INTRODUCTION

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The length of the simple sequence tracts present at the telomere is tightly regulated by coordinated mechanisms of telomere lengthening and shortening, leading to a steady-state equilibrium (20, 63, 66, 73). In the yeast Saccharomyces cerevisiae, individual telomeres consist of irregular TG_1-3 sequences, with each complete telomeric tract maintained within 50 bp of a genetically determined mean size (63, 73). In yeast, as in most eukaryotes, the enzyme responsible for telomere lengthening is telomerase, a ribonucleoprotein reverse transcriptase consisting of a catalytic protein subunit, encoded by the EST2 gene (36, 37, 41), and an RNA species, encoded by the TLC1 gene (64). The RNA component of telomerase contains the template for the addition of telomeric repeats onto G-rich single-stranded substrates (20, 21, 64). In cells lacking telomerase, recombination among subtelomeric or telomeric sequences can also result in telomere lengthening (69, 70).

The mechanisms that counteract the possibly unlimited lengthening by telomerase are less well understood. Recent studies have provided evidence for several non-mutually exclusive mechanisms for maintaining a genetically set equilibrium of sizes. First, the major telomere binding protein, Rap1p, associating with the telomeric tract sequence motif GGTGTGTGGGTGT (14), is directly involved in achieving telomere length homeostasis. Rap1p activity is mediated in part through recruitment of positive and negative regulators of telomere addition to the Rap1p C-terminal 165 amino acids (10, 25, 30, 52, 77). The number of bound Rap1p C termini, or of factors associating with the C termini, is counted through an as-yet-unknown mechanism, until an optimal stable length is reached (44, 61). According to this model, shorter telomeres are elongated via telomerase to wild-type sizes, while overelongated telomeres gradually lose telomeric repeats until the wild-type size is regained. In this way, a homeostasis between addition and loss of telomeric sequences could be established (43, 44, 72). Second, Rap1p interacts directly or indirectly with telomerase to form a cap against the uncontrolled addition of telomeric tracts (29, 65). Third, the telomeric single-strand binding protein Cdc13p recruits multiple complexes to the telomeric 3' single-stranded overhang, where they act to maintain a balance between telomere elongation and loss at the extreme terminus (9, 17, 18, 40, 56, 60).

In addition to these mechanisms, we have proposed that the rapid truncation of overelongated telomeres, termed telomeric rapid deletion (TRD), negatively regulates telomere length (35). This mechanism is distinct from the slow attrition of telomeric sequences observed after increases in telomere tract size (43). TRD is observed in wild-type RAP1 strains containing a subset of telomeres that range in size from 400 to 3,000 bp greater than that of wild type, with the vast majority (>80%) of deletion events at an individual telomere reducing telomeric tracts to wild-type size (termed complete deletions). For the remaining events (<20%), deletions lead to intermediate sizes (termed incomplete deletions). TRD occurs at the relatively high rate of 3 × 10³ events/cell division/telomere (35).

Loss of the major recombination protein, RAD52, diminishes the TRD rate, implicating recombination in TRD (35). In addition, hpr1 cells, which display an elevated rate of recombination between direct repeats (1), also show an increase in TRD, raising the possibility that TRD may be an intrachromatid recombination event. However, no definitive mechanistic conclusions could be drawn from these initial genetic studies.

The frequency of complete rapid deletion events at an individual telomere depends upon the lengths of other telomeres in the cell (35). Cells that contain an increased number of wild-type length telomeres have a corresponding increase in complete deletions. Based on these results, we have proposed two components of TRD: a recombinational process between imperfect (homeologous) repeats and a "yardstick" that measures telomere lengths relative to one another.

The yeast and human Ku heterodimer, involved in nonhomologous end joining, also associate with telomeres in vivo and in vitro (3, 19, 32, 42). Interestingly, loss of either of the subunits of the yeast Ku heterodimer (yKu70 or yKu80) confers a large increase in the TRD rate (58), in nucleolytic degradation (58), and in the length of terminal 3' single-stranded overhangs (19, 58). The yku70 and yku80 alleles also display a global decrease in telomere tract size (6, 59). These data suggest that the yKu heterodimer is part of a telomeric cap that guards against potentially deleterious processes such as promiscuous recombination and end degradation.

yKu acts together with a trimeric complex consisting of Mre11p, Rad50p, and Xrs2p (the MRX complex) in the nonhomologous end-joining pathway (8, 23, 42, 76). However, the role of the Mre11p-Rad50p-Xrs2p (MRX) complex is far more complicated and is additionally required for mitotic homologous recombination, induced by ionizing radiation (47), double-strand break formation (7, 27), and meiotic recombination (23). At telomeres, each MRX component plays a positive role in telomere addition (6, 50), which is mediated through the telomerase pathway (23, 34, 50). Further indicating a role of the MRX complex at telomeres, human Rad50, Mre11, and Nbs1 (the presumed Xrs2p homologue) associate with the telomere in vivo (78). However, the role of the MRX complex in TRD remains unknown.

We investigate here the mechanism of telomere rapid deletion. Through a combination of physical and genetic assays, we provide evidence that TRD is an intrachromatid recombination process, with sequences near or at the extreme terminus invading distal tract sequences. Our studies also indicate that the MRX complex plays a critical role in the TRD process.

	MATERIALS AND METHODS

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Plasmids. Plasmid AD3ARUGT-IV [pVIIL URA3()tel] contains VIIL sequences, a "flipped" URA3 gene transcribing toward subtelomeric sequences [designated by ()], and an 80-bp poly(TG_1-3) seed sequence (64). pVR AD3ARUGT [pVR URA3()] was derived by ligating the 2.8-kb HindIII fragment of pVR URA3()tel (16) into the unique HindIII site of pVIIL URA3()tel. pVR URA3() in which the URA3 sequences are transcribed toward subtelomeric sequences was identified by Southern analysis. pADADE2 was used for labeling of VIIL telomeres with the URA3/ADE2 marker, as previously described (16).

Yeast media and strains. Rich (YPD [yeast extract-peptone-dextrose]), synthetic complete (SC), and SC omission media were prepared by standard methods. YPAD is identical to YPD except for supplementation with 30 mg of adenine/ml.

View larger version (30K): [in this window] [in a new window]   FIG. 1.   Incorporation of HaeIII sites into elongated telomeres. (A, left panel) rap1-17 cells containing newly generated URA3/ADE2-marked VIIL telomeres were derived from a URA3()-marked VIIL telomere by recombination with a homologous fragment containing the URA3/ADE2 sequences and 80 bp of telomeric seed sequence. Transformation was conducted in the presence of a plasmid borne copy of TLC1-1. The TLC1-1 RNA contains a HaeIII site within its template region. The telomeric seed sequence was elongated to sizes greater than that of the wild type, which is typical of the telomeres present in rap1-17 cells. As a consequence, HaeIII sites were introduced into the URA3/ADE2-marked VIIL telomere, as well as into other unmarked chromosomal telomeres, during the addition of telomeric repeats. After loss of the plasmid borne TLC1-1 gene, the rap1-17 strains containing the elongated URA3/ADE2 HaeIII-marked telomere were mated to an isogenic wild-type strain, and wild-type RAP1 spore colonies containing elongated telomeres were identified. These spore colonies represent the MBH series of strains. (A, left panel) Elongated URA3()-marked VIIL telomeres were grown in the presence of TLC1-1 RNA to generate HaeIII sites within both marked and unmarked chromosomal telomeres in rap1-17 cells. Incorporation of HaeIII sites at multiple positions is presumably due to repetitive cycles of deletion and elongation typical of rap1-17 telomeres. After loss of the mutant telomerase RNA, the plasmid-borne copy of rap1-17 was replaced with a wild-type copy of RAP1 through a plasmid shuffle, giving rise to the MUH series of strains (A, right panel). Dark striped regions, the URA3 gene; light striped regions, the ADE2 gene; chromosomal sequences, gray lines; telomeric sequences, black lines; <CEN, direction toward the centromere; arrow, direction of transcription. (B) Restriction maps of the genomic and telomeric ADE2 genes in the strains used in this study (35). WT refers to the strain CZY1/RAP1 carrying a wild-type length URA3/ADE2-marked telomere. Fragment lengths are shown for NdeI (N), NdeI/HaeIII, and HaeIII (H) sites. Each URA3/ADE2-marked VIIL telomere contains one of the three HaeIII sites. The position of a HaeIII site in the telomere was determined by the size of the NdeI/HaeIII fragment after subtracting the length of subtelomeric sequences (1.1 kb). These sites were positioned at 150 bp (HaeIII 150), 250 bp (HaeIII 250), and 750 bp (HaeIII 750) from the subtelomeric junction. The relative positions of HaeIII 150, HaeIII 250, and HaeIII 750 telomeres are shown in the expanded view. The 3.6- and 1.1-kb ADE2 probes used in the study (top and bottom, respectively) are also shown. Designations were described in the Fig. 1A legend. TEL> refers to the direction toward the telomere. (C, left panel) DNA isolated from MBH22-7b colonies, containing the URA3/ADE2 HaeIII 250-marked VIIL telomere, was digested with NdeI (N) or with NdeI and HaeIII (NH) and then subjected to Southern blotting with the 3.6-kb ADE2 probe indicated in panel B. Progenitor refers to the original marked elongated telomere after digestion of DNA isolated from several MBH22-7b subclones with NdeI (lanes 2, 4, and 6). In some MBH22-7b colonies (lane 8), the progenitor had been deleted before subculturing was done for individual colonies. WT refers to NdeI-cleaved DNA derived from CZY1/RAP1. , Restriction fragments derived from the telomeric copy of ADE2; x, restriction fragments derived from the internal ADE2 locus. Size markers (in kilobases) are shown on the left. The positions of the Progenitor, the TRD product, and the NdeI/HaeIII fragment carrying the HaeIII 250 (H250) site are also indicated. M refers to the molecular mass marker. (C, right panel) As a control, DNA was isolated from CZY1/RAP1 and digested with NdeI or with both NdeI and HaeIII. Note that digestion of CZY1/RAP1 generates an internal 1.45-kb NdeI/HaeIII fragment that overlaps with the telomeric fragment. For presentation purposes, the autoradiogram is a composite of two portions of a single gel, with the junction borders indicated by the vertical line on top of the gel. Similar confirmations of HaeIII incorporation were carried out in MBH11-8b and MBH93-6d, carrying the HaeIII 150- and the HaeIII 750-marked telomeres, respectively.

TRD assays. Rapid deletion was measured by a change in the size of the telomeric fragment relative to the elongated progenitor. To this end, the progenitor and truncated forms of URA3/ADE2 or URA3 VIIL()-marked VIIL telomeres were digested with NdeI and ApaI, respectively, and subjected to Southern analysis.

Site retention assay. The site retention assay measures the presence or absence of the HaeIII site in the marked VIIL telomeres after a screen for cells containing partial deletion events (i.e., a subset of incomplete deletions that are smaller than the distance between the HaeIII site and the extreme terminus). In the case of the URA3() HaeIII-marked VIIL telomeres, ApaI and ApaI/HaeIII double digests were used to determine telomere length, the distance between the HaeIII site and the extreme terminus, and the retention or loss of the HaeIII site after Southern blot hybridization with URA3 sequences as a probe. A similar protocol was used with strains containing URA3 (or ura3)/ADE2 HaeIII-marked VIIL telomeres, except that the telomeric length was determined by digestion with NdeI. NdeI/HaeIII double digests were used to measure the distance from the HaeIII site to the extreme terminus and the degree of HaeIII site retention after Southern blots were probed with ADE2 sequences.

Site transfer assay. The site transfer assay measures the movement of a HaeIII site in elongated URA3/ADE2 HaeIII 250- and ura3/ADE2 HaeIII 750-marked VIIL telomeres to a heterologous wild-type-length URA3()-marked VR telomere lacking HaeIII sites in strains YP17-24c and DJ204-10b, respectively.

Site acquisition assay. Conversely, we determined whether the VIIL telomere is capable of acquiring HaeIII sites from heterologous telomeres. To this end, we first crossed AJL 437-1d and MBH9 to give rise to the strain AJL528. The diploid was sporulated, and two spore colonies, AJL 528-1b, and AJL 528-2b, were isolated. These strains carry 50% elongated telomeres with a wild-type length ura3/ADE2 VIIL telomere. A total of 221 independent subclones were selected and subsequently subcultured in batches of 10 to 11 as described above.

2

Statistical analysis. Chi-square analysis was used for comparisons between expected and observed values in site transfer, retention, and acquisition assays. The rank sum (Mann-Whitney) test was employed to compare the degree of overlap between the distributions of wild-type and mutant cell values.

	RESULTS

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A physical system to study telomeric dynamics. We have previously proposed that TRD is the consequence of intrachromosomal recombination. However, further tests of this hypothesis have been limited by the absence of physical markers in telomeric simple sequences. To overcome this obstacle, we introduced HaeIII restriction enzyme sites at defined positions within URA3()-marked and URA3/ADE2-marked telomeres on the left arm of chromosome VII (VIIL [35]). This was accomplished by the use of an allele of TLC1, TLC1-1, that acts as a template for the HaeIII sequence (64). Transient expression of TLC1-1 was performed in cells containing both the VIIL marked telomere and the rap1-17 mutation, which confers overelongated telomeres (Fig. 1A; see Materials and Methods). HaeIII sites were kept to a minimum by the coexpression of wild-type and mutant telomerase RNAs. Based on HaeIII digestion of genomic DNA, we estimate that 50% of the telomeres incorporated one or more HaeIII sites (data not shown).

TRD is an end-mediated process. To test whether TRD is mediated through intrachromatid recombination, as postulated previously (35), or through other intra- or interchromosomal pathways, we designed an assay that explores the relationship between the degree of deletion and the retention of the HaeIII site. This site retention assay takes advantage of incomplete TRD events that do not delete to wild-type sizes (see Materials and Methods; Fig. 2A). A subset of incomplete deletions, termed partial deletions in this study, form the basis of the site retention assay. Partial deletions are operationally defined here as incomplete deletions in which the size of the deletion is smaller than the distance from the HaeIII site to the extreme terminus (Fig. 2B, subclones 7, 8, 9, and 12). The behavior of larger incomplete deletions (Fig. 2B, subclones 11 and 16) can be explained by a plethora of mechanisms and are therefore not included in this analysis. Partial deletion products are capable of deletion to wild-type telomeric tract size in a manner indistinguishable from the progenitor (Fig. 2B, right panel). Hence, partial deletions are likely to be mechanistically related to complete deletions in the wild-type TRD pathway (see Discussion).

View larger version (58K): [in this window] [in a new window]   FIG. 2.   Site retention patterns associated with TRD. (A) Site retention was assayed in partial deletions, representing incomplete deletions that are smaller than the distance between the HaeIII site and the terminus. A terminal deletion pattern (left panel) maintains the HaeIII site. A complex deletion TRD pattern (right panel) would lead to both site loss and retention. The shadings of lines and boxes are described in the legend to Fig. 1A. (B, left panel) MBH91-3 (P), which contains both elongated and deleted forms of the URA3/ADE2 HaeIII 750-marked VIIL telomere, was subcultured, and individual colonies were isolated (lanes 7 to 16). DNAs isolated from these colonies were digested with NdeI to determine the extent of the deletion and with NdeI and HaeIII to measure the retention of the site. The resulting Southern blot was probed with the 1.1-kb NdeI/BamHI ADE2 restriction fragment of pL909. Size markers (in kilobases) are shown to the left of the autoradiogram. Below each lane, the telomeric fragment size (NDEI), the NdeI/HaeIII telomeric fragment size (NDEI/HAEIII), and the presence or absence of the HaeIII site (HAEIII RETENTION) are depicted. HaeIII 750 (H750) denotes the position of the terminal NdeI/HaeIII fragment. x, Restriction sites from the genomic ADE2 locus; NA, not applicable due to absence of HaeIII sites. Other designations are described in the legend to Fig. 1. Subclones 11 and 16 have lost the HaeIII site as a consequence of incomplete deletions larger than the distance between the HaeIII site and the extreme terminus. Partial deletions, on the other hand, (subclones 7, 8, 9, and 12) all displayed the HaeIII 750 site. (B, right panel) DNAs isolated from three subclones of MBH21-20b were subjected to digestion with NdeI (N), and subsequent Southern blots were probed with the 3.6-kb BamHI fragment containing the ADE2 gene. Lane 1, the 3.1-kb precursor representing a telomere tract length of 2.0 kb; lane 2, the 2.5-kb partial deletion product representing a 1.4-kb telomeric tract; lane 3, the 1.35-kb complete deletion product representing a tract length of 300 bp. All other designations are indicated in the legend to Fig. 1.

Absence of interchromosomal exchange in TRD. To distinguish between these possibilities, we designed a "site transfer" assay to measure the movement of a HaeIII site between telomeres during rapid deletion. Site transfer measures the ability of the VR URA3()-marked telomere that lacks a HaeIII site telomere to acquire a HaeIII site from a heterologous HaeIII-marked VIIL telomere during rapid deletion (Fig. 3A; see Materials and Methods). In most cells, the marked VIIL telomere was the only elongated and HaeIII-marked telomere. To carry out site transfer, we first identified cells carrying a VIIL telomere which had undergone a deletion to the wild-type size (Fig. 3B, right panel). Transfer of HaeIII sites in these cells was subsequently measured by the ability of the VR-marked telomere to be cleaved by HaeIII. Site transfer would be expected to occur in a nonreciprocal translocation, but not in an intrachromatid deletion (Fig. 3A).

View larger version (63K): [in this window] [in a new window]   FIG. 3.   Relationship between TRD and HaeIII site transfer. (A) Site transfer is assayed by measuring the frequency of HaeIII site movement from the elongated URA3/ADE2 HaeIII 250-marked VIIL telomere to the URA3()-marked VR telomere in strain YP17-24c. The shading of lines and boxes has been described in Fig. 1A. (B, left panel) DNA was isolated from 20 batch cultures, each containing 10 independent colonies. DNA was digested with ApaI and HaeIII and probed with a 1.7-kb PstI fragment of pRS316 containing the URA3 gene. The telomeric fragments (VR) were detected in each batch. The position of a unique HaeIII site transferred to the VR telomere (as well as the length of the VR telomere after transfer) is unpredictable and depends on the mechanism leading to the site transfer (gray arrows to the right of the panel). The 1.8-kb fragment represents the left junction of the chromosomal URA3 gene, while the smaller fragments represent two 350-bp HaeIII sites that are common to both telomeric and genomic copies of URA3. The positions of internal URA3 sequences are designated by "i." Fragments originating from the URA3() telomeric fragment are denoted by a hatched mark. Size markers are shown on the left. (B, right panel) DNAs from the same samples were digested with NdeI and Southern blots were probed with the 3.6-kb BamHI fragment containing the ADE2 gene. All samples contained deleted URA3/ADE2-marked VIIL telomeres. WT refers to CZY1/RAP1. Other designations are defined in Fig. 1C.

The Rap1p C terminus does not influence TRD. The 165-amino-acid Rap1p C terminus acts as a negative regulator of telomere addition. The rap1-17 mutant that lacks this domain displays multiple and frequent cycles of rapid loss and overelongation of telomeric tract sequences (30). However, it remains unknown whether this rapid loss of telomeric sequences proceeds through the wild-type mechanism for TRD. We therefore sought to determine the influence of the Rap1p C-terminal domain on TRD by examining the behavior of TRD in rap1-17 strains (30, 35).

Rad50p and Mre11p are positive regulators of TRD. The MRX complex participates in a multiplicity of homologous mitotic and meiotic recombinational pathways, nonhomologous end joining, and telomere size control (6, 7, 8, 23, 27, 28, 42, 47, 50, 76). At least one component of this complex, Rad50p, is known to influence the abrupt elongation of telomeres by telomere/telomere recombination in cells that lack telomerase (type II recombination [69]).

View larger version (47K): [in this window] [in a new window]   FIG. 4.   rad50 and mre11 mutations inhibit TRD. (A) Diagram of the restriction maps from the progenitor and deleted ura3/ADE2-marked VIIL telomeres. Line and box shadings are defined in Fig. 1A. (B, left panel) DNA was isolated from a wild-type spore colony (YPR50-14c) and from rad50 spore colonies (YPR50-15c and YPR50-15d) after two rounds of liquid subculturing, corresponding to 20 generations of growth (0 to 2, indicated below each lane). All strains contain the elongated ura3/ADE2-marked VIIL telomere. The DNA was digested with NdeI, and the resulting Southern blots were hybridized with the 3.6-kb BamHI fragment of pL909 carrying the ADE2 gene. The expected mobility of the telomeric fragment in rad50 cells is indicated on the right. Note that an incomplete deletion also accumulates during subculturing of wild-type cells. (B, right panel) DNA was isolated from a wild-type spore colony (YPM11-1b) and from mre11 spore colonies (YPM11-3d and YPM11-6a), containing the elongated ura3/ADE2-marked VIIL telomere, after two rounds of liquid subculturing as described above. DNA was digested and subjected to Southern analysis as described in the text. The expected mobility of the telomeric fragment in mre11 cells is indicated on the right. Size markers (in kilobases) are shown on the left of each panel. Vertical bars above the autoradiograms indicate the positions of lanes from the same gel that were spliced for presentation purposes. C refers to the strain CZY1/RAP1. All other designations were described in the legend to Fig. 1C.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this investigation, we present the first mechanistic insights into TRD, a process that is likely to be a major participant in telomere size control. Our studies have been based on the development of a physical assay for TRD, in which HaeIII sites are introduced into the telomeric tract. The presence of telomeric HaeIII sites does not interfere with telomeric functions including silencing, size control, and TRD (Fig. 1C; also data not shown).

Two physical criteria were used to investigate TRD. The first criterion was the relationship between the loss of sequences and the retention of the HaeIII site. The site retention assay relies on the characteristics of partial TRD events, i.e., incomplete deletions that are smaller than the distance from the HaeIII site to the extreme terminus. Partial deletion follows the same pathway as complete deletions. The evidence for this is twofold: (i) partial deletion species subsequently delete to wild-type-length telomere size (Fig. 2B, right panel), and (ii) complete TRD occurs more frequently as the percentage of wild-type-length telomeres increases (35). Thus, the size of the deletion is dependent on the size of the majority of telomeres rather than being an obligatory intermediate step in TRD. Hence, partial TRD is likely to be the consequence of sizing relative to aberrantly elongated telomeres.

The second criterion is the movement of HaeIII sites between two marked telomeres during rapid deletion. We found no evidence for site transfer in our studies. For statistical evaluation of our data, we made the simplifying assumption that the VIIL telomere associates with VR and other telomeres at similar rates. One important difference, however, is the presence of subtelomeric elements in natural, but not artificially marked, telomeres. Previous studies have revealed the presence of selected groups of telomeres that exhibit preferentially high rates of interchromosomal recombination between conserved Y' subtelomeric elements (38). Subtelomeric regions that share the greatest degree of homology have the highest rates of recombination, suggesting that the degree of homology dictates the probability of subtelomeric recombination (38). If these preferentially interacting telomeres are functionally sequestered from the marked telomeres, site transfers between the VR and VIIL telomeres may occur at a higher frequency than expected. Hence, the actual significance of the difference between observed and expected values may be greater than we reported. Therefore, associations based on subtelomeric homology are unlikely to interfere with the ability to observe HaeIII site transfer. The lack of ura3/ADE2-marked VIIL telomere site acquisition from heterologous HaeIII-marked elongated telomeres further argues that interchromosomal exchange is not likely to be responsible for TRD.

The presence of a terminal pattern of site retention and the absence of site transfer between nonhomologous telomeres, lacking subtelomeric homology, effectively rule out interchromosomal recombination. These include those processes involving crossing over (see, e.g., references 5 and 24), double-strand break repair (68) and break-induced replication (53). The data are also inconsistent with intrachromosomal events such as sister chromatid exchange (57), single-strand annealing (26, 53), and other mechanisms of internal deletion (Table 2). Rather, the data support a mechanism of intrachromatid loss of telomeric sequences initiated at the extreme terminus. An exonucleolytic activity is an unlikely explanation of our data, given that this activity would be expected to generate heterodispersed telomeric fragments that decrease in size during growth, rather than the discrete TRD products that we observe. In addition, our physical data cannot be explained by endonuclease cleavage, unless cleavage occurs in the absence of DNA repair. We note that any recombinational basis for TRD must involve a homeologous interaction between telomeric sequences, given the limited sequence homology present in telomeric tracts.

TRD is independent of the C terminus of Rap1p. This finding strongly suggests that TRD is also independent of components of telomeric chromatin, including both negative (Rif1p and Rif2p) and positive (Sir3p and Sir4p) regulators of telomere size (10, 25, 52, 77). Additionally, TRD is not linked to the overall structure of subtelomeric regions, since loss of the C-terminal tail and Sir proteins results in an open chromatin state in subtelomeric regions (15, 31).

TRD is likely to be a mechanism that acts in concert with other elements of the telomere sizing system to achieve and maintain homeostasis. In particular, the Rap1p counting mechanism (44, 61), size regulation through Rap1p-telomerase interactions (29, 65), or Cdc13-mediated homeostatic mechanisms (9, 17, 18, 40, 56, 60) present at the terminal 3' overhang may maintain the size of the TRD product. It is noteworthy that the Rap1p C terminus is essential for the Rap1p counting mechanism, but dispensable for TRD, suggesting that the two processes are mechanistically distinct.

Participation of the MRX complex in TRD. The strong inhibitory effect of rad50 and mre11 on TRD, coupled with the diminution of TRD in rad52 cells (35; data not shown) supports a recombinational model for this process. However, the relationship between Rad50p stimulation and Rad52p stimulation of TRD is unclear, and the presence of Rad50p-dependent Rad52p-independent deletions remains a distinct possibility.

TRD and homeologous recombination. Unlike most homeologous recombination, TRD is not constrained by mismatch repair enzymes (35, 53). Mutations in MSH2, MSH3, or PMS1 have no effect on the rate of TRD (35). This apparent discrepancy has several possible explanations. First, the effect of mismatch repair enzymes on homeologous sequences is dependent on the degree of homology (11). While mismatches of 10% are strongly repressed by Msh2p and Msh3p, DNA sequences having lower levels of homology are independent of mismatch repair. Second, cells may have multiple pathways for homeologous recombination, as suggested by the presence of a homeologous recombination pathway independent of mismatch-repair in pms1 cells (4). Third, the chromatin structure at telomeres may influence the telomeric homology requirements or the role of mismatch repair enzymes. For example, among telomeric sequences are multiple copies of the highly conserved 13-bp binding site for Rap1p (14, 74), which, in the context of telomeric chromatin, may play a unique role in telomere-telomere pairing. If Rap1p is involved, however, it must be independent of the Rap1p C terminus, since the rap1-17 mutation does not influence TRD. Similarly, a specific structure formed at telomeres, such as a t-loop (22) or a G-quadraplex structure (75), may act in cis to promote TRD. Distinguishing among these possibilities is a major goal for the future.

A model for telomeric recombination. The genetic and physical studies presented here suggest that TRD is a 3' end-mediated intrachromatid recombinational process (Fig. 5). Specifically, the components of the MRX complex may directly or indirectly prepare the 3' telomeric terminus for strand invasion. The recombinationally proficient 3' extreme terminus would subsequently invade distal telomeric DNA, leading to an excision of the intervening sequences through branch migration, D-loop degradation, and resolution of the Holliday junction (Fig. 5). This may result in the transient formation of linear or circular degradation products, depending upon the precise nature of resolution, which would be quickly lost from the population. One intriguing feature of this model is that multiple cycles of both semiconservative replication primed from the invading strand and subsequent reinvasion could give rise to amplified telomeric DNA. This interrelationship raises the possibility that TRD and type II events may be mechanistically related. Interestingly, the proposed TRD intermediate (Fig. 5, step 2) is virtually identical in structure to the stable human t-loops involved in telomere cap function (22). The function and evolutionary relationship between TRD and t-loops, however, remains unknown.

View larger version (19K): [in this window] [in a new window]   FIG. 5.   Model for telomeric rapid deletion. We propose that the 3' single strand from the telomeric terminus (A) invades distal telomere tract sequences, leading to the formation of a looped structure (B). (C) After branch migration, the displaced strand forms both a D-loop and Holliday junction. After nicking of the D-loop (yellow arrow), degradation of the D-loop (D), and resolution of the outer strands of the Holliday junction (green arrows), both the TRD product and a linear excision product are produced (E). As shown, we propose that the Ku heterodimer regulates the degree of capping, which is likely to reduce the rate of TRD. Hence, yKu may essentially control the number of telomeres capable of rapid deletion. In this model, the Rad50p and Mre11p components of the MRX complex act to regulate the initial strand invasion either through chromosome condensation or the preparation of ends for recombination. Red line, the leading strand proceeding 5' to 3' toward the terminus; blue line, the complementary strand. Dependent upon the direction of resolution and the formation and stability of the D-loop, the intermediate can also give rise to circular forms of the terminal fragment.