Title: TRF2 promotes, remodels and protects telomeric Holliday junctions
Abstract: Article5 February 2009free access TRF2 promotes, remodels and protects telomeric Holliday junctions Anaïs Poulet Anaïs Poulet Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Rémi Buisson Rémi Buisson Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Cendrine Faivre-Moskalenko Cendrine Faivre-Moskalenko Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire de Physique, CNRS UMR5672, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Mélanie Koelblen Mélanie Koelblen Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Simon Amiard Simon Amiard Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, FrancePresent address: Université Clermont-Ferrand, UMR6247 CNRS, 24 av. des Landais, 61177 Aubière, France Search for more papers by this author Fabien Montel Fabien Montel Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire de Physique, CNRS UMR5672, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Santiago Cuesta-Lopez Santiago Cuesta-Lopez Université de Lyon, Laboratoire de Physique, CNRS UMR5672, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Olivier Bornet Olivier Bornet Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France Search for more papers by this author Françoise Guerlesquin Françoise Guerlesquin Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France Search for more papers by this author Thomas Godet Thomas Godet Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Julien Moukhtar Julien Moukhtar Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire de Physique, CNRS UMR5672, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Françoise Argoul Françoise Argoul Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire de Physique, CNRS UMR5672, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Anne-Cécile Déclais Anne-Cécile Déclais Cancer Research UK, Nucleic Acids Structure Research Group, MSI/WTB Complex, University of Dundee, Dundee, UK Search for more papers by this author David M J Lilley David M J Lilley Cancer Research UK, Nucleic Acids Structure Research Group, MSI/WTB Complex, University of Dundee, Dundee, UK Search for more papers by this author Stephen C Y Ip Stephen C Y Ip Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK Search for more papers by this author Stephen C West Stephen C West Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK Search for more papers by this author Eric Gilson Corresponding Author Eric Gilson Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Marie-Josèphe Giraud-Panis Marie-Josèphe Giraud-Panis Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Anaïs Poulet Anaïs Poulet Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Rémi Buisson Rémi Buisson Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Cendrine Faivre-Moskalenko Cendrine Faivre-Moskalenko Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire de Physique, CNRS UMR5672, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Mélanie Koelblen Mélanie Koelblen Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Simon Amiard Simon Amiard Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, FrancePresent address: Université Clermont-Ferrand, UMR6247 CNRS, 24 av. des Landais, 61177 Aubière, France Search for more papers by this author Fabien Montel Fabien Montel Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire de Physique, CNRS UMR5672, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Santiago Cuesta-Lopez Santiago Cuesta-Lopez Université de Lyon, Laboratoire de Physique, CNRS UMR5672, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Olivier Bornet Olivier Bornet Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France Search for more papers by this author Françoise Guerlesquin Françoise Guerlesquin Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France Search for more papers by this author Thomas Godet Thomas Godet Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Julien Moukhtar Julien Moukhtar Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire de Physique, CNRS UMR5672, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Françoise Argoul Françoise Argoul Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire de Physique, CNRS UMR5672, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Anne-Cécile Déclais Anne-Cécile Déclais Cancer Research UK, Nucleic Acids Structure Research Group, MSI/WTB Complex, University of Dundee, Dundee, UK Search for more papers by this author David M J Lilley David M J Lilley Cancer Research UK, Nucleic Acids Structure Research Group, MSI/WTB Complex, University of Dundee, Dundee, UK Search for more papers by this author Stephen C Y Ip Stephen C Y Ip Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK Search for more papers by this author Stephen C West Stephen C West Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK Search for more papers by this author Eric Gilson Corresponding Author Eric Gilson Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Marie-Josèphe Giraud-Panis Marie-Josèphe Giraud-Panis Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France Search for more papers by this author Author Information Anaïs Poulet1,2,‡, Rémi Buisson1,2,‡, Cendrine Faivre-Moskalenko2,3, Mélanie Koelblen1, Simon Amiard1,2, Fabien Montel2,3, Santiago Cuesta-Lopez3, Olivier Bornet4, Françoise Guerlesquin4, Thomas Godet1,2, Julien Moukhtar2,3, Françoise Argoul2,3, Anne-Cécile Déclais5, David M J Lilley5, Stephen C Y Ip6, Stephen C West6, Eric Gilson 1 and Marie-Josèphe Giraud-Panis1,2 1Université de Lyon, Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR5239, Ecole Normale Supérieure de Lyon, Lyon, France 2Université de Lyon, Laboratoire Joliot-Curie, CNRS USR3010, Ecole Normale Supérieure de Lyon, Lyon, France 3Université de Lyon, Laboratoire de Physique, CNRS UMR5672, Ecole Normale Supérieure de Lyon, Lyon, France 4Institut de Biologie Structurale et Microbiologie, CNRS, Marseille, France 5Cancer Research UK, Nucleic Acids Structure Research Group, MSI/WTB Complex, University of Dundee, Dundee, UK 6Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK ‡These authors contributed equally to this work *Corresponding author. Laboratoire de Biologie Moléculaire de la Cellule, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, France. Tel.: +33 472 728453; Fax: +33 472 728080; E-mail: [email protected] The EMBO Journal (2009)28:641-651https://doi.org/10.1038/emboj.2009.11 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The ability of the telomeric DNA-binding protein, TRF2, to stimulate t-loop formation while preventing t-loop deletion is believed to be crucial to maintain telomere integrity in mammals. However, little is known on the molecular mechanisms behind these properties of TRF2. In this report, we show that TRF2 greatly increases the rate of Holliday junction (HJ) formation and blocks the cleavage by various types of HJ resolving activities, including the newly identified human GEN1 protein. By using potassium permanganate probing and differential scanning calorimetry, we reveal that the basic domain of TRF2 induces structural changes to the junction. We propose that TRF2 contributes to t-loop stabilisation by stimulating HJ formation and by preventing resolvase cleavage. These findings provide novel insights into the interplay between telomere protection and homologous recombination and suggest a general model in which TRF2 maintains telomere integrity by controlling the turnover of HJ at t-loops and at regressed replication forks. Introduction The termini of eukaryotic chromosomes are composed of specialised nucleoprotein structures called telomeres that are essential for the protection of chromosome ends against degradation and illicit repair. In vertebrates, telomeric DNA contains several kilobases of tandemly repeated 5′ TTAGGG motifs, terminated by a 3′ oriented single-stranded G-rich tail. In human telomeres, two proteins, TRF1 and TRF2 (TTAGGG repeat factors 1 and 2), specifically recognise the telomeric double-stranded sequence, whereas POT1 binds to the 3′ overhang (Liu et al, 2004a; de Lange, 2005). Although TRF1, TRF2 and POT1 (together with TIN2, TPP1 and Rap1) form a complex called shelterin or telosome (Liu et al, 2004a; de Lange, 2005), they can also have an independent role(s) at telomeres. For instance, TRF2 protects telomeres against checkpoint recognition and recombination (van Steensel et al, 1998). These protective functions of TRF2 are thought to result from both a TRF2-dependent folding of telomeres into a lasso-like structure called the t-loop (Griffith et al, 1999; Stansel et al, 2001) and the ability of TRF2 to regulate various DNA transactions and enzymatic activities (Bae and Baumann, 2007; Gilson and Geli, 2007). Four-way DNA structures such as Holliday junctions (HJs) or chickenfeet are structural intermediates in several processes (recombination, repair or replication). Telomeres present a special challenge for managing four-way DNA structures because of (i) the presence of resident telomeric proteins; (ii) inherent difficulties during their replication, which might lead to the formation of a high number of chickenfeet (Fouché et al, 2006b; Gilson and Geli, 2007; Verdun and Karlseder, 2007); (iii) the telomerase-independent mechanisms of telomere maintenance (alternative lengthening of telomeres) that involve recombination (Dunham et al, 2000) and (iv) the hypothetical presence of an HJ at the foot of the t-loop formed by migration of the single-strand 3′ tail inside the D-loop (Stansel et al, 2001). The deletion of t-loops was proposed to be a key event for the creation of extrachromosomal telomeric DNA circles, and for telomere shortening in cells overexpressing TRF2 (Ancelin et al, 2002; Karlseder et al, 2002; Wang et al, 2004). This process, named t-loop homologous recombination (t-loop HR, Wang et al, 2004) depends upon the RAD51 paralog XRCC3 and two TRF2-interacting factors, the XPF protein and the Werner helicase (Zhu et al, 2003; Li et al, 2008) and is inhibited by the N-terminal basic domain of TRF2 (named B) (Wang et al, 2004). In agreement with a key role played by B in the regulation of recombination, Griffith and coworkers showed recently by electron microscopy that this domain binds specifically to a plasmid-based HJ containing telomeric repeats (Fouché et al, 2006a). These data suggest a model of t-loop HR regulation based on a dual role of TRF2, both as an activator by its B-independent capacity to mediate t-loop formation and as an inhibitor through the binding of B on the HJ structure present at the foot of the t-loop. The mechanism by which TRF2 controls four-way DNA junctions is expected to be of paramount importance for telomere protection, as aberrant recombination of t-loop or stalled replication forks can lead to a severe loss of telomeric DNA and ultimately to cell growth arrest and genome instability. In this report, we demonstrate that the basic domain of TRF2 not only binds to telomeric HJs but also opens their centre, favours their migration and prevents their resolution by resolvases from different sources. We discuss models for the role of TRF2 in the formation, stabilisation and resolution of telomeric HJ and the implications for chromosome end protection. Results TRF2 reduces the resolution of the telomeric HJ by human, yeast and bacterial resolvases To investigate the effect of TRF2 on HJ resolution, we constructed a synthetic semimobile telomeric junction tHJ by incubation of four 54 nt oligonucleotides, each containing two human-type telomeric 5′TTAGGG repeats surrounded by two heterologous 21 nt long nontelomeric sequences (Supplementary Figure 1). Thus, tHJ harbours a 12 nt homologous region at its centre, allowing the junction branch point to migrate through 13 distinct positions (from −6 to +6, Supplementary Figure 2A). This junction is readily recognised by TRF2, to give several distinct complexes that can be visualised by EMSA (top gel, Supplementary Figure 2C). These results indicate that TRF2 forms stable complexes with HJ DNA, as observed previously with double-strand DNA. Deletions of the C-terminal Myb-like telobox domain (TRF2ΔM, Supplementary Figure 2B) or the N-terminal B domain (TRF2ΔB, Supplementary Figure 2B) do not seem to modify the nature of the complexes seen by EMSA, but binding affinity is substantially reduced for the TRF2ΔB mutant (TRF2 concentration at half binding increases from 20 to 75 nM). Deletion of the Myb-like domain only marginally affects junction binding (half binding occurs at 30 nM for TRF2ΔM). Competition experiments performed with TRF2, TRF2ΔB and TRF2ΔM show that the B domain is responsible for the binding preference of TRF2 for the structure of the junction but does not exhibit sequence specificity (Supplementary Figure 3). Thus, efficient binding of TRF2 to tHJ requires its N-terminal B domain, in agreement with the results obtained by Fouché et al (2006a). To examine whether the binding of TRF2 could affect the processing of HJs, we analysed the effect of TRF2 on HJ cleavage by the human GEN1 HJ resolving activity. GEN1 has been recently identified as the protein responsible for the resolving activity formerly known as ResA (Ip et al, 2008). It was shown that GEN1 cleaves HJ specifically and with perfect symmetry, as observed with other resolving activities such as T4 endonuclease VII, T7 endonuclease I, RuvC and CceI (Ip et al, 2008). When tHJs were treated with a catalytically active N-terminal fragment of GEN1 purified from Escherichia coli (GEN11–527, which resolves HJs in a manner that is identical to that catalysed by the full-length protein), we observed efficient cleavage (Supplementary Figure 4). Denaturing PAGE analysis revealed that GEN11–527 cleaves the HJ by introduction of symmetrically related nicks in pairs of opposite strands. In strands 1 and 3, cleavage occurs in G-rich regions, whereas in strands 2 and 4, the nicks are observed in C-rich regions. The efficiency of resolution of the tHJ by GEN11–527 is reduced by preincubation with TRF2, reaching an inhibition of >80% for the highest concentration of TRF2 (Figures 1 and 2, Supplementary Figure 5). This inhibition is found to depend on the N-terminal B domain of TRF2, as deletion of this domain totally removes the inhibition of cleavage (Figures 1 and 2, Supplementary Figure 5). All cleavage sites are affected in an equal manner for sites on the G-rich strands and on the 3′ side of the C-rich strands. However, sites located on the 5′ side of the latter strands seem more resistant to TRF2-inhibition (asterisks in Figure 2 and Supplementary Figure 5). This behaviour may depend on the Myb-like domain of TRF2, as its deletion harmonises the cleavage profiles. Overall, these data show that the B-dependent binding of TRF2 on a telomeric HJ has a marked effect on its cleavage by this human HJ resolving enzyme and might provide important insight into how TRF2 could control the processing of HJs. To determine whether this inhibition is a general feature of TRF2 or is specific to GEN1, we investigated the effect of TRF2 on three archetypal enzymes representing the three major groups of resolving enzymes (Rafferty et al, 2003; Déclais and Lilley, 2008): Endonuclease I of phage T7 (abbreviated as T7 Endo I) of the nuclease family, the yeast mitochondrial CceI enzyme of the integrase family and the unclassified RusA resolving enzyme. T7 Endo I cleaves HJs 1 nt 5′ to the junction centre (Déclais et al, 2006) and cleaves tHJ on all strands at 12 different positions corresponding to 12 of the 13 possible positions of the branch point (Supplementary Figures 6 and 7). The absence of cleavage for the most 5′ terminal positions as well as the presence of favoured cleavage sites could be explained by some sequence selectivity of the enzyme (Picksley et al, 1990; Déclais et al, 2006). CceI exhibits sequence specificity for cleavage (White and Lilley, 1996), and its main site (3′ side of a CT dinucleotide) can be found in the two C-rich strands of the telomeric junction (Supplementary Figure 8 and data not shown). The same strands will be cleaved by RusA, which also presents sequence specificity (5′ side of a CC dinucleotide) (Giraud-Panis and Lilley, 1998), although, in this case, one putative site seems to be less prone to cleavage (Supplementary Figure 8). Figure 1.TRF2 protects tHJ from cleavage by GEN1 in a B domain-dependent manner. (A) A quantity of 5 nM of tHJ labelled on strand 1 was incubated with increasing amounts of TRF2 before cleavage with GEN11–527. Concentrations of TRF2 used were 10, 20, 50, 100 and 200 nM. Lane 1 shows the uncleaved junction, and in lane 8 only 200 nM of TRF2 was added. Positions of GEN1 major cleavage sites (assigned by comparison with the result of a Maxam and Gilbert A+G sequencing reaction, Supplementary Figure 4) and the cleavage profile are represented on the left side. (B) Same experiment as in (A) with TRF2ΔB. (C) Comparison of cleavage profiles for GEN1 alone (grey line) or in the presence of 200 nM of TRF2 (black line) or TRF2ΔB (dotted line). (D) Graph representing the variations in percentage of the intensities of each cleavage band (ΔI%) as a function of TRF2 concentration (closed squares) or TRF2ΔB (open squares). The values correspond to average ΔI were calculated using the four major cleavage bands. Error bars correspond to standard errors in these values. Download figure Download PowerPoint Figure 2.TRF2 protects tHJ from cleavage by GEN1 in a B domain-dependent manner. (A) A quantity of 5 nM of tHJ labelled on strand 4 was incubated with increasing amounts of TRF2 (10, 20, 50, 100 and 200 nM) before cleavage with GEN11–527. Lane 1 shows the uncleaved junction, and in lane 8 only 200 nM of TRF2 was added. Positions of GEN11–527 major cleavage sites and the cleavage profile are represented on the left side. Note the distinct behaviour obtained for the 5′ terminal cleavage site (*). (B) Same experiment as in (A) with TRF2ΔB. (C) Comparison of cleavage profiles for GEN11–527 alone (grey line) or in the presence of 200 nM of TRF2 (black line) or of TRF2ΔB (dotted line). (D) Graph representing the variations in percentage of cleavage intensity (ΔI%) as a function of the concentration of TRF2 (closed and grey squares) or TRF2ΔB (open and grey triangles). Grey symbols correspond to the behaviour of the 5′ terminal cleavage site (*). Other symbols with their corresponding error bars represent the average behaviour and the corresponding standard errors of all other major cleavage sites. (E) A quantity of 5 nM of tHJ labelled on strand 1 was incubated with 200 nM of TRF2 or of TRF2ΔM before cleavage with GEN11–527. The first lane shows the uncleaved junction, and in the last two lanes only telomeric proteins were added. Numbers below the gel represent the variations in cleavage intensity (ΔI%) in each sample containing the telomeric protein compared with the sample only containing the enzyme. Negative numbers show protection. (F) Same experiment as in (E) with strand 4. *Marks the position of the 5′ terminal cleavage site. Download figure Download PowerPoint Remarkably, TRF2 impairs the action of all three enzymes (Supplementary Figures 6–8), similarly to that observed with GEN1. In the case of CceI, cleavage of both strands is inhibited and all sites are equally affected (Supplementary Figure 8 and data not shown). For RusA, inhibition is stronger on strand 4 but, as for CceI, all cleavages are equally impaired within one strand. The effect of TRF2 on T7 Endo I is more complex and more resembles that observed on GEN1 cleavage. Although the inhibition can be observed on four centrally located positions (black symbols in graph and profile in Supplementary Figures 6 and 7), other positions show an increase in T7 Endo I activity (again 5′ located sites, grey symbols) or even the absence of an effect (open symbols). It is worth noting that, as for GEN1, removing the Myb-like domain results in the loss of the 5′ cleavage activation. Inhibition of T7 Endo I, RusA and CceI cleavage, like that of GEN1, is mainly dependent on the B domain of TRF2 (Supplementary Figures 6–8). Therefore, TRF2 binding to tHJ through its Myb-like domain is not sufficient per se to mask the cleavage sites of these enzymes. In further agreement with a specific role of B, TRF1 (which does not contain an N-terminal basic domain) does not inhibit the cleavage by T7 Endo I, CceI or RusA (Supplementary Figure 9). As expected, because TRF1 and TRF2 share a nearly identical Myb-like domain, activation of Endo I cleavage at 5′ positions is visible for the TRF1-bound junction (Supplementary Figure 9A). Overall, we conclude that TRF2 prevents cleavage by the human GEN1 enzyme and three archetypal resolvases that recognise and cleave HJ in different ways and represent the three major types of known resolving enzymes. This is achieved through the recognition of the junction centre by the B domain. An assay to measure the rate of formation and migration of a telomeric HJ Next, we analysed the influence of TRF2 on the rate of branch migration through a telomeric sequence. For this purpose, we adapted an in vitro assay originally designed by Panyutin and Hsieh (1994). A HJ is obtained through the annealing of two homologous double-stranded DNA (S1 and S2) containing four TTAGGG repeats located 22 bp away from the 3′ terminus of the homologous region. They both end by single-stranded tails presenting complementary sequences between each other (Figure 3A, Supplementary Figure 10). Once formed, this junction can spontaneously migrate by random walking, with a rate that will depend on the assay conditions and DNA sequence (Panyutin and Hsieh, 1994; McKinney et al, 2005). Finally, an irreversible dissociation step leads to fully hybridised duplex products (P1 and P2). Disappearance of the labelled S1 substrate and appearance of the junction and of the P1 product can be followed by gel electrophoresis of the sample (Supplementary Figure 10B), allowing quantification of all species. The presence of the single-stranded tails in S1 and the hybridisation state of P1 were both verified by cleavage with the HindIII and BamHI restriction enzymes (H and B, respectively, Supplementary Figure 10C). In an additional control, we noted that when using an S2 substrate containing a 6-bp heterologous sequence (hS2, Supplementary Figure 10D), the junction was still formed but migration prevented, and thus no product was formed (Supplementary Figure 10E). Figure 3.TRF2 greatly increases the junction formation but slows down migration. (A) Reaction scheme of the migration assay and corresponding rates k1 and k2. (B) Migration assay performed in the absence of telomeric proteins. Numbers above indicate the time points corresponding to each sample. (C) Variations of the percentage of the S1, junction and P1 species through time corresponding to the experiment shown in (B). The lines represent the fitting curves obtained with the rates calculated using the experimental data. (D) Migration assay performed in the presence of 300 nM of TRF1. (E) Same as (C) for TRF1. (F) Migration assay performed in the presence of 100 nM of TRF2. (G) Same as (C) for TRF2. The insert shows an enlarged part of the graph corresponding to the early time points. Download figure Download PowerPoint A kinetic analysis for junction formation and migration was performed. To simplify the analysis, the migration and final dissociation steps were associated in a single step described by k2 (Figure 3A), as performed previously by Panyutin and Hsieh (1994). Both k1 and k2 were obtained by fitting the experimental data to the three equations presented in Materials and methods. In the absence of telomeric proteins (Figure 3B and C), the different steps are slow (Table I). Although the slowness of the annealing process can be explained by the low concentration of S1 used (2 nM), the migration/dissociation step is far slower than what could be expected from previous results on random DNA. As a comparison, Panyutin et al measured a half time of 20 min for the substrate of a strand-exchange reaction on a 956-bp DNA fragment (30°C, 100 mM NaCl). In the case of our 88 bp substrate, 40% of the junction still remains 50 min after disappearance of the substrate (30°C, 50 mM NaCl). This slowness suggest that the junction spends a significantly larger amount of time in the telomeric sequence compared with random DNA, a phenomenon already observed by Fouché et al (2006b). Table 1. Annealing and migration/dissociation rates of the HJ in the presence of telomeric proteins Components k1 M−1 s−1 k2 s−1 DNA alone 6.2±0.4 × 103 1.2±0.1 × 10−4 TRF1 9±2 × 104 4±0.5 × 10−5 TRF2 >107 3±1 × 10−5 TRF2ΔB 2.9±0.3 × 106 3±2 × 10−5 TRF2ΔM 1.1±0.2 × 106 4±1 × 10−4 B peptide 2.8±0.5 × 104 6±2 × 10−4 TRF2AΔB 1±0.2 × 105 6±3 × 10−5 TRF2 accelerates the rate of junction formation We measured the rate of junction formation in the presence of saturating amounts of TRF1, TRF2 and truncated forms of TRF2 (Figure 3D–G, Supplementary Figure 11 for longer time points with TRF2 and Table I). When TRF1 is added, the annealing rate is increased by about 10-fold; this is probably due to the capacity of TRF1 to form paired synapses in DNA (Griffith et al, 1998), thereby helping to bring the substrates together. With TRF2, the annealing step is strikingly accelerated (k1 is more than 100 times higher in the presence of TRF2 than TRF1). TRF2ΔB and TRF2ΔM also increase annealing suggesting that both domains participate in this step. Likewise, we observed that a chemically synthesised B peptide corresponding to residues 1–45 of TRF2 can also accelerate the annealing (about 5 times), although not a