct or indirect) through its interaction with the MRN complex. Since TRRAP has been described as a stable component of several distinct multisubunit complexes with chromatin remodeling and DNA damage recognition activities, it is conceivable that the 400-kDa TRRAP protein can serve as a molecular platform to bring these activities together. Altogether, our results suggest that TRRAP can function as a molecular link between transcriptional coactivation, chromatin remodeling, and an essential DNA metabolism such as DSB repair.