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]).

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 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.

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.

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.

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.