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TRF2/RAP1 and DNA–PK mediate a double protection against joining at telomeric ends

2010; Springer Nature; Volume: 29; Issue: 9 Linguagem: Inglês

10.1038/emboj.2010.49

1460-2075

Oriane Bombarde, Céline Boby, Dennis Gómez, Philippe Frit, Marie‐Josèphe Giraud‐Panis, Éric Gilson, Bernard Salles, Patrick Calsou,

Chromosomal and Genetic Variations

Article20 April 2010free access TRF2/RAP1 and DNA–PK mediate a double protection against joining at telomeric ends Oriane Bombarde Oriane Bombarde CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, France Search for more papers by this author Céline Boby Céline Boby CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, FrancePresent address: INRA, UMR85 Physiologie de la Reproduction et des Comportements, CNRS, UMR6175, Université François Rabelais de Tours, Haras Nationaux, 37380 Nouzilly, FrancePresent address: GreD, CNRS, UMR 6247, INSERM, U931, Faculté de Médecine, 63001 Clermont Ferrand, France Search for more papers by this author Dennis Gomez Dennis Gomez CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, France Search for more papers by this author Philippe Frit Philippe Frit CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, France Search for more papers by this author Marie-Josèphe Giraud-Panis Marie-Josèphe Giraud-Panis Laboratory of Biology and Pathology of Genomes, University of Nice, UMR 6267 CNRS U998 INSERM 28 avenue Valombrose Faculté de Médecine 06107 Nice Cedex 2, France Laboratoire Joliot-Curie, CNRS USR3010, Université de Lyon, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Eric Gilson Eric Gilson Laboratory of Biology and Pathology of Genomes, University of Nice, UMR 6267 CNRS U998 INSERM 28 avenue Valombrose Faculté de Médecine 06107 Nice Cedex 2, France Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Université de Lyon, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Bernard Salles Corresponding Author Bernard Salles CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, France Search for more papers by this author Patrick Calsou Corresponding Author Patrick Calsou CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, France Search for more papers by this author Oriane Bombarde Oriane Bombarde CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, France Search for more papers by this author Céline Boby Céline Boby CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, FrancePresent address: INRA, UMR85 Physiologie de la Reproduction et des Comportements, CNRS, UMR6175, Université François Rabelais de Tours, Haras Nationaux, 37380 Nouzilly, FrancePresent address: GreD, CNRS, UMR 6247, INSERM, U931, Faculté de Médecine, 63001 Clermont Ferrand, France Search for more papers by this author Dennis Gomez Dennis Gomez CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, France Search for more papers by this author Philippe Frit Philippe Frit CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, France Search for more papers by this author Marie-Josèphe Giraud-Panis Marie-Josèphe Giraud-Panis Laboratory of Biology and Pathology of Genomes, University of Nice, UMR 6267 CNRS U998 INSERM 28 avenue Valombrose Faculté de Médecine 06107 Nice Cedex 2, France Laboratoire Joliot-Curie, CNRS USR3010, Université de Lyon, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Eric Gilson Eric Gilson Laboratory of Biology and Pathology of Genomes, University of Nice, UMR 6267 CNRS U998 INSERM 28 avenue Valombrose Faculté de Médecine 06107 Nice Cedex 2, France Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Université de Lyon, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Bernard Salles Corresponding Author Bernard Salles CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, France Search for more papers by this author Patrick Calsou Corresponding Author Patrick Calsou CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France Université de Toulouse, UPS, IPBS, Toulouse, France Search for more papers by this author Author Information Oriane Bombarde1,2, Céline Boby1,2, Dennis Gomez1,2, Philippe Frit1,2, Marie-Josèphe Giraud-Panis3,4, Eric Gilson3,5, Bernard Salles 1,2 and Patrick Calsou 1,2 1CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France 2Université de Toulouse, UPS, IPBS, Toulouse, France 3Laboratory of Biology and Pathology of Genomes, University of Nice, UMR 6267 CNRS U998 INSERM 28 avenue Valombrose Faculté de Médecine 06107 Nice Cedex 2, France 4Laboratoire Joliot-Curie, CNRS USR3010, Université de Lyon, Ecole Normale Supérieure de Lyon, Lyon, France 5Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Université de Lyon, Ecole Normale Supérieure de Lyon, Lyon, France *Corresponding authors. CNRS, IPBS, UMR5089, Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, 31077 Toulouse, Cedex4, France. Tel.: +33 561 175 970 or +33 561 175 936; Fax: +33 561 175 933; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2010)29:1573-1584https://doi.org/10.1038/emboj.2010.49 Present address: INRA, UMR85 Physiologie de la Reproduction et des Comportements, CNRS, UMR6175, Université François Rabelais de Tours, Haras Nationaux, 37380 Nouzilly, France Present address: GreD, CNRS, UMR 6247, INSERM, U931, Faculté de Médecine, 63001 Clermont Ferrand, France PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info DNA-dependent protein kinase (DNA-PK) is a double-strand breaks repair complex, the subunits of which (KU and DNA-PKcs) are paradoxically present at mammalian telomeres. Telomere fusion has been reported in cells lacking these proteins, raising two questions: how is DNA–PK prevented from initiating classical ligase IV (LIG4)-dependent non-homologous end-joining (C-NHEJ) at telomeres and how is the backup end-joining (EJ) activity (B-NHEJ) that operates at telomeres under conditions of C-NHEJ deficiency controlled? To address these questions, we have investigated EJ using plasmid substrates bearing double-stranded telomeric tracks and human cell extracts with variable C-NHEJ or B-NHEJ activity. We found that (1) TRF2/RAP1 prevents C-NHEJ-mediated end fusion at the initial DNA–PK end binding and activation step and (2) DNA–PK counteracts a potent LIG4-independent EJ mechanism. Thus, telomeres are protected against EJ by a lock with two bolts. These results account for observations with mammalian models and underline the importance of alternative non-classical EJ pathways for telomere fusions in cells. Introduction Loss of chromosome fragments can lead to apoptosis or initiate carcinogenesis by means of improper expression or silencing of key genes controlling cell proliferation. Thus, signalling and repair of DNA double-strand breaks (DSB) are essential for genome stability (O'Driscoll and Jeggo, 2006). Mammals have evolved two main DSB repair mechanisms, homologous recombination (HR) and non-homologous end-joining (NHEJ) (Pardo et al, 2009). HR uses strand exchange at the break site mainly with the sister chromatid during S and G2 phases of the cell cycle. NHEJ, on the other hand, rejoins the two ends of the break throughout the cell cycle and is the predominant mechanism in G1. The main NHEJ reaction, hereafter named classical NHEJ (C-NHEJ), relies on recognition, protection and bridging of the DNA ends by the DNA-dependent protein kinase complex (DNA–PK). This complex is composed of the DNA binding KU70/KU80 heterodimer, which recruits the serine-threonine kinase catalytic subunit (DNA–PKcs) (for a review, see Mahaney et al, 2009). DNA–PK also activates end-processing enzymes such as the Artemis nuclease (Goodarzi et al, 2006) and is required for the stable recruitment of the XRCC4/DNA ligase IV (LIG4)/Cernunnos–XLF complex that catalyses the final ligation step (Drouet et al, 2005; Wu et al, 2007). Recently, evidence has accumulated for an alternative or backup NHEJ pathway (hereafter named B-NHEJ), which accounts for residual end-joining (EJ) of DSB in cells that are deficient in components of C-NHEJ (for reviews, see Nussenzweig and Nussenzweig, 2007; Haber, 2008; McVey and Lee, 2008). B-NHEJ is repressed by C-NHEJ and preferentially uses DNA microhomology for EJ (Guirouilh-Barbat et al, 2007; Schulte-Uentrop et al, 2008). Data from our and other laboratories have implicated XRCC1/DNA ligase III and PARP-1 in B-NHEJ (Audebert et al, 2004; Wang et al, 2005; Robert et al, 2009); in addition these proteins are involved in the repair of base damage and single-strand breaks. Efficient cellular mechanisms for coping with genomic DSB present a challenge to linear chromosomes that must prevent unwanted signalling, joining or recombination at their ends. In most eukaryotes, chomosome ends are composed of special nucleoprotein structures called telomeres. In humans, the telomeric DNA comprises typically 10–15 kb of T2AG3 duplex repeats oriented 5′ to 3′ towards the chromosome end, followed by a 3′ single-stranded extension composed of these same repeats over 50–500 nucleotides. The T2AG3 repeats associate with shelterin, a complex of proteins: Tin2 bridges the TRF1 and TRF2/RAP1 complex that is bound to the double-stranded (ds) telomeric repeats whereas the POT1/TPP1 complex is attached to the G-rich tail (reviewed in Palm and de Lange, 2008). The 3′ overhang folds back and invades the duplex telomeric repeat to form the so-called T loop (Griffith et al, 1999) most likely because of the DNA unwinding properties of TRF2 (Amiard et al, 2007). A minimal telomere length is needed, probably required for shelterin assembly. This is maintained either by the telomerase activity or alternative recombination-based mechanisms (reviewed in Verdun and Karlseder, 2007; Cesare and Reddel, 2008). Some of these proteins participate in DSB signalling avoidance at telomeres; it has been shown that TRF2 is necessary to prevent ATM activation whereas POT1 in association with TPP1 is responsible for ATR inhibition (Denchi and de Lange, 2007). They also have a role in preventing end fusion; TRF2 is essential for inhibition of telomere fusion in cells (van Steensel et al, 1998; Celli and de Lange, 2005; Celli et al, 2006), most likely by anchoring RAP1 on telomeres (Sarthy et al, 2009)). The extent of end fusion on loss of TRF2 is far more pronounced than for POT1 deficiency (Hockemeyer et al, 2006). In addition, the G-rich tail is not required for TRF2/RAP1-mediated inhibition of EJ on telomeric DNA in vitro (Bae and Baumann, 2007). Some human telomeres whose length is incompatible with T-loop formation also appear to escape fusion in cells (Baird et al, 2003; Xu and Blackburn, 2007). These results suggest that duplex telomeric repeats and the associated proteins are a major contribution to EJ avoidance at telomeres. Surprisingly, both KU and DNA–PKcs components of the C-NHEJ machinery are present on telomeres (Hsu et al, 1999; d'Adda di Fagagna et al, 2001). This observation underlines the paradoxical protection and fusion functions of these proteins at telomeres (reviewed in Fisher and Zakian, 2005; Riha et al, 2006). The near complete absence of chromosome fusions on inhibition or loss of TRF2 in an LIG4-deficient background clearly argues for a predominant role of the C-NHEJ mechanism for telomeric fusions under these shelterin destabilization conditions (Smogorzewska et al, 2002; Celli and de Lange, 2005). Under these conditions, telomeric fusion has been shown to predominate in G1 phase of the cell cycle (Konishi and de Lange, 2008). Similarly, LIG4 is responsible for the fusion of sister telomeres in cells deficient in the telomeric poly(adenosine-diphosphate ribose) polymerase tankyrase-1 (Hsiao and Smith, 2009). However, significant chromosome end fusions occur on TRF2 loss in the absence KU, although 10-fold less frequently than in wild-type cells (Celli et al, 2006). In addition, spontaneous chromosomal end fusions are also promoted by a deficiency in either KU (Bailey et al, 1999; Hsu et al, 1999, 2000; Samper et al, 2000; d'Adda di Fagagna et al, 2001; Espejel et al, 2004; Li et al, 2007) or DNA–PKcs (Gilley et al, 2001; Goytisolo et al, 2001), where, at least in the absence of KU, the core shelterin complex appears broadly normal (Celli et al, 2006). Moreover, both DNA–PKcs and LIG4 have been shown to be dispensable for chromosomal fusions arising in telomerase-deficient mouse cells (Maser et al, 2007), in line with the report of KU- and LIG4-independent telomere fusions on attrition in fission yeast (Wang and Baumann, 2008). However, another group has reported that KU and DNA–PKcs are necessary for fusions in telomerase-deficient mouse cells (Espejel et al, 2002a, 2002b). These observations raise several questions: (1) which of the end-recognitions or the DNA ligation steps in the C-NHEJ mechanism is blocked at telomeres? (2) Does a DNA–PK-independent and LIG4-mediated or a B-NHEJ alternative EJ mechanism operate at chromosome ends under special conditions? (3) What is the connection between both the shelterin and the NHEJ proteins at telomeres and this DNA–PK-independent EJ mechanism? To address these questions, we have used an EJ assay with plasmid substrate bearing at one end ds telomeric tracks, defined as the minimal structure protecting from EJ (Bae and Baumann, 2007). The joining reaction was catalysed by human cell extracts under controlled conditions enabling either C-NHEJ or B-NHEJ. We show that TRF2/RAP1 and DNA–PK complexes protect telomeres by two complementary mechanisms. The former prevents C-NHEJ-mediated end fusion at the initial DNA–PK end-binding step whereas the latter counteracts a potent LIG4-independent EJ mechanism that promotes ligation in the absence of DNA–PK. This double-lock protection accounts for observations with mammalian models and underlines the importance of alternative non-classical EJ pathways for telomeres fusion in cells. Results Inhibition of C-NHEJ-mediated joining of telomeric ds DNA ends relies on an impaired DNA–PKcs activation An in vitro assay with cell extracts and DNA substrates was used to mimick the effect of telomeric DNA on EJ mediated by the NHEJ apparatus. As ligation substrate, the pUCtelo2 plasmid containing 648 bp of 5′-T2AG3 repeats was used (Amiard et al, 2007), which closely matches the natural mean telomere length (Figure 1A). Appropriate restrictions of pUCtelo2 produced linear plasmids with a ds telomeric tract plus 1 bp at one end, at the 5′ end (pT5′), at the 3′end (pT3′) or ending with a 31 bp non-telomeric sequence (pT3′H). The EJ reaction obtained with the HeLa extracts was sensitive to the addition of a DNA–PKcs-specific inhibitor or antibodies directed against XRCC4, showing that it was true C-NHEJ (Supplementary Figure S1A, lanes 1 and 3, respectively). When introduced into the EJ assay, 30–31% of a control pBS substrate digested with the same restriction enzyme as pUCtelo2 was converted into dimers and multimers, whatever the DNA end (Figure 1B, lanes 1 and 5; Supplementary Figure S1B, lanes 1–3). In contrast, only 15–18% of the pT3′ was rejoined and only dimers were observed (Figure 1B, lanes 2 and 6; Supplementary Figure S1B, lane 5). However, the pT5′ and the pT3′H substrates were as efficiently rejoined as the control pBS (Supplementary Figure S1B, lanes 4 and 6, respectively). All the ligation events were sensitive to NU7026 (Figure 1B, lane 9; Supplementary Figure S1B, lanes 7–9). These results show that the C-NHEJ inhibition mediated by the telomeric tract was restricted to the T2AG3-3′ orientation (pT3′) and was released by the addition of non-telomeric 31 bp at this end. Figure 1.DNA substrates construction and EJ and kinase assays with telomeric DNA. (A) Construction scheme of the plasmids and DNA fragments used. pUCtelo2 plasmid contains a 648 bp telomeric sequence inserted between EcoRI and BamHI sites as detailed in the upper part of the figure. Track 1: non-biotinylated plasmids. pUtelo2 was digested with the indicated enzymes to produce linearized plasmid bearing a telomeric sequence at the 5′ end (pT5′), the 3′ end (pT3′) or moved at various distance inward from the 3′ end (pT3′X, pT3′S, pT3′H). Track 2: biotinylated plasmids. Biotinylation followed by appropriate restriction produced the same 3′ended-telomeric plasmids but containing biotin at the opposite end (biopT3′, biopT3′X, biopT3′S). Track 3: biotinylated fragments. Biotinylation followed by appropriate restriction produced fragments with the telomeric sequence at various distance inward from the 3′ end (fT3′, fT3′X, fT3′S, fT3′H) and containing biotin at the opposite end. The control plasmid (biopC) corresponds to the pUCtelo2 plasmid without telomeric sequence and biotinylated at the 5′ end. A control non-telomeric 502 bp fragment biotinylated or not at the 5′-end was amplified by PCR from pBluescript-KS-II(−) (see Materials and methods). (B) EJ assay catalysed under standard reaction conditions with the indicated plasmids and HeLa extracts, in the presence or not of streptavidin or DNA–PK-specific inhibitor NU7026. DNA ligation products were separated by agarose gel electrophoresis followed by SYBR-Green staining. pBS stands for pBluescript-KS-II(−). Ligation efficiency (% of multimers versus monomer) was 30.2, 17.9, 13.7, 2.7 for lanes 1–4 (without streptavidin) and 31, 15.2, 12.7, 1.6 for lanes 5–8 (with streptavidin), respectively (C) DNA–PK assay catalysed under standard conditions with the indicated DNA fragments and HeLa extracts, in the presence or not of streptavidin or DNA–PK-specific inhibitor NU7026. DNA–PK peptide substrate was isolated by polyacrylamide denaturing gel electrophoresis followed by auto-radiography of the gel. (D) Quantification of independent experiments as shown in (C) (n=3). Relative DNA–PKcs activity was calcultated as the % of radiolabel incorporation in the peptide substrate obtained with fT3′ fragment as activating DNA compared with the incorporation obtained with fT3′H fragment, after subtraction in each case of the background incorporation obtained without DNA. Error bars correspond to s.e.m. Download figure Download PowerPoint Having validated our in vitro assay for EJ inhibition by telomeric DNA as reported earlier (Bae and Baumann, 2007), we restricted the accessibility of ligation only to one end by constructing another series of substrates, bearing a biotin residue at the end opposite to the telomeric end (Figure 1A, biopT plasmids). A biotinylated non-telomeric plasmid with a smilar length and free end was used as control (Figure 1A, biopC plasmid, track 3). As shown in Figure 1B, blocking the non-telomeric end by a biotin residue was sufficient to prevent the residual dimer formation on the pT3′ substrate and led to an extensive EJ inhibition, which was very similar with or without streptavidin in the reaction (87 and 80%, respectively, as quantified from Figure 1B). This shows that a long T2AG3-3′ tract mimicking a natural ds chromosome end is refractory to NHEJ-mediated ligation in vitro. DNA–PKcs activity is necessary for efficient EJ of DSB in vitro and in cells (Meek et al, 2008). Therefore, we checked the effect of telomeric tracts at a DNA end on the activation of DNA–PKcs in vitro. The telomeric fragment was isolated from pUCTelo2 and biotin was incorporated at one end to restrain the possibility of DNA–PKcs activation either at the 3′ telomeric end (fT3′) or the 3′ telomeric end extended with a 31 bp non-telomeric sequence (fT3′H) (Figure 1A). DNA–PKcs activity on a standard peptide substrate was assessed in the NHEJ competent HeLa extracts under the same buffer conditions as the EJ reaction and with the various activating DNA fragments biotinylated at one end, with or without streptavidin (Figure 1C). Peptide phosphorylation was completely abolished with NU7026 showing that this reaction requires DNA–PKcs activity. In the absence of streptavidin, all added fragments promoted an activity above the background obtained with the extracts alone, indicating that DNA–PKcs activation was dependent on the exogenous DNA ends provided in the assay. On streptavidin addition, the DNA–PKcs activity promoted by the fT3′ fragment dropped markedly whereas it remained unchanged with the other DNA fragments. As quantified in Figure 1D, DNA–PKcs activation targeted on the T2AG3-3′ extremity (fT3′+streptavidin) was reduced by more than 80% when compared with the activation obtained with a 31 bp non-telomeric DNA extension added to the telomeric tract (fT3′H). The latter fragment yielded the same DNA–PKcs activation level as a control DNA fragment (Figure 1C), indicating that 31 non-telomeric bps were sufficient to completely overcome the inhibitory effect of telomeric repeats at the 3′ end on DNA–PKcs. To check more precisely the distance from the end that is required for the inhibitory effect on NHEJ of telomeric repeats at the 3′ end, the 5′ biotinylated substrates biopT3′X and biopT3′S were constructed, bearing 7 or 13 non-telomeric bps added 3′ to the telomeric sequence, respectively (Figure 1A). As shown in Supplementary Figure S2A, NHEJ was repressed on 3′X ends as strongly as on 3′ telomeric plasmids (lanes 1, 2 and 4, 5) but was efficient on 3′S substrates, leading to multimers without biotin (pT3′S plasmid, lane 3) and to dimers with biotin (biopT3′S plasmid, lane 6). Thus, under these conditions, the 3′ telomeric tract did not inhibit NHEJ-mediated EJ at this end beyond a 13 bp distance. We then assessed the effect of the telomeric tract distance from the 3′ end on DNA–PKcs activity. Supplementary Figure S2B shows that the strong kinase inhibition observed at the fT3′ fragment (lane 2) was already absent when the telomeric tract was moved 7 bp inward from the 3′ end (lane 3, fT3′X substrate). TRF2/RAP1 complex mediates C-NHEJ inhibition at telomeric ends through hindrance at the KU loading and DNA–PK activation steps Binding of TRF2/RAP1 complex to ds telomeric DNA has a major role in EJ inhibition (Bae and Baumann, 2007). To investigate the molecular basis of NHEJ inhibition at ds telomeric DNA ends under our in vitro conditions, we immuno-depleted HeLa extracts for TRF2 and RAP1 proteins (Figure 2A). After immuno-depletion, RAP1 was undetectable whereas a faint amount of TRF2 was still present and TRF1 concentration remained unchanged. We failed to selectively immuno-deplete TRF1 from the extracts because of a cross-reactivity with TRF2 of immuno-precipitant anti-TRF1 antibodies (Supplementary Figure S3), as reported (Bae and Baumann, 2007). When immuno-depleted extracts were assayed with the telomeric ends and control substrates, EJ was largely re-established at the telomeric DNA end of the biopT3′ plasmid (Figure 2B and C). A decrease in the overall EJ activity on the control substrate was observed, probably because of extract manipulations during immuno-depletion. EJ remained sensitive to NU7026 (Figure 2B, lane 5) and was undetectable in the absence of LIG4 (Supplementary Figure S4B, lane 8), indicating that telomeric ends ligation still relied on C-NHEJ in the absence of TRF2/RAP1 complex. Figure 2.TRF2/RAP1complex mediates C-NHEJ inhibition at telomeric ends through hindrance at the KU loading and DNA–PK activation steps. (A) Western blotting analysis of HeLa protein extracts after immuno-depletion as indicated. Protein samples were denatured and separated on 8% SDS–PAGE gel followed by electrotransfer on membrane and blotting with the antibodies as indicated. (B) EJ assay catalysed under standard reaction conditions with the indicated plasmids and HeLa extracts depleted as specified, in the presence or not of NU7026. DNA ligation products were separated by agarose gel electrophoresis followed by SYBR-Green staining. (C) Quantification of independent experiments as shown in (B) (n=3). Relative ligation efficiency was calcultated as the % of ligation obtained in each case related to the ligation obtained on the control biopC plasmid with the control IgG-depleted extracts. Error bars correspond to s.e.m. Black bars correspond to control IgG-depleted extracts and grey bars to TRF2/RAP1-depleted extracts. Absolute ligation efficiency was 4.6±0.8% s.e.m. for IgG-depleted extracts on bioPC plasmid. (D) Pull-down experiment under standard conditions with HeLa extracts mixed with the indicated biotinylated DNA fragments. Salt molarity during washing of the beads is specified. The initial amount of protein used during the pull-down experiment was loaded as input. Protein samples were denatured and separated on 8% SDS–PAGE gel followed by electrotransfer on membrane and blotting with the antibodies as indicated. (E) Pull-down experiment as in (D) but with immuno-depleted HeLa extracts as indicated and washing under high salt conditions. C stands for the 502 bp non-biotinylated control DNA fragment. Download figure Download PowerPoint Under the salt conditions used here, KU binding to DNA ends is necessary for DNA–PK activation (Hammarsten and Chu, 1998). As DNA–PK activity was impaired at telomeric ends, we have perfomed pull-down experiments with streptavidin beads and biotinylated DNA fragments to assess KU loading at these ends with HeLa extracts proficient or deficient in TRF2/RAP1. The reaction contained ATP to allow DNA–PKcs activation, auto-phosphorylation (Chan and Lees-Miller, 1996) and detachment from the probe in case of proper activation, as shown earlier (Calsou et al, 1999). As binding of KU to the telomeric probe might occur indirectly through interaction with TRF2 or TRF1 proteins (Hsu et al, 1999; Song et al, 2000), we first tested the effect of the salt molarity during the washing step (Figure 2D). As expected, RAP1 was specifically pulled down with the telomeric probe and resisted high salt washing (lanes 3 and 5). At the lower salt concentration, no difference was observed for KU and DNA–PK pull-down between non-telomeric and telomeric probes (lanes 2 and 3). In contrast, a marked difference between the two probes was observed for the two proteins under a higher salt molarity: KU failed to accumulate at the telomeric probe whereas DNA–PKcs was mainly pulled down on this probe (lanes 4 and 5). This pattern is reminiscent to what we have observed under conditions of kinase inhibition that led to a blocked DNA–PK complex at the DNA end (Calsou et al, 1999). The higher salt conditions were therefore chosen to perform a pull-down with both probes and control and TRF2/RAP1-depleted extracts in parallel (Figure 2E). Again, RAP1 was specifically pulled down with the telomeric probe but, as expected, was absent in pull-down from the depleted extracts. In contrast, TRF1 was equally pulled down on the telomeric probes, whether TRF2/RAP1 was present or not (lanes 4 and 6). Regarding KU and DNA–PKcs, the same pattern as in Figure 2D was found with the control extract, consistent with the presence of a blocked DNA–PK complex at the telomeric-ended probe (lanes 3 and 4). In contrast, KU was fully pulled down on the telomeric probe from TRF2/RAP1-depleted extracts (lane 6) and DNA–PKcs was equally pulled down with both the probes in the absence of TRF2/RAP1, suggesting equal kinase activity on both probes. At ds telomeric ends, TRF2/RAP1 complex, but not TRF1 alone, is likely responsible for an hindrance of the DNA–PK loading on DNA necessary for proper DNA–PKcs activation that in turn impairs LIG4-dependent EJ at these ends. KU and DNA–PKcs are the main factors that prevent B-NHEJ from operating on telomeric ends Various instances of telomere fusions independent of C-NHEJ have been described in cells (Bailey et al, 1999; Hsu et al, 1999, 2000; Samper et al, 2000; Gilley et al, 2001; Goytisolo et al, 2001; d'Adda di Fagagna et al, 2001; Espejel et al, 2004; Li et al, 2007; Maser et al, 2007). We, therefore, decided to characterize this EJ activity in vitro. To prevent C-NHEJ, we used extracts from a human pre-B-cell line, N114P2, with targeted disruption in both LIG4 alleles (Grawunder et al, 1998), and from its parental line, Nalm-6 as control. As expected, no LIG4 was detected in N114P2 extracts when compared with extracts from the parental Nalm-6 cell (Figure 3A; Supplementary Figure S4A). Likewise as shown in Supplementary Figure S4B, no ligation activity was observed with LIG4− extracts on control plasmid (lanes 2 and 4), whereas LIG4+ extracts ligated the control plasmid but not the telomeric substrate (lanes 1 and 5). As already observed with HeLa extracts (Figure 2B), immuno-depletion of TRF2/RAP1 proteins from LIG4+ extracts overcame the ligation inhibition on telomeric plasmid (Supplementary Figure S4B, compare lanes 5 and 7), whereas TRF2/RAP1-depleted LIG4− extracts still did not ligate this substrate (compare lanes 6 and 8), indicating that C-NHEJ is involved. Figure 3.KU and DNA–PKcs prevent B-NHEJ at telomeric ends. (A) Western blotting analysis of Nalm-6 and N114P2 protein ex