DNA-end capping by the budding yeast transcription factor and subtelomeric binding protein Tbf1
2011; Springer Nature; Volume: 31; Issue: 1 Linguagem: Inglês
10.1038/emboj.2011.349
ISSN1460-2075
AutoresVirginie Ribaud, Cyril Ribeyre, Pascal Damay, David Shore,
Tópico(s)Plant Molecular Biology Research
ResumoArticle27 September 2011free access DNA-end capping by the budding yeast transcription factor and subtelomeric binding protein Tbf1 Virginie Ribaud Virginie Ribaud Department of Molecular Biology, NCCR Program 'Frontiers in Genetics', University of Geneva, Geneva, Switzerland Search for more papers by this author Cyril Ribeyre Cyril Ribeyre Department of Molecular Biology, NCCR Program 'Frontiers in Genetics', University of Geneva, Geneva, Switzerland Search for more papers by this author Pascal Damay Pascal Damay Department of Molecular Biology, NCCR Program 'Frontiers in Genetics', University of Geneva, Geneva, Switzerland Search for more papers by this author David Shore Corresponding Author David Shore Department of Molecular Biology, NCCR Program 'Frontiers in Genetics', University of Geneva, Geneva, Switzerland Search for more papers by this author Virginie Ribaud Virginie Ribaud Department of Molecular Biology, NCCR Program 'Frontiers in Genetics', University of Geneva, Geneva, Switzerland Search for more papers by this author Cyril Ribeyre Cyril Ribeyre Department of Molecular Biology, NCCR Program 'Frontiers in Genetics', University of Geneva, Geneva, Switzerland Search for more papers by this author Pascal Damay Pascal Damay Department of Molecular Biology, NCCR Program 'Frontiers in Genetics', University of Geneva, Geneva, Switzerland Search for more papers by this author David Shore Corresponding Author David Shore Department of Molecular Biology, NCCR Program 'Frontiers in Genetics', University of Geneva, Geneva, Switzerland Search for more papers by this author Author Information Virginie Ribaud1, Cyril Ribeyre1, Pascal Damay1 and David Shore 1 1Department of Molecular Biology, NCCR Program 'Frontiers in Genetics', University of Geneva, Geneva, Switzerland *Correspondence to: [email protected] The EMBO Journal (2012)31:138-149https://doi.org/10.1038/emboj.2011.349 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 Telomere repeats in budding yeast are maintained at a constant average length and protected ('capped'), in part, by mechanisms involving the TG1−3 repeat-binding protein Rap1. However, metazoan telomere repeats (T2AG3) can be maintained in yeast through a Rap1-independent mechanism. Here, we examine the dynamics of capping and telomere formation at an induced DNA double-strand break flanked by varying lengths of T2AG3 repeats. We show that a 60-bp T2AG3 repeat array induces a transient G2/M checkpoint arrest, but is rapidly elongated by telomerase to generate a stable T2AG3/TG1–3 hybrid telomere. In contrast, a 230-bp T2AG3 array induces neither G2/M arrest nor telomerase elongation. This capped state requires the T2AG3-binding protein Tbf1, but is independent of two Tbf1-interacting factors, Vid22 and Ygr071c. Arrays of binding sites for three other subtelomeric or Myb/SANT domain-containing proteins fail to display a similar end-protection effect, indicating that Tbf1 capping is an evolved function. Unexpectedly, we observed strong telomerase association with non-telomeric ends, whose elongation is blocked by a Mec1-dependent mechanism, apparently acting at the level of Cdc13 binding. Introduction Telomeres, the protein–DNA complexes that constitute the ends of linear eukaryotic chromosomes, ensure the complete DNA replication of chromosome ends and protect these ends from recombination, degradation, and DNA damage checkpoint activation, the so-called 'capping' function (reviewed recently in Palm and de Lange, 2008; Lydall, 2009; de Lange, 2009; Wellinger, 2010). The molecular basis for these two essential telomere functions can be found in the telomere DNA sequences themselves, which are comprised of simple DNA repeats, T2AG3 in all vertebrates and most higher eukaryotes, and TG1−3 in the well-studied budding yeast Saccharomyces cerevisiae. These repeat sequences generate binding sites for factors (TRF1 and TRF2 in higher eukaryotes and Rap1 in the budding yeast) that act as platforms for the assembly of a complex set of proteins (referred to as the 'shelterin' complex in metazoans) that carry out both telomerase recruitment/activation and capping functions at chromosome ends. The extent to which the duplex DNA telomere-repeat binding proteins play a direct role in these processes is still poorly understood. In Saccharomyces cerevisiae, at least two different protein complexes have been implicated in telomere protection. The first of these capping complexes to be characterized was the Cdc13, Stn1, Ten1 (CST) complex, a putative structural homologue of the more ubiquitous RPA hetero-trimer (Gao et al, 2007), which binds to the GT-rich single-stranded overhang at telomeres (Lin and Zakian, 1996; Nugent et al, 1996). Inactivation of any one of the three CST components causes extensive and specific telomere DNA degradation, primarily of the 5′-end strand, and a checkpoint-dependent G2/M cell-cycle arrest (Garvik et al, 1995; Grandin et al, 1997, 2001). Interestingly, loss of CST function only leads to telomere damage following DNA replication in cells with high CDK activity (Vodenicharov and Wellinger, 2006). The CST complex may act by specifically blocking telomere association of the Mec1 (homologue of mammalian ATR) kinase, a key transducer in the DNA damage checkpoint pathway (Hirano and Sugimoto, 2007). A second telomere-capping mechanism involves the Rap1 protein and two Rap1-interacting factors, Rif1 and Rif2 (Hardy et al, 1992; Wotton and Shore, 1997; Negrini et al, 2007; Marcand et al, 2008; Hirano et al, 2009), whose target may be the Tel1 (ATM) kinase, through direct binding of Rif2 to the Xrs2 component of the DNA end-binding MRX complex (Hirano et al, 2009). MRX, consisting of Mre11, Rad50, and Xrs2 (NBS1 in mammals), is a highly conserved complex that binds rapidly to accidental DNA double-strand breaks (DSBs) and plays key roles in both repair and checkpoint pathways. Experiments examining the effect of telomeric sequences adjacent to a DSB suggest in addition that Rap1 possesses capping functions independent of the two Rif proteins, as well as the CST complex (Negrini et al, 2007). Finally, the yeast Ku heterodimer, a conserved, ubiquitous DNA end-binding protein, plays an important role in telomere capping outside of S phase by blocking the initiation of DNA resection (Bonetti et al, 2010; Vodenicharov et al, 2010). Both CST and Rap1–Rif protein complexes also regulate telomerase action at telomeres (reviewed in Bianchi and Shore, 2008 and Shore and Bianchi, 2009). Several lines of genetic and biochemical evidence point to a critical interaction between Cdc13 and the essential telomerase holoenzyme subunit Est1 in the recruitment and/or activation of telomerase at chromosome ends (Pennock et al, 2001; Taggart et al, 2002; Bianchi et al, 2004; Chan et al, 2008). Telomerase action at individual telomeres is regulated by a mechanism that senses the length of the TG-repeat tract such that short telomeres have a higher probability of being elongated by telomerase in a given cell cycle than do longer ones (Teixeira et al, 2004). Studies employing chromatin immunoprecipitation (ChIP) to examine protein association at individual telomeres in vivo as a function of TG-tract length suggest that the association of telomerase holoenzyme with ends is regulated by telomere length and that this effect is driven by increased binding of Tel1 kinase (Bianchi and Shore, 2007; Sabourin et al, 2007). A recent study (Gao et al, 2010) suggests, contrary to expectation, that Tel1 does not modulate the Cdc13–Est1 interaction, and its target or targets responsible for TG-tract length-dependent recruitment and/or activation of telomerase remain to be determined. Nevertheless, in vitro biochemical studies point to a role for the Cdc13–Est1 interaction in activation of a telomere-bound enzyme (DeZwaan and Freeman, 2009). Interestingly, the Rap1-bound Rif1 and Rif2 proteins are implicated in a TG-tract length-dependent mechanism that regulates MRX complex binding at DNA ends, and through this the recruitment of Tel1, which requires an interaction with the Xrs2 component of MRX (Negrini et al, 2007; Hirano et al, 2009; McGee et al, 2010). These findings suggest that the Rap1–Rif complex may employ related mechanisms to control both telomere end protection and telomerase recruitment or activation. Previous studies have indicated that T2AG3 repeats can participate in telomerase regulation when present adjacent to native TG1−3 tracts in budding yeast. Such chimeric telomeres have been generated either by de novo telomere formation with seed sequences consisting of T2AG3 repeats (Alexander and Zakian, 2003; Brevet et al, 2003), or by replacement of the endogenous TLC1 telomerase template RNA with a mutated allele that directs the synthesis of T2AG3 repeats onto the native TG1−3 ends (Henning et al, 1998; Alexander and Zakian, 2003; Brevet et al, 2003). Strains with the altered telomerase RNA, so-called 'humanized telomerase' strains, have also been used to generate novel telomeres that contain only T2AG3 repeats (Alexander and Zakian, 2003). TG-tract length regulation by T2AG3-repeat sequences has been shown to involve an endogenous and essential yeast protein, Tbf1 (Brigati et al, 1993), which binds to T2AG3 repeats through an SANT/Myb type DNA-binding domain that is related to that of both TRF1 and TRF2 (Bilaud et al, 1996). Curiously, telomere length regulation by Tbf1 is partially inhibited by Tel1 (Berthiau et al, 2006) and independent of either Rap1 or the Rif proteins (Alexander and Zakian, 2003). The mechanism(s) by which Tbf1 regulates telomere length is unknown. Furthermore, it is unclear how, or even the extent to which Tbf1 can cap telomeres in yeast. Interestingly, strains carrying a humanized telomerase template RNA appear to be in a chronic state of checkpoint activation, raising the question of whether Tbf1 possesses a capping function (di Domenico et al, 2009). Here, we exploit a simplified system, involving de novo telomere formation at an induced DNA DSB, to explore both the capping and telomerase regulatory functions of Tbf1 in yeast. Our data provide strong evidence that Tbf1 can efficiently block checkpoint activation at a DSB flanked by sufficiently long T2AG3-repeat arrays, through a mechanism strikingly similar to that of Rap1. In contrast, we show that shorter T2AG3-repeat arrays, though efficiently elongated by telomerase, more closely resemble an uncapped DSB. Our data also reveal a remarkably robust association of telomerase enzyme at non-telomeric DSBs and thus help to define situations in which telomerase activity is regulated at a step or steps following recruitment. Finally, this work strongly supports the idea (Berthiau et al, 2006; Arneric and Lingner, 2007) that Tbf1 may play a key 'backup' role in promoting the healing of telomeres that have experienced a catastrophic loss of terminal TG1−3 repeats. Results Telomerase elongation of Tbf1 site arrays (T2AG3 repeats) is length dependent In order to examine the dynamics of telomere formation at arrays of vertebrate-like (T2AG3) telomere repeats, we took advantage of a de novo telomere formation assay first described by Gottschling and colleagues that employs a galactose-inducible HO endonuclease gene (Diede and Gottschling, 1999). T2AG3 repeat telomere 'seed' sequences of either 60 or 230 bp in length were placed on the centromere-proximal side of an HO recognition site at cassettes placed near the left end of Chr. VII and the right end of Chr. V, respectively (Figure 1A). Galactose induction of HO in these strains triggers the production of a DSB with T2AG3 telomere repeat tracts at one end. We observed on a Southern blot that the short 60-bp T2AG3 tract is rapidly elongated following HO induction while the 230-bp tract, which is very similar in length to T2AG3-only telomeres generated in a humanized telomerase yeast strain (210–240 bp; Alexander and Zakian, 2003), is maintained at a constant length (Figure 1B). Because we did not modify the TLC1-encoded telomerase template RNA, telomerase adds TG1−3 repeats onto the T2AG3 ends, whose subsequent conversion to duplex DNA can be monitored by ChIP of Rap1 (Supplementary Figure S1), which has been shown previously to be a sensitive proxy for telomerase-mediated elongation at a DSB (Hirano et al, 2009; Zhou et al, 2011). Significantly, Tbf1–Myc binding remains remarkably constant following the induction of HO, even in cultures grown to saturation overnight, suggesting that there is little or no degradation of the T2AG3 tracts (Figure 1C; see below). Figure 1.Regulated elongation of short T2AG3 seed sequences by yeast telomerase is Tbf1 dependent. (A) Schematic representation of the modified subtelomeric regions of Chr. VII-L and Chr. V-R. (B) Southern blots monitoring HO cleavage and elongation of the indicated T2AG3 tract ends in a wild-type (TBF1) strain. An internal loading control ('INT'), a fragment arising before HO cutting ('U'), and a fragment derived from 'U' following HO digestion ('C') are marked. (C) Analysis by ChIP of the binding of Tbf1–myc in wild-type strains after HO induction. Results are reported as average fold enrichment (bar) and standard deviation (lines) relative to an internal control sequence within the PDI1 gene on Chr. III (see Materials and methods for details). (D) Southern blots monitoring HO cleavage and elongation of the indicated T2AG3 tract ends in a tbf1-Δi mutant strain. Download figure Download PowerPoint To test the role of Tbf1 in telomere healing at the T2AG3 ends, we repeated the experiment described above in strains carrying the tbf1-Δi allele, which lacks an internal region (amino acids 327–403) immediately upstream of the C-terminal SANT/Myb-like DNA-binding domain (Berthiau et al, 2006). The tbf1-Δi protein binds DNA but is defective in length regulation of T2AG3-containing telomeres and apparently unable to support viability in humanized telomerase yeast strains (Berthiau et al, 2006). In tbf1-Δi cells, we observed elongation of both the short and the long T2AG3 tracts (Figure 1D), indicating that Tbf1 is indeed required for the regulation of TG1−3-repeat addition observed in wild-type cells. We confirmed by qPCR ChIP that the mutant tbf1-Δi protein is still able to bind T2AG3 tracts in vivo (Supplementary Figure S1). These results show that short T2AG3 arrays are recognized as telomeres and elongated by the yeast telomerase in a manner similar to that of short TG1−3 seeds. Likewise, a long T2AG3 tract, similar to a long TG1−3 array (Negrini et al, 2007), is maintained at a constant length, in this case due to a regulatory function provided, at least in part, by the binding of Tbf1. Short T2AG3 arrays induce a transient G2/M arrest, but long arrays are capped To determine if T2AG3 sequences are able to 'cap' the DSB, and thus prevent activation of a DNA damage checkpoint response, we used an assay developed by Weinert and colleagues (Michelson et al, 2005) to examine directly cell-cycle progression at the single-cell level. Since the stability and checkpoint status of the DNA end distal to the elongating telomeric end in the HO cut experiments is still controversial (Hirano and Sugimoto, 2007), and in any event would be expected to induce a checkpoint response immediately following induction of the break, we modified the set-up described in Figure 1 by placing identical T2AG3 tracts on the distal side of the HO site, oriented in the opposite direction (Figure 2A, right panel). The behaviour of these constructs (with either head-to-head 60 or 230 bp T2AG3 arrays) was compared with that of two controls strains, one with no HO cleavage site and the other with no telomere-like sequences flanking the HO site. Figure 2.Short T2AG3 tracts elicit a checkpoint delay and are actively resected, while long T2AG3 ends are capped. (A) Percent of large-budded cells (G2/M-arrested cells) after HO cleavage (left panel) for strains (wild-type or vid22 ygr071c double mutants) containing the wild-type or modified Chr. VII-L constructs indicated in the right panel. Single and triple black arrowheads indicate 60 and 230 bp T2AG3 tracts, respectively, flanking an HO site (orange bar). The open arrowheads indicate the native telomere, located ∼13 kb from the HO site cassette. (B) Percent of single-stranded DNA measured by QAOS at either short (subtelomeric region of Chr. VII-L) or long (subtelomeric region of Chr. V-R) T2AG3 telomeric ends, or ends containing no TG-repeat sequence (DSB; the distal, telomere-proximal end of the HO site on Chr. VII-L) after HO cleavage. (C) ChIP analysis of Mec1–Myc and Rfa1–Myc association at short or long T2AG3 ends, or at non-TG (DSB) ends in wild-type cells. Fold enrichment reported as described in Figure 1. Download figure Download PowerPoint As expected (Michelson et al, 2005), the majority of cells from the strain containing no T2AG3 tracts flanking the HO site remained blocked in G2/M throughout the course of the experiment (7 h following galactose induction of HO), whereas most cells lacking the HO site had already traversed G2/M by 4.5 h. Remarkably, cells in which the HO site was flanked by 230 bp of T2AG3-repeat sequence passed through G2/M with kinetics similar to cells lacking the HO site (average restart time of 4.2 h versus 3.7 h), suggesting that the exposed 230 bp T2AG3 arrays were only very transiently recognized as DNA damage (Figure 2A). Furthermore, virtually all of these cells (82/84 or 97.6%) survived induction of the DSB (which was confirmed by Southern blotting; Supplementary Figure S2B), suggesting that stable telomere formation at the break was highly efficient. This behaviour is similar to that observed in cells carrying long arrays of native TG1−3 repeats (CR and DS, unpublished results). Interestingly, cells in which the HO site was flanked by a short (60 bp) T2AG3-repeat array displayed an intermediate phenotype, where passage through G2/M was clearly delayed, such that by 7.5 h only ∼50% of the cells examined had proceeded to the next cell cycle. Although these short T2AG3 tracts appeared to be efficiently elongated as judged by Southern analysis (Supplementary Figure S2B), reduced survival of these cells (83%; Supplementary Figure S2E) suggests that a small but significant fraction failed to be healed, and remained permanently arrested. Taken together, these data indicate that the short, elongating T2AG3 repeats were initially recognized as DNA damage that induced a G2/M checkpoint arrest. We presume that the elongation of these arrays in most cells (see Figure 1B) gradually converts them to a state that no longer promotes checkpoint activation. Consistent with an initial capping defect at the 60-bp T2AG3-repeat ends, we detected a considerable amount of single-stranded DNA (ssDNA) upstream of these ends (Figure 2B). Since the probes used to monitor the ssDNA are internal to the T2AG3 sequences (>1.2 kb from HO site), the assay detects resection events that proceed well beyond the repeats themselves. Thus, although the population of short T2AG3 tracts is undergoing telomerase-mediated 3′-end elongation (Figure 1B), they are also being subjected to extensive 5′-nucleolytic attack. In contrast, and consistent with the cell-cycle arrest data, we measured little or no ssDNA at the long T2AG3 repeats following HO induction (Figure 2B). In line with these resection data, we detected by ChIP significant recruitment of Rfa1 protein, a subunit of the trimeric ssDNA-binding RPA complex, and Mec1, the yeast ATR checkpoint kinase, at both the short T2AG3 tract and non-TG ends, but little or no recruitment of either protein at the long T2AG3 array (Figure 2C). These results reinforce the conclusion that the long T2AG3-repeat tract is hidden from the DSB checkpoint machinery, whereas the short tract is not. Tbf1 forms a stable complex with two BED domain-containing proteins, Vid22 and Ygr071c (Krogan et al, 2006), and these two proteins co-localize with Tbf1 at a large number (∼100) of promoter binding sites (Preti et al, 2010; CR and DS, unpublished data). Therefore, we asked if Vid22 and Ygr071c play a role in capping at T2AG3 array-containing ends by repeating the cell-cycle assay in strains where both VID22 and YGR071c genes were deleted. Interestingly, the capping function of the long T2AG3 array was unaffected by the double mutation (Figure 2A), despite the fact that Tbf1 binding at the end was considerably reduced, as measured by ChIP (Supplementary Figure S2A). Similarly, the weak capping function at the short T2AG3 array ends was not obviously affected by mutation of these two genes, though cell survival was reduced to <70% (Supplementary Figure S2E), probably reflecting a decrease in the efficiency of elongation, and thus stable telomere formation. Taken together, these data indicate that although Vid22 and Ygr071c promote more stable chromatin association of Tbf1, an effect observed also at promoter binding sites for Tbf1 (Preti et al, 2010), they are not required for Tbf1-mediated end protection. We also tested the effect of the tbf1-Δi mutation and of two mutations that affect the telomerase pathway: cdc13-2, and tlc1-Δ48. Only the tbf1-Δi mutation had a significant effect on capping at the long T2AG3 ends, leading to an ∼30-min delay in the cell-cycle arrest assay (Supplementary Figure S2D). The short array ends still displayed a prolonged arrest in these mutant backgrounds, as expected, and survival in these mutants was further reduced, compared with the vid22-Δ ygr071c-Δ double mutant (Supplementary Figure S2C and E). Interestingly, none of these mutations had as severe an effect on survival as did mre11-Δ (Supplementary Figure S2E). Other subtelomeric or SANT/Myb domain DNA-binding proteins do not cap DNA ends The ability of multiple Tbf1 molecules to cap DSBs, and to support their elongation by telomerase, prompted us to ask if other SANT/Myb domain-containing proteins, or factors known to bind at subtelomeric regions, would behave similarly. We, thus, constructed binding site arrays for Bas1 (TGACTCTG), an Myb-related transcription factor involved in purine and histidine biosynthesis, Reb1 (CCGGGTAAC), an SANT/Myb domain transcription factor that binds to many promoters, but also to subtelomeric sites, and Abf1 (GTCACTCTAGACG), another ubiquitous general regulatory factor (GRF) similar to Rap1 and Reb1, with both promoter and subtelomeric binding sites. Unlike the other factors tested (including Rap1 and Tbf1), Abf1 does not contain a SANT/Myb domain, but instead binds to DNA through an unusual bipartite Zn-finger domain. We generated binding site arrays of either 9 or 24 tandem copies for each of these three new factors, which were placed adjacent to the HO sites at the previously described cassettes near the telomeres of chromosome V-R and chromosome VII-L, respectively. None of these new sequence arrays were maintained after induction of the HO endonuclease, despite strong association of their respective DNA-binding proteins, as measured by ChIP (Supplementary Figure S3; data not shown). Southern blots revealed the rapid disappearance of the restriction fragment containing the arrays following HO digestion, with no evidence for elongation of the ends seen, in contrast to what was observed for the Tbf1 site arrays, or previously with TG1−3 tracts (Diede and Gottschling, 1999; Negrini et al, 2007). These results suggest that DSB end capping and extension by telomerase are not general features conferred by extended arrays of DNA-binding proteins. The negative result obtained with Reb1 arrays is particularly significant since Reb1, like Tbf1, has been directly implicated in telomere length regulation both by insertion of its binding sites immediately upstream of TG1−3 ends or by protein-tethering experiments at individual telomeres (Berthiau et al, 2006). Telomerase is excluded from long T2AG3 arrays but associates equally well with both short array or non-TG containing ends To begin to characterize the capping effect of T2AG3 tracts at the molecular level, we first examined the repeat-length dependence of telomerase association with these ends by ChIP, monitoring Myc-tagged versions of both the catalytic subunit Est2 and the associated Est1 protein. Telomerase was shown previously to crosslink more strongly to short versus long TG1−3 seed sequences at a DSB (Negrini et al, 2007), and the same relationship holds for native telomeres of different lengths (Bianchi and Shore, 2007; Sabourin et al, 2007). As expected, both Est1 and Est2 were detected at the short T2AG3 tract, while the long tract displayed weak binding of both proteins (Figure 3, upper panels). These results are in accord with the Southern blot observations, which showed that only the short tract is elongated (Figure 1B). In contrast, both telomerase subunits associate at very similar levels to the short and long T2AG3 tracts in cells carrying the tbf1-Δi mutation (Figure 3, bottom panels). Again, this situation is consistent with the observed elongation of both ends (Figure 1D). These data demonstrate directly that Tbf1, like Rap1, can regulate telomerase recruitment at DNA ends in a manner dependent upon the number of molecules bound immediately adjacent to that end, as suggested by previous studies (Alexander and Zakian, 2003; Brevet et al, 2003; Berthiau et al, 2006). Figure 3.Yeast telomerase is not recruited at long T2AG3 ends in wild-type cells, but is bound to these ends in a tbf1-Δi mutant strains. Analysis by ChIP of Est1–Myc and Est2–Myc binding after HO induction at 60 bp T2AG3, 250 bp T2AG3, or non-TG ends (as marked), in wild-type cells (top panels) or in tbf1-Δi mutant strains (bottom panels). HO constructs, PCR probes, ChIP methods, and statistical analysis were as described in Figures 1 and 2, and in Materials and methods. Download figure Download PowerPoint We also checked the binding of telomerase at the distal (telomere-proximal) side of the DSB at chromosome VII-L, even though this end does not contain TG-repeat sequences of any kind. Surprisingly, we detected strong binding of Est1 and Est2 at this end, similar to that observed at the elongating 60 bp T2AG3 end (Figure 3). However, this DSB end did not appear to be elongated, as expected, as judged by the absence of a slower migrating species on Southern blots and the failure to observe any Rap1 recruitment to this side of the DSB, which would have indicated the addition of TG1−3 repeats (data not shown). To validate these observations, we repeated the experiment using strains containing either Bas1 repeats or 300 bp of lambda DNA in place of the T2AG3 tracts, in this case upstream (on the centromere-proximal side) of the HO cut site. Again, we observed significant telomerase association following HO cutting at both types of ends, despite the fact that Southern blotting showed that they were not elongated (Supplementary Figure S3C and data not shown). Long T2AG3 tracts inhibit Mre11 binding, which promotes telomerase association at short tracts To investigate the underlying cause of reduced telomerase association at long T2AG3 tracts we turned to Mre11 protein. Mre11, a component of the conserved MRX (Mre11/Rad50/Xrs2) complex, is required for proper 3′ G-rich single-strand overhang production at telomeres (Diede and Gottschling, 2001; Larrivee et al, 2004), as well as normal recruitment of telomerase (Goudsouzian et al, 2006). MRX also binds rapidly to newly formed DSBs where it plays a key role in both non-homologous DNA-end joining and homologous recombination (reviewed in Rupnik et al, 2010). Given its role at both telomeres and accidental DSBs, we asked whether T2AG3 tracts influence in cis the association of Mre11 with a DSB. Notably, we observed severely reduced Mre11 binding at an end carrying the 230-bp T2AG3 array, compared with either a 60-bp T2AG3 end or a non-TG repeat containing end (DSB), where binding of Mre11 was equally strong (Figure 4A). We note that this reduction in Mre11 association at the long T2AG3 ends might also be sufficient to explain their relatively low levels of ssDNA and Rfa1 or Mec1 binding (Figure 2). These data indicate that long T2AG3 arrays block both the DNA-damage response and telomerase pathways at a very early step (MRX end recruitment), as was observed previously for long TG1−3 tracts (Negrini et al, 2007). Figure 4.Mre11 is required for efficient telomerase recruitment. (A) Analysis by ChIP of the binding of Mre11–Myc after HO induction in a wild-type strain. (B) Analysis by ChIP of the binding of Est1–Myc and Est2–Myc after HO induction in mre11-Δ. (C) Southern blot monitoring cleavage and elongation at both short (60 bp, left panel) and long (230 bp, right panel) T2AG3 tracts in mre11-Δ strains. HO constructs, PCR probes, ChIP methods, and statistical analysis were as described in Figures 1 and 2, and in Materials and methods. Download figure Download PowerPoint Given these findings, we next asked whether Mre11 is required for telomerase recruitment at the short T2AG3 seed sequence or a DSB. Indeed, we found that association of both telomerase subunits is very strongly reduced at these ends in mre11-Δ cells compared with wild type, nearly to levels observed at the long T2AG3 arrays (Figure 4B). Consistent with these findings, Southern blot analysis and measurement of Rap1 binding by ChIP indicated that neither the short T2AG3 seed nor the D
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