In vivo ChEC analyses of the chromatin organization at Y’-telomere junctions
The in vivo ChEC method (for Chromatin Endogenous Cleavage), based on fusions of specific DNA-binding proteins of interest with the catalytic domain of micrococcal nuclease (MNase), was initially developed and applied to map the DNA-binding sites of the certain proteins on genomic sequences [41,42,43]. In short, MNase-dependent DNA cuts are induced in permeabilized yeast cells in vivo by addition of calcium ions. The localization of the induced cleavages by diverse techniques, i.e., Southern blot or high-throughput sequencing, allows the identification of high-affinity binding sites of the protein of interest. On the other hand, purified MNase has been used to detect protein-bound DNA sequences, for example nucleosomal DNA in vitro [44]. In this way, MNase has been used extensively to analyse protein positioning on DNA, and as in the case mentioned, nucleosome positioning. We reasoned that identification of MNase-protection patterns on DNA by performing ChEC experiments with expressed nuclear MNase could provide not previously available information on in vivo chromatin organization. To overexpress free nuclear MNase, we expressed it fused to a nuclear localization sequence (NLS) from the inducible gal1-10 promoter, used as control in previous studies [41, 42]. We also overexpressed MNase fused to the Gal4 DNA-binding domain (GBD). Given the absence of known Gal4-binding sites in our regions of interest, i.e., subtelomeres and telomeres, we similarly consider this GBD-MN a non-targeted nuclear protein. In addition, we analyzed MNase-dependent DNA cuts induced by MN-Rap1 [42, 43]. Rap1 being an abundant telomeric protein, it will yield a high local concentration of MNase at telomeres and provide a demarcation point for the transition between telomeric repeats bound by Rap1 and subtelomeric sequences [43]. Finally, to map subtelomeric nucleosomes on the same regions, we used a previously developed H2A-MN construct [41, 45]. A limitation of the ChEC method is that on areas with multiple closely spaced potential cut sites, conclusions on the dynamics of individual sites are difficult to draw. Also, it may not always be possible to know whether a cut is caused by a bound or an unbound fused protein [42].
We first focused on Y’ elements, representing approximately half of all yeast chromosomal ends (Additional file 1: Fig. S1a; [10]). First, we mapped MNase-dependent cut sites reflecting MNase accessible DNA sites on Y’-terminal restriction fragments (Y’-TRF) by Southern blotting with a Y’-specific probe proximal to the XhoI site that is conserved on all Y’ elements (YPX probe) (Fig. 1a, b). The combination of results obtained with MN-Rap1, GBD-MN and NLS-MN yielded five MNase-dependent cut sites in the Y’-TRF region analysed (indicated by arrowheads in Fig. 1b–d). Cleavage efficiencies of MNase-sensitive sites varied depending on the specific MN-fused protein and will be discussed below. Unexpectedly, spacing between some of these MNase-dependent cuts were inconsistent with the presence of phased nucleosomes upstream of telomeres, as was suggested in previous studies [4, 29]. Two MNase-protected DNA regions, between cutting sites II and III (106 ± 15 bp) and between III and IV (129 ± 11 bp), are significantly shorter than the 146 bp expected and minimally required in the case of phased nucleosomes (Fig. 1d and Additional file 1: Fig. S1b). Furthermore, between the first two MNase-dependent cuts (cutting sites I and II) is an area with a high density of binding sites for the Tbf1 and Reb1 proteins, two general regulatory factors (Fig. 1d and Additional file 1: Fig. S1a). If all those sites were bound by the cognate proteins, the first approximately 200 bp of the Y’ element proximal to telomeric repeats could be devoid of nucleosomes. To test this hypothesis, we fused MN to a canonical histone core protein, H2A, and determined the in vivo ChEC-induced cleavage pattern in this area. The experiment was performed on ice (4 °C) or at 30 °C, which allowed an assessment of cleavages occurring with different digestion kinetics (Additional file 1: Fig. S1c, d). The very partial H2A-MN cleavage at 4 °C indeed is necessary to detect MNase-cutting sites that are distant from the probe used. Ca2+-induced H2A-MN cleavage on ice during 15 min resulted in a partial digestion with visible mono-, di-, tri-, and tetra-nucleosome sized DNA fragments as assessed by whole genomic DNA analysis (Additional file 1: Fig. S1c lane 4). A comparable extent of cleavage is observed after 1 min of Ca2+-induced H2A-MN at 30 °C (Additional file 1: Fig. S1c lane 6). The resulting cleavage pattern at 4 °C analyzed with the YPX probe is very similar to that induced by GBD-MN and NLS-MN (compare Fig. 1c left with Fig. 1b). However and remarkably, while cleavage at cut site I is very efficient by the MN-Rap1 protein (Fig. 1b right), it is undetectable in the H2A-MN strain and only very weakly observable with the GBD-MN and NLS-MN proteins (Fig. 1b, c, dotted line arrowhead). This result thus is consistent with an absence of a nucleosome proximal to telomeric repeats. The complete H2A-MN digestion obtained after 5 min of Ca2+-induced H2A-MN at 30 °C allowed a direct assessment of whether there was a nucleosome in this area. Southern analysis with the YPX probe shows only mononucleosome-sized fragments near the probe (Fig. 1c, lane 8 (*)) similar to the ethidium bromide stain for bulk chromatin (Additional file 1: Fig. S1c, lane 8). In contrast, Southern analysis with a probe complementary to the stretch of Tbf1- and Reb1-binding sites on the Y’-TRF (YTR probe, Fig. 1a, e) yielded only a very low signal for the I–II fragment, annotated 2 on Fig. 1e and most of the signal remained in a smear near 600 bp that corresponds to the terminal fragment from site II to the end (annotated 1, Fig. 1e, lane 8), inconsistent with positioning of a nucleosome. Indeed, quantification of the gels indicates that only 24.3 ± 1.8% of the total signal corresponds to the I–II fragment after 5 min of Ca2+-induced H2A-MN at 30 °C (Fig. 1e, f). These results suggest two possible arrangements for chromatin on terminal Y’-sequences as depicted in Fig. 1g. In both models, the 200 bp of Y’ sequences proximal to the terminal repeats (DNA between cut sites I and II) are not occupied by a nucleosome. Most likely, these sequences are bound by Tbf1 and Reb1 proteins. In addition, the next short protected area between sites II and III covers the verified Y’-ACS and thus could be bound by the origin recognition complex (ORC, see discussion below for a more complete description).
As mentioned above, the subtelomere–telomere junction appeared to be differentially accessible to MN-fused proteins. A band corresponding to a cleavage at this position (MNase dependent cut site I) appeared early in the Ca2+-induced MN-Rap1 time course (2 min) and is the main cleavage product (Fig. 1b, c). To estimate and compare MNase accessibility to the Y’-telomere junction according to MN-fused proteins, we determined the ratio of cleavage products detected at the Y’-junction (% signal from band I) on cleavage products detected around the Y’-ACS (% signal from bands II and III) for each MN-fused protein (Fig. 1h). In contrast to MN-Rap1 (Ratio cleavage I/II + III: 0.87 ± 0.26), a very low accessibility of this subtelomere–telomere junction cutting site is observed with untargeted nuclear MNases, i.e., GBD-MN, NLS-MN, or H2A-MN (Fig. 1b, c, h) (Ratio cleavage I/II + III: 0.14 ± 0.08 (GBD-MN); 0.09 ± 0.003 (NLS-MN), 0.10 ± 0.06 (H2A-MN)) suggesting an inaccessibility of the Y’-telomere junction to free nuclear proteins and H2A.
The above results confirm the suitability of in vivo ChEC to analyse chromatin organization at subtelomere–telomere junctions. Furthermore, the results strongly suggest that the most distal portion of the subtelomeric regions of telomeres with Y’-elements, i.e., approximately half of the telomeres, are bound by Reb1 and Tbf1 proteins but lack a nucleosome.
In vivo ChEC analyses for the chromatin organization at X-telomere junctions
We applied the same procedures to analyse the chromatin organization of two X-telomere junctions: those at the TEL03L and TEL06R chromosomal ends. A TEL03L-specific probe identified eight MNase-dependent cut sites on the TEL03L-TRF (Fig. 2a–c). Two MNase-protected DNA regions, namely those observed between cutting sites I and II (approximately 106 bp) and between cutting sites V and VI (87 ± 10 bp), are too short to be able to accommodate a positioned nucleosome (Fig. 2d, e). Interestingly, between cutting sites I and II, a high density of potential Tbf1- and Reb1-binding sites is found, suggesting that the last 100 bp of the TEL03L-X element is protected by bound Tbf1 and Reb1 (Fig. 2d). Note that in strains without additional mutations as in Fig. 2, the MNase-cutting site II is detected only with H2A-MN and is relatively weak (black arrow on Fig. 2c), but it is readily detectable in strains with mutations in SIR complex genes (see below). The spacing between cutting sites II and III (152 ± 12 bp) and between III and IV (187 ± 16 bp) being congruent with the size of DNA protected by nucleosomes (Fig. 2e), we favor a model where these areas are associated with nucleosomal particles, despite a few potential Tbf1- and Reb1-binding sites identified (Fig. 2d, f). Interestingly, the cut site IV (average location: 804 ± 21 bp from the HindIII site) is close to an Abf1-binding site (centered at 783 bp from the HindIII site), suggesting that at this telomere, an MNase-sensitive DNA area is induced by Abf1 bound to its cognate site (Fig. 2d). The results also suggest a nucleosome positioned upstream of the Abf1-binding site (between MNase-sensitive sites IV and V) and ORC binding on the TEL03L-ACS (between cutting sites V and VI) (Fig. 2d, f). With complete H2A-MN digestion, we observed mostly mono-nucleosome size fragments (*) (Fig. 2c lane 4) as expected with the presence of nucleosomal arrays close to the probe used.
These results suggest that as shown above for the Y’-telomere junction, the region proximal to the TG repeats of X-only telomeres is devoid of nucleosomes. This hypothesis was assessed by Southern blotting H2A-MN ChEC samples with a telomeric probe (Additional file 1: Fig. S2). Note that this probe will reveal all telomeric bands, the X-only and Y’-telomeres at the same time. In the case of a positioned nucleosome abutting the terminal telomeric TG repeats and complete Ca2+-induced H2A-MN digestion, we expected the appearance of a smear centered on the average TG repeat length (300 ± 75 bp). However, the experiment yielded two distinct smears, one centered approximately at 600 bp (annotated 1 on the gels of Additional file 1: Fig. S2) and the other one approximately at 450 bp (annotated 3 on the same gels, Additional file 1: Fig. S2 lane 8). In contrast, when using MN-Rap1 and an induction time of 5 min, the expected smear close to 300 bp indeed became detectable (annotated 4 on Additional file 1: Fig. S2, left, lane 12). These findings are inconsistent with a nucleosome positioned next to TG-repeats on any chromosome end.
Given that the non-nucleosomal regions in Y’ or X(TEL03L) upstream of the telomeric repeats show potential binding sites for Tbf1 and Reb1 (Fig. 1c, d), we decided to analyse an X-only telomere, TEL06R, that lacks such potential binding sites for Tbf1 and Reb1 proximal to the TG repeats (see Additional file 1: Fig. S3a). ChEC analysis as above with a TEL06R-specific probe identified five preferential MNase-dependent cut sites on the TEL06R-TRF (Fig. 3a–d). Except for the fragment between cut sites I and II (spacing of 143 ± 9 bp), all MNase-protected DNA fragments were larger than 146 bp, arguing for positioned nucleosomes on those fragments (Additional file 1: Fig. S3b). Mono-nucleosome sized fragments (*) visible with complete H2A-MN digestion (Fig. 3b) confirm nucleosome arrays close to the probe used. As for the X(TEL03L)-telomere junction, we did not detect an H2A-MNase-dependent cut at the X(TEL06R)-telomere junction (MNase cut site I, Fig. 3b). Moreover, on X(TEL06R) the DNA region between MNase cut site I and II appears to be poorly protected from being cut by MNase. Indeed, with GBD-MN or MN-Rap1, a non-negligible diffuse cutting signal is observed between these two preferential MNase cut sites if compared to MN-Rap1-induced cuts in the corresponding area of TEL03L (Fig. 3c, Additional file 1: Fig. S3c). In specific, after 5 min of Ca2+-induced GBD-MNase or MN-Rap1, the bands corresponding to preferential MNase cut sites I and II on TEL06R are almost completely replaced by a very diffuse signal (Fig. 3c, Additional file 1: Fig. S3c). On TEL03L, a 5-min induction of MN-Rap1 still yields a sharp band with virtually no background cutting on either side (Additional file 1: Fig. S3c).
Moreover, the X(TEL06R)-telomere junction appears to be more accessible to free MNase than the X(TEL03L)-telomere junction (Fig. 1h). As previously discussed, to compare MNase accessibility to the subtelomere–telomere junction, we determined the ratio of cleavage detected at cut site I (subtelomere–telomere junction) versus cleavages detected around the X-ACS (Cleavages V and VI for X(TEL03L)-telomere, and cleavage III for X(TEL06R)-telomere). Compared to TEL03L or a Y’ telomere, this ratio increased sevenfold (GBD-MN) and ninefold (NLS-MN) for TEL06R (Fig. 1h). This site is also more accessible for MN-Rap1, but the relative increase is slightly less dramatic (about 3.5-fold increase for TEL06R as compared to TEL03L, Fig. 1h).
These results argue that on TEL06R, proximal to the TG-repeats, there is an area of increased MNase accessibility and that is potentially bound only by one Reb1 protein and one Abf1 protein. By extension, here again there may be no nucleosome abutting the TG repeats. From these observations, we propose a model of the chromatin organization of X(TEL06R)-TRF, depicted in Fig. 3e, which despite the clear differences of this telomeric X compared to other telomeric X elements, resembles a general model for X- and Y’-telomeres.
A common thread of all the findings reported to here on the terminal sequences of Y’- and X-telomeres is a strong and general MN-sensitivity very close to the ACS sequences, which could be attributed to ORC-binding (see Figs. 1b–g, 2b–f and 3b–e). To verify whether these observed patterns here are consistent with the well-established chromatin organization on a known ACS locus near an ARS sequences or particular for subtelomeric elements, we analyzed the TRP1ARS1 locus near centromere IV in an analogous fashion (Fig. 3f–h). The ARS1 locus contains well-documented ACSs and probably is the origin of replication with the most detailed information on chromatin organization and ORC binding (for example, [46, 47]). Hence, it is well established that ORC binds to this ACS and causes a nucleosome-free region starting in G2 all the way to the next S-phase [47]. The analyses of that locus with our technology are very consistent with these findings: a nucleosome-free area on the ACS region (cut site II to cut site III in Fig. 3f, g), and this area is accessible for cutting by free MN-Rap1 late in the time-course (Fig. 3f). Left and right from that short fragment are strongly positioned nucleosomes (Fig. 3f), as expected from ORC binding [47]. These characteristics therefore very closely parallel the results for the ACS sites on the X- as well the Y’-telomeres (Figs. 1, 2, 3). Altogether, these results strongly suggest that the sequences around the subtelomeric ACSs are ORC-bound, nucleosome-free fragments.
Chromatin organization at the subtelomere–telomere junctions is independent of telomere length and SIR2
To test if short telomeres induce changes in chromatin organization at subtelomere–telomere junctions, we analyzed MN-Rap1 ChEC patterns in yku80Δ cells of the three subtelomere–telomere junctions analyzed above. In addition to a short telomere phenotype and altered physical DNA ends, yku80Δ cells also suffer from telomere capping defects and a loss of TPE [48,49,50,51,52]. Despite expecting a lower amount of MN-Rap1 proteins at telomeres, MN-Rap1 ChEC patterns for the Y’-TRFs were virtually identical in YKU80 and yku80Δ cells (Fig. 4a and Additional file 1: Fig. S4a). Similarly, all previously identified MNase-sensitive sites for TEL03L-TRF were identical in YKU80 and yku80Δ cells (Fig. 2b, c and 4b). Remarkably, the previously determined MNase cut site II detected only with H2A-MN in wt cells becomes readily detectable with MN-Rap1 in yku80Δ cells (Fig. 4b and Additional file 1: Fig. S4b). These observations suggest that on TEL03L too, the absence of the Yku complex has no impact on the location of MNase-protected DNA fragments despite increased accessibility of the previously described MNase-sensitive site II (black arrow in Fig. 4b and Additional file 1: Fig. S4b). In a similar manner, we observed that MNase-sensitive site locations remained unchanged between YKU80 and yku80Δ cells when analysing TEL06R-TRF (Fig. 4c).
In addition, we tested if SIR2-dependent histone tail deacetylation is implicated in the chromatin organization at the subtelomere–telomere junctions. Sir2 being recruited to telomeres via an Sir4-mediated interaction with Rap1, we analyzed MN-Rap1 ChEC pattern in sir4Δ cells [15, 35]. Overall, MN-Rap1 ChEC patterns obtained on DNA in sir4Δ cells are indistinguishable from the ones obtained in yku80Δ cells. For example, the MN-Rap1 ChEC pattern in the absence of the Sir4 protein is unchanged for Y’-TRF (Fig. 4d). For TEL03L-TRF and TEL06R-TRF, in sir4Δ cells, we detected MNase cut sites at the previously characterized MNase-sensitive sites (Fig. 4e, f). Moreover, as detected in yku80Δ cells, in sir4Δ cells we observed an increase in accessibility of MNase cutting site II to MN-Rap1 for the TEL03L X-telomere junction (Fig. 4e).
We conclude from the above results that the locations of MNase-sensitive sites and, by extension, the locations of nucleosomes and proteins bound to subtelomeres are independent of telomere length and Sir2-dependent histone tail deacetylation.
Chromatin organization at internal X–Y’ junctions analyzed by in vivo ChEC
Given the subtelomeric organization of the X- and Y’-elements (see introduction), an X-element can abut either on a terminal TG repeat tract, such as on telomeres TEL03L and TEL06R, or abut to the distal end of a Y’-element. We thus wondered whether the chromatin organization on an X-telomere junction was different of that on an X–Y’ junction. Thus, we analyzed the ChEC pattern of two X–Y’ junctions, those in TEL05R and TEL16R. Whereas some X–Y’ junctions show TG repeats between the X element and Y’ [2], we confirmed by sequencing that, as expected from the available data on published databases, these two X–Y’ junctions do not have TG repeats between the X and Y’ element. However, these X–Y’ junctions show very distinct differences in size, spacing between the X-ACS and the X-Abf1 sites and in the number of potential Tbf1- and Reb1-binding sites (Additional file 1: Fig. S5a). The X element on TEL05R shows an organization and features similar to most X–Y’ areas with a 221-bp spacing between the X-ACS and the Abf1 sites and ends distally in a relatively high density of potential Tbf1- and Reb1-binding sites (Additional file 1: Fig. S5a). Note that overall, the X(TEL05R) is also similar to the X(TEL03L) previously analyzed in the X-only context (Additional file 1: Fig. S3a). To analyze the X–Y’ region of TEL05R by the ChEC method, we used a probe complementary to a subtelomeric region located at 1281 bp from the start of the X element (Fig. 5a). The analyzed fragment (AF) corresponding to this probe is obtained by genomic DNA digestion with PvuI and in addition to the X element, encompasses the first 4467 bp of a Y’(long) element (Fig. 5a). Upstream of the X element start we identified three MNase-sensitive sites (S-I, S-II and S-III on Fig. 5b, c). Moreover, we identified four MNase-sensitive sites on the X(TEL05R) element that are cut by all MN-fused proteins tested: GBD-MN, NLS-MN, H2A-MN and MN-Rap1 (Fig. 5b, c). As previously observed for the X(TEL03L)-TRF, two MNase-sensitive sites flank the X(TEL05R)-ACS located at 1325 bp from the PvuI site, generating a very short protected area of about 85 bp only (X-I to X-II in data summary in Fig. 5d). The cut site X-III centered at 1594 ± 19 bp from the PvuI site is close to the X-Abf1 site (1545 bp from PvuI site). Interestingly, we detected six preferential MNase-sensitive sites on Y’(TEL05R): one shared by all MN-fused proteins tested (cut site Y’-I), one detected only with MN-Rap1 (cut site Y’-II), and four detected with GBD-MN and NLS-MN only (see Fig. 5d). The cut site Y’-I is located at 70 ± 20 bp from the X–Y’ junction and is correlated with a potential Abf1-binding site centered at 75 bp from XY’ junction. The efficient cleavages over the X element with MN-Rap1 (Fig. 5c) strongly suggest a relatively high local concentration of MN-Rap1 over X–Y’ junctions.
The X(TEL16R) shows a spacing between the X-ACS and X-Abf1 site of 192 bp, shorter than the one of X(TEL05R) and with only one potential Tbf1-binding site within the X element (Additional file 1: Fig. S5a). In fact, X(TEL16R) is quite similar to X(TEL06R) which ends at telomeric repeats (see above, Additional file 1: Fig. S3a). According to published sequences, TEL16R harbours a X–Y’(short) junction. It should be noted that we found that in our strains (W303 background), TEL16R shows two consecutive Y’ elements (scheme depicted in Additional file 1: Fig. S5c). Nonetheless, we sequenced the XY’ junction and found no point mutations compared to the sequence from the standard S288C strain background. With our probe hybridizing at 1306 bp from the X element start, and genomic digestion of ChEC samples with BamHI and XhoI, we identified six MNase-sensitive sites (Additional file 1: Fig. S5d, e). Non-specific bands are observed with this probe (dotted line in Additional file 1: Fig. S5d), but the intensity of these bands again is negligible compared to intensity of the band corresponding to our fragments of interest (AF in Additional file 1: Fig. S5c, d). Three of the detected cut sites are located in the subtelomeric region upstream of the X element start site, two are located over the X element and one in Y’. Overall, these MNase-sensitive sites on the X(TEL16R) junction fragment are very similar to those described above for the X(TEL06R) element (see Additional file 1: Fig. S5e). The Y’-I cut site detected with GBD-MN and NLS-MN is located at 65 ± 4 bp from the X–Y’ junction. These data highlight the involvement of X-ACS and Abf1-bound DNA in chromatin organization of X elements, irrespective of whether the X element is terminal or not.
Again and as for the TEL05R above, while no TG repeats with potential Rap1-binding sites are present at the TEL16R X–Y’ junction, a remarkably efficient cutting by MN-Rap1 of the X-element sequences is observed. Detailed sequence analyses revealed certain potential Rap1-binding elements on both of these X-elements (Fig. 5d, Additional file 1: Fig. S5b, e). The efficient MN-Rap1 cutting at these sites on internal X-elements therefore could be due to direct Rap1 binding. We tested this prediction by ChIP experiments (Fig. 5e). Indeed, we could detect a specific Rap1 signal for both X-elements on TEL05R and on TEL16R by ChIP. As expected, a strong signal was also found for terminal Y’-sequences and the single Rap1 bound at the HMR-E element (Fig. 5e) [53]. These results therefore strongly suggest that Rap1 binds directly on X-elements, even on non-terminal ones such as those on TEL05R and TEL16R.
No cis-telomere fold-back detected by in vivo ChEC
Given the readily detectable MN-Rap1-mediated cut sites as well as direct Rap1 binding in the above two internal X-elements, we tested for the presence or absence of a telomere fold-back structure [29, 35, 37,38,39,40]. The model predicts that the distal telomeric TG repeat tract folds back and associates with the internal X-element, bringing the terminal Rap1 molecules in close contact with the X-sequences. This “telomere fold-back model” is supported by several indirect experiments but has not yet been demonstrated directly [35,36,37,38, 40]. In an X–Y’ context, the telomere foldback loops out the entire Y’ element in order for the terminal repeat tract to associate with the X-element [29]. However, previous experiments also showed that this cis-telomere foldback is strictly dependent on the SIR complex, i.e., the observed telomere-X interactions were lost in the absence of the Sir proteins [35, 36]. These findings predicted that if the MN-Rap1-dependent cuts detected over the X-element in an X–Y’ context are due to the canonical foldback mechanism, they will be dependent on the SIR proteins. Unexpectedly, the ChEC patterns on the X(05R)–Y’ junction obtained in sir3Δ and sir4Δ cells are indistinguishable from those obtained with cells that are SIR+ (Fig. 6a). In addition, as established by ChIP, the direct Rap1 binding at these X-elements also is independent from the Sir proteins (Fig. 5e), just as the binding of Rap1 on sequences at the ends of Y’-elements. Note that a Sir-dependent Rap1 binding was detectable in our experiments for the HMR-E element (Fig. 5e). Moreover, the yKU complex is also thought to be implicated in the telomere foldback structure [36, 39, 40]. However, our results show an extremely similar MN-Rap1 ChEC pattern of the X(05R)–Y’ junction in yku80Δ and YKU80 cells (Fig. 6b). In contrast and as expected [43, 54], the efficiency of MN cuts over X(05R)-Y’ is significantly decreased in sir4Δ when ChEC patterns obtained from strains expressing the Yku70-MN fused protein were analyzed (Additional file 1: Fig. S6a). We conclude that the MN-Rap1-mediated cleavages on the X-element in an X–Y’ context are independent of the SIR and yKU complexes and that Rap1 does bind to these elements directly in a Sir-independent fashion.
The telomere foldback model also predicts that cleavages on an X element analyzed with MN-Rap1 would be dependent on the presence of telomeric repeats on the same DNA molecule. Therefore, we analyzed the MN-Rap1 ChEC pattern on a replicative circular plasmid encompassing the X–Y’ junction from the same TEL05R as above (Fig. 6c, d). In the case of a telomere foldback, we expected a loss of MN-Rap1-induced cleavages over the X element on the plasmid. In contrast, we observed four MNase cut sites over the X element in both endogenous and plasmid context (X–I, X–II, X–III, X–IV on Figs. 5b, d and 6d, e). Comparison of the localization of these cleavages with respect to the X-element start at the endogenous site or the plasmid X(05R)–Y’ junction (Fig. 6e) shows a very analogous cutting pattern with no significant variation of the localization of MN-Rap1-dependent cuts over the X-element in an X–Y’ junction context. At the endogenous locus, two cleavages were detected in the first 200 bp of the Y’ element from TEL05R chromosomal end (Y’–I and Y’–II on Fig. 5b, d). On the p05RA plasmid, the cut Y’-II is observed whereas the cut Y’-I is confounded with a non-specific band (dotted line, Fig. 6d). Nevertheless, we conclude that preferential cuts induced by MN-Rap1 over an X-element in an X–Y’ context are independent of the presence of TG repeats or a physical DNA end on the same DNA molecule.