INTRODUCTION

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Telomeres, the ends of eukaryotic chromosomes, are critical for maintaining chromosome stability and genome integrity (2, 8, 60). Telomeres are composed of particular DNA sequences which are rich in TG and arranged in species-specific repeated motifs. Telomeres are capped by proteins that bind to these repeating DNA sequences (6, 20). This apparently serves at least two distinct purposes. First, some of these telomeric proteins presumably form complexes that regulate telomerase activity and, hence, the length of telomeric tracts (31, 43). Some telomeric proteins have also been implicated in the physical protection of chromosome ends (38), in preventing recombinational events that would otherwise frequently occur between repeating telomeric sequences (33, 34, 53), and in keeping off DNA repair enzymes (14). Indeed, telomeres represent naturally occurring DNA double-strand breaks that, contrary to those resulting from accidental damage, do not need to (and must not) be repaired. Surprisingly, however, yeast Ku proteins, as well as proteins of the Mre11-Rad50-Xrs2 complex, which have been implicated in DNA repair by nonhomologous end joining have also been implicated in telomere maintenance (3, 4, 7, 11, 12, 27, 28, 39, 44, 46, 47). Moreover, yKu70 and yKu80 have been found to localize at the telomeres (18, 37).

The repeating TG-rich telomeric DNA sequences are mostly double stranded. However, during S phase only, telomeres display a short (ca. 35- to 50-nucleotide) single-stranded DNA extension that marks the very end of the telomere (57, 58). Single-stranded telomeric DNA is thought to represent the site of anchoring of telomerase, which is composed of the evolutionary conserved Est2 reverse transcriptase enzyme and of the TLC1 RNA template (31, 42). However, recent experiments suggest that telomerase-dependent elongation of de novo ends does not appear to involve single strandedness and does not require significant degradation prior to addition of newly synthesized telomeric DNA (9). Est1 and Est3 represent two subunits of the telomerase complex (25, 29, 55), which although not required for in vitro telomerase catalytic activity (32), are nevertheless stable components of the enzyme and regulate its activity in vivo through physical association with Est2 and TLC1 (25, 61).

Cdc13 was the first identified single-strand DNA-binding telomeric protein in Saccharomyces cerevisiae and, consequently, its status as a candidate for most of telomeric functions has become prominent (5, 14, 30, 45). The isolation of the cdc13-2/est4-1 allele, which confers a strong deficit in telomerase activity (29), as well as the recent finding that a fusion protein made of Cdc13 plus Est1 could bypass telomerase-defective mutations in either protein, strongly suggests that interactions between Cdc13 and Est1 represent the mechanism by which a number of regulators can control telomerase recruitment (10). Indeed, physical association between Cdc13 and Est1 has been revealed recently (48). Cdc13 has also been shown to bind Pol1 in vivo, and it has been proposed that Cdc13 might coordinate regulation at the telomere ends of G-strand lengthening by telomerase, via Est1, and C-strand resynthesis by polymerase  (48). In addition, the observation that the temperature-sensitive cdc13-1 allele displays abnormal accumulation of single-stranded DNA specifically at telomeric regions of chromosomes argued that Cdc13 might be a major telomeric capping protein (14). It has also been observed that when Cdc13 was defective, in cdc13-1 cells, the absence of either yKu70 or yKu80, which was otherwise dispensable, impaired growth (44, 46). The yKu proteins, which bind to double-stranded telomeric DNA, have been proposed to be involved in establishing the proper terminal DNA structure of chromosomes in cooperation with telomerase (18, 46). In addition to its nonessential function in the recruitment of telomerase at telomere ends (45), Cdc13 has an essential function that has not yet been clearly defined. cdc13-1 mutant cells are temperature sensitive and present a first cell cycle arrest at restrictive temperatures of growth (14).

In the present study, we have isolated several new mutant alleles of CDC13 which confer either abnormal telomere elongation or, on the contrary, telomere shortening. Telomere elongation in these novel cdc13 alleles was found to be more affected by mutations in either YKU70 or YKU80 than by mutations in TEL1 or RAD50, therefore implicating the yeast Ku proteins in the telomerase-loading function of Cdc13. This was supported by observing telomere elongation as a direct result of the expression of a Cdc13-yKu70 fusion protein, which is comparable in length to that produced by a Cdc13-Est1 fusion. We also present evidence, based on overexpression of Stn1 or expression of Stn1-Cdc13 fusions, that Stn1, a protein that associates with Cdc13 by two-hybrid interaction (17), is an inhibitor of telomerase recruitment via Cdc13. We propose that Cdc13 is both a positive and a negative regulator of telomerase recruitment, which establishes differential interactions with other telomeric proteins, and that the balance between these two opposing effects principally relies on interactions with yKu70 or yKu80 and with Stn1.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Strains, plasmids, and media. General plasmids and media used in this study were as described previously (17). Disruptions of TLC1, EST1, RAD50, YKU70, or YKU80 in wild-type, cdc13-1, or cdc13-109i strains were achieved following transformation of linearized tlc1::LEU2 (52), est1::URA3 (55), rad50::hisG-URA3-hisG (41), yku70/hdf1::URA3 (47), or yku80::TRP1 (see below) DNA fragments. The correct disruptions were detected on Southern blots. In some cases, these disruption strains were further mixed with other mutations by genetic crosses. The tel1::kanMX4 (strain record number 3114; Research Genetics, Inc., Huntsville, Ala.) and rad52-7::LEU2 (strain record number XS560-1C-1D1; Yeast Genetic Stock Center, Berkeley, Calif.) disruption strains were backcrossed five times against the genetic background used in our lab (17). The cdc13::TRP1 disruption plasmid was constructed by inserting TRP1 at the BamHI site of CDC13, at nucleotide 1349, and the yku80::TRP1 disruption plasmid was constructed by inserting TRP1 between the XbaI and AccI sites (nucleotides 84 to 1762) of YKU80. The cdc13-109 integrated allele was constructed by the pop-in-pop-out method. To do this, cdc13-109 was cloned into a URA3 integrative plasmid (YIp211) which was then used to transform a wild-type strain. The Ura⁺ transformants were then grown on 5-fluoro-orotic acid (5-FOA) plates to counterselect for cells that, together with the URA3 marker gene, had lost one copy of the CDC13 gene (either the wild-type or the mutant copy). Telomere length was then monitored on Southern blots in several of these Ura cells after a few generations of growth (see below). Only half of these colonies displayed elongated telomeres, with the other half exhibiting telomeres of wild-type size. Cells with elongated telomeres were selected, and the presence of only one copy of the CDC13 gene was verified by Southern blot. This suggested that cells with elongated telomeres had integrated the cdc13-109 allele at the CDC13 locus, while cells with wild-type telomeres had, on the contrary, evicted the cdc13-109-URA3 integrated construct. Integration of the cdc13-69 allele at the CDC13 locus was performed using the same methodology as for cdc13-109.

Construction and selection of cdc13 alleles and of stn1-63. CDC13 open reading frame (ORF) plus 300 bp upstream of the ATG was amplified by PCR under mutagenic conditions, as described previously for STN1 (17) in standard PCR buffer supplemented with MnCl₂, using standard Taq polymerase (Gibco-BRL). Following two rounds of PCR mutagenesis, using the products of the first reaction as a template for the second reaction, the PCR products were cleaned and used directly to transform a cdc13-1 strain, together with a single-copy, centromeric, plasmid, YCp111-LEU2 (15), made linear by digestion and carrying CDC13 flanking regions at each extremity (gap repair method). The 5' fragment of these flanking regions consisted of the 964 bases before the start codon plus the 57 bases after, while its 3' fragment comprised the 690 bases before the stop codon plus the 830 bases after. Cells were then plated onto leucine-lacking (Leu) medium and incubated at 25°C until colonies of transformants developed. Because the objective of CDC13 mutagenesis was to uncover non-temperature-sensitive alleles deregulated in telomere length control, transformants were then replica plated on Leu medium at 32 and 34°C, temperatures of growth restrictive for cdc13-1. This allowed us to select for mutagenized CDC13 plasmids capable of sustaining growth of the cells bearing them in the absence of functional endogenous Cdc13 protein, because the Cdc13-1 protein is inactivated at temperatures higher than 28°C in our genetic background (17). Among several thousands of such colonies growing at 32 or 34°C, 121 were picked out randomly and separately grown for further analysis of the length of their telomeric tracts (see below). The most interesting YCp111-borne cdc13 alleles, in terms of telomere length deregulation, were then recovered from the original cdc13-1 recipient strain and used to retransform the cdc13-1 strain. Genomic DNA from transformants grown for about 100 generations was then prepared, and the lengths of the telomeric tracts were analyzed by Southern blotting, as explained below.

Measurement of telomere length. Genomic DNA was prepared as described previously (17), digested with XhoI and separated by electrophoresis in a 0.9% agarose gel in Tris-borate-EDTA. After denaturation, DNA was transferred onto nitrocellulose membrane and immobilized by baking at 80°C for 1 h under a vacuum (1). The membrane was then prehybridized in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate-1% nonfat milk and hybridized with a 270-bp TG_1-3 ³²P-labeled probe representing S. cerevisiae telomeric sequences. Results were analyzed using a Storm PhosphorImager (Molecular Dynamics).

Construction of fusion proteins. Cdc13-Est1, Cdc13-69-Est1, Cdc13-yKu70, Cdc13-yKu80, Cdc13-Stn1-63, and Cdc13-231-Stn1 in-frame fusion proteins were constructed by cloning in a single-copy, centromeric, plasmid the entire CDC13 ORF (or the cdc13-69 or cdc13-231 ORFs) plus upstream promoter sequences in front of the EST1, YKU70, YKU80, or STN1 (wild-type or mutant) ORF (which included their natural stop codons). The yKu80-Cdc13 fusion protein was constructed by cloning in a single-copy plasmid the entire YKU80 ORF plus upstream promoter sequences in front of CDC13, so as to keep a continuous reading frame. The CEN plasmids used in the present study are of the YCplac series and are single-copy plasmids (15), just like the CEN plasmid, pRS415 (51), used by Evans and Lundblad (10). Telomere elongation by the Cdc13-Est1 fusion protein was not due to increased protein expression because overexpression of CDC13 or EST1 had no effect on telomere length (10, 17). Moreover, the functionality of the Cdc13-Est1 fusion protein was attested to by its ability to assume the essential function of Cdc13 (rescue of cdc13-1 cells at a restrictive temperature or of cdc13 cells) and to rescue the senescence phenotype of an est1::URA3 mutant.

Epitope tagging, Western blot detection, and band shift experiments. The 10 cdc13 mutant DNAs corresponding to the mutant strains described in the present study were subcloned into a YEp195-GAL1 (2µ, URA3) plasmid under the control of the strong, inducible, GAL1 promoter. In this plasmid the natural stop codon of the cdc13 mutant genes was eliminated so as to obtain a continuous reading frame between the Cdc13 protein and a 2HA-6His epitope tag (16). This allowed visualization of the Cdc13 mutant proteins by Western blotting using monoclonal anti-hemagglutinin antibody (12CA5; Boehringer). The presence of this epitope tag also allowed purification of the Cdc13 mutant proteins by Ni chromatography directed against the His₆ part of the tag, using Qiagen reagents. After transfer to nitrocellulose membrane (Schleicher & Schuell), proteins were detected using an enhanced chemiluminescence system (ECF; Amersham) coupled with detection in a Storm PhosphorImager (Molecular Dynamics).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

New telomere-shortening and telomere-elongating cdc13 alleles. To better understand the role of Cdc13 at telomeres, we set out to generate new alleles of CDC13 using PCR amplification under mutagenic conditions (see Materials and Methods) and reintroduce them into a conditional system provided by a cdc13-1 mutant strain (14). cdc13-1 cells stop growing at temperatures above 23 to 25°C (14) or above 27 to 28°C in our genetic background (17). Among several thousand transformants of cdc13-1 capable of growth at 32 or 34°C, 121 were picked randomly for further study. Genomic DNA was prepared, and the lengths of the telomeric tracts were measured by probing total XhoI-digested genomic DNA with a ³²P-labeled TG_1-3 telomeric probe, as described in Materials and Methods. Only mutant strains with substantial changes in telomere length were selected for further analysis, namely, cdc13-7, cdc13-109, cdc13-231, and cdc13-276, which are telomere-elongating alleles, and cdc13-23, cdc13-30, cdc13-69, cdc13-243, cdc13-273, and cdc13-280, which are telomere-shortening alleles (Fig. 1A).

View larger version (56K): [in this window] [in a new window]   FIG. 1.   Telomere lengthening associated with the cdc13 alleles relies on telomerase activity and requires Est1. (A) Telomere Southern blot analysis of novel non-temperature-sensitive cdc13 mutant alleles generated by PCR mutagenesis (see Materials and Methods) reveals the possibility of a large panel of telomere size deregulation when CDC13 is mutated (lanes 2 to 14) compared to a wild-type strain (lane 1). Here, the cdc13 alleles (names of which are indicated on top of the figure) were present on a single-copy centromeric plasmid in a cdc13-1 mutant. These mutant strains were grown at 32°C to inactivate Cdc13-1. (B) Augmentation of telomerase activity is responsible for telomere elongation in the new cdc13 mutant strains described here. TLC1, the RNA subunit of telomerase, was disrupted directly in a cdc13-109i mutant or in a cdc13-1 mutant harboring YCp-cdc13-276. The resulting strains no longer displayed the telomere elongation conferred by the cdc13-109 and cdc13-276 mutations, as measured here after approximately 50 generations (compare lane 2 to lane 3 and lane 5 to lane 6). Telomere lengths in wild-type strains (lanes 1 and 4) are shown for comparison. (C) Telomere elongation in the cdc13 mutants necessitates the presence of Est1, a regulator of telomerase. The EST1 gene was genetically disrupted in cdc13-109i or in cdc13-1 YCp-cdc13-276 and telomere size measured after approximately 50 generations. Absence of telomere lengthening in the resulting double mutants (compare lane 2 to lane 3 and lane 4 to lane 5) was observed. (D) The Cdc13-109 and Cdc13-231 mutant proteins can sustain growth on their own when expressed from a single-copy plasmid in a strain disrupted for CDC13 (cdc13::TRP1). Both Cdc13-109 (lane 3) and Cdc13-231 (lane 4) conferred telomere lengthening compared to a wild-type strain (lane 1) or a cdc13 disruptant expressing wild-type CDC13 (lane 2). (E) Expression of the temperature-sensitive cdc13-1 allele from an episomal plasmid, at the restrictive temperature of 34°C, had no incidence on the cdc13-109-associated telomere lengthening (compare lanes 2 and 3).

Telomere elongation in the new cdc13 mutants depends on telomerase and requires Est1. Because both telomerase-dependent and telomerase-independent mechanisms, in the latter case relying on homologous recombination, can regulate telomere size (33, 34), we next asked which one of these two mechanisms affected telomere regulation in the telomere-elongating cdc13 mutants described here. To do this, we introduced a null mutation in TLC1, the RNA subunit of telomerase essential for telomerase activity (52), into the cdc13-109i mutant (see Materials and Methods) and measured telomere size in the resulting double mutants. The typical telomere elongation observed in the cdc13-109i was not observed in the cdc13-109i tlc1 mutants (Fig. 1B). These results were confirmed using the cdc13-1 YCp-cdc13-276 strain (Fig. 1B). Because the homologous recombination mechanisms controlling telomere length are entirely dependent on Rad52, we then analyzed telomere size deregulation in cdc13-109i rad52 or cdc13-1 YCp111-cdc13-276 rad52 mutant strains and found that they displayed telomeres elongated to the same extent as that in the corresponding RAD52⁺ strains (data not shown).

The telomere-elongating Cdc13 mutant proteins still associate with telomeric DNA. To determine whether the telomere length phenotype could be merely due to a defect of the Cdc13 mutant proteins in binding telomeric DNA (5, 26, 30), band shift experiments were performed using Cdc13-2HA-6His mutant proteins purified by Ni chromatography (see Materials and Methods). The Cdc13-109 and Cdc13-231 proteins were still be able to bind telomeric DNA (Fig. 2A). The specificity of these interactions was evidenced by visualizing supershifts when a monoclonal anti-HA antibody was added to the reaction (Fig. 2A). Careful examination of the intensities of the DNA-protein bands revealed that the Cdc13-109 and Cdc13-231 mutant proteins were only partially competent in binding telomeric DNA compared to wild-type Cdc13 (Fig. 2).

View larger version (58K): [in this window] [in a new window]   FIG. 2.   The telomere-elongating Cdc13 mutant proteins described in this study are still able to bind telomeric sequences. (A) The in vitro binding of wild-type Cdc13 and of telomere-elongating Cdc13 mutant proteins to specific TG1-3 telomeric DNA sequences was measured by assessing gel retardation of Cdc13-2HA-6His proteins (see Materials and Methods). The same reaction mixtures were incubated in parallel with a monoclonal anti-HA antibody, which generated a supershift of the complex attesting of the specificity of the DNA-protein binding reaction. Binding of the Cdc13-109-2HA-6His and Cdc13-231-2HA-6His proteins was somewhat weaker than that of wild-type Cdc13-2HA-6His, while Cdc13-7-2HA-6His and Cdc13-276-2HA-6His did not bind telomeric DNA. Unlabeled arrows indicate the position of the DNA-protein and DNA-protein-antibody complexes. (B) Immunoblot analysis, using a monoclonal anti-HA antibody, of crude extracts from cells expressing Cdc13-2HA-6His proteins under the control of the inducible GAL1 promoter was performed essentially to assess the presence of the Cdc13 mutant proteins used in band shift experiments. This analysis revealed that, in fact, Cdc13-7 and Cdc13-276 were not produced as entire proteins. Sequence analysis of these cdc13 alleles revealed the presence of a stop codon, generated during mutagenesis, upstream of the natural stop codon (see text).

Telomere elongation in the telomere-elongating cdc13 mutants requires both yKu70 and yKu80. We reasoned that the telomere-elongating Cdc13 mutant proteins might be deregulated in their interaction with another telomeric protein. To test this hypothesis, telomere-elongating alleles of CDC13 were introduced into mutant strains known to exhibit abnormally short telomeres: rad50, tel1, yku70/hdf1, and yku80/hdf2. Rad50 is part of the Mre11-Rad50-Xrs2 telomeric complex that has been previously shown to function in so-called DNA nonhomologous end-joining (NHEJ) and in telomere maintenance (21, 28, 44). Tel1 is a telomeric protein that has been implicated in telomere length regulation and shares homology with the human ATM proteins (19, 35, 49). The yeast Ku proteins have been implicated in NHEJ, in heterochromatin organization, in telomere silencing, and in telomere maintenance (4, 7, 11-13, 18, 22, 37, 39).

cdc13-109i yku70

cdc13-109i yku80

yku70

yku80

tel1

yku70/yku80

View larger version (54K): [in this window] [in a new window]   FIG. 3.   Mutations in YKU70 or YKU80 have a larger suppressing effect on cdc13-109- or cdc13-276-associated telomere elongation than mutations in RAD50 or TEL1. (A) cdc13-109i tel1 (lane 1), cdc13-109i yku70 (lane 7), cdc13-109i rad50 (lane 10), and cdc13-109i yku80 (lane 14) double mutants were grown for approximately 100 generations, and their telomere lengths were compared to those in the corresponding single mutants (neighboring lanes in each panel for each of the four mutations) and in wild-type cells (lanes 2, 5, 9, and 13). See the text for interpretation of the data. (B) cdc13-1 rad50, cdc13-1 tel1, cdc13-1 yku70, and cdc13-1 yku80 double mutants were propagated at 25°C and transformed with a single-copy plasmid harboring the cdc13-276 allele. The resulting triple mutants were grown at 34°C to inactivate Cdc13-1, and the sizes of their telomeres were measured after approximately 100 generations (lanes 3, 6, 10, and 14) and compared to those in cdc13-1 rad50, cdc13-1 tel1, cdc13-1 yku70, or cdc13-1 yku80 (lanes 2, 5, 8, and 13), respectively, at 34°C or to that in wild-type isogenic strains (lanes 1, 4, 7, and 11). Telomere lengths in cdc13-1 cells grown at 25°C (lane 12) and in cdc13-1 cells harboring cdc13-276 on a single-copy plasmid, grown at 34°C (lane 9), served as negative and positive controls, respectively. Note that cdc13-1 yku70 YCp-cdc13-276 (lane 10) and cdc13-1 yku80 YCp-cdc13-276 cells (lane 14) no longer displayed the long telomere phenotype characteristic of the cdc13-276 allele (lane 9), contrary to cdc13-1 rad50 YCp-cdc13-276 (lane 3) and cdc13-1 tel1 YCp-cdc13-276 (lane 6) which did. This interpretation is confirmed by visualizing the dots in lane 10, which highlight two bands corresponding to two non Y' telomeres. These were also present in wild-type cells (lane 7) and yku70 cells (lane 8) but were absent from cells harboring the cdc13-276 mutation alone (lane 9). This confirmed that the telomere structure in the cdc13-1 YCp-cdc13-276 yku70 mutant no longer resembled that in cdc13-1 YCp-cdc13-276 but was instead similar to that in wild-type cells. The same held true for yku80 (lane 14) but not for rad50 (lane 3) and tel1 (lane 6) cells. See the text for further explanations. Conditions of strain culturing were as described above.

A Cdc13-yKu70 fusion protein induces abnormal telomere lengthening. Because the genetic interactions described above were only indicative of a potential functional relationship between Cdc13 and either yKu70 or yKu80, we decided to adopt a complementary approach. One of our hypotheses to explain these interactions relied on the existence of a physical interaction between either yKu70 or yKu80 and Cdc13. Because attempts to detect physical association between Cdc13 and yKu70 in a two-hybrid system failed (unpublished results), we decided to use a method recently applied with success in the analysis of interactions between Cdc13 and Est1 (10), which consists in the expression of fusion (hybrid) proteins. We first expressed a Cdc13-Est1 fusion protein from a single-copy centromeric plasmid under the control of the CDC13 promoter (see Materials and Methods) and observed that it produced telomere elongation in a CDC13⁺ EST1⁺ strain, an effect that was much more pronounced in a cdc13-1 background at 34°C or in cdc13::TRP1 than in a wild-type background (Fig. 4A, lanes 2, 8, and 11), as described recently (10).

View larger version (37K): [in this window] [in a new window]   FIG. 4.   A Cdc13-yKu70 fusion protein produces telomere elongation that is dependent on Est1. (A) Expression of CDC13-EST1 or CDC13-YKU70 hybrid gene from a single-copy centromeric plasmid under the control of the CDC13 promoter for approximately 100 generations, either in a cdc13-1 strain grown at 34°C to inactivate Cdc13-1 (left panel), in a wild-type background (middle left panel), or in a cdc13-1 background (middle right panel) provoked dramatic telomere elongation (lanes 2, 5, 8, 9, 11, and 12) compared to controls (cdc13-1 cells grown at 25°C, lane 1; wild-type cells, lanes 7 and 10). A fusion consisting of the telomere-shortening Cdc13-69 mutant protein and of either wild-type Est1 or wild-type yKu70 also produced telomere elongation in cdc13 (lanes 16 and 17) or cdc13-1 at 34°C (lanes 3 and 6), therefore indicating rescue of the short telomere phenotype conferred by Cdc13-69 (lane 15). A Cdc13-Stn1 fusion (lane 13) served as a negative control. (B) Telomere elongation provoked by the Cdc13-yKu70 fusion no longer took place in the absence of Est1 (lane 6; compare to the est1 [lane 5] and wild-type [lane 4] strains). Lanes 1 and 3, in which CDC13 or EST1 alone were expressed, provided additional controls for these protein fusion experiments. See the text for further explanations. (C) Telomere elongation provoked by the Cdc13-yKu70 no longer took place in yku80 cdc13-1 cells (lane 1; compare to the YKU80+ cdc13 cells expressing the same fusion (lane 2) and to the wild-type [lane 3] and yku80 [lane 4] strains). This effect was observed both in a cdc13 background (lane 1) and a CDC13+ background (lane 5).

cdc13-1 yku70

yku80

yku80 cdc13

YKU80⁺ cdc13

Novel nonsenescent telomere-shortening cdc13 alleles are severely defective in DNA-binding activity. None of the telomere-shortening cdc13 alleles described in this study (see Fig. 1A), namely, cdc13-23, cdc13-30, cdc13-69, cdc13-243, cdc13-273, and cdc13-280, provoked senescence (29, 33, 34) in contrast to cdc13-2/est4-1 cells or tlc1 cells (data not shown). To further characterize these novel telomere-shortening Cdc13 mutant proteins, we performed band shift experiments to measure their ability to bind telomeric DNA (Fig. 5A). All six telomere-shortening Cdc13 mutant proteins were severely defective in binding telomeric DNA (Fig. 5A). Among these, Cdc13-69-2HA-6His Cdc13-243-2HA-6His, and Cdc13-273-2HA-6His retained some DNA binding activity, as confirmed by observing a supershifted band upon addition of anti-HA antibody during the reaction, while the Cdc13-23-2HA-6His, Cdc13-30-2HA-6His, and Cdc13-280-2HA-6His proteins were almost completely defective in binding telomeric DNA (Fig. 5A).

View larger version (38K): [in this window] [in a new window]   FIG. 5.   In vitro binding of telomere-shortening Cdc13 mutant proteins to specific TG1-3 telomeric DNA sequences. (A) Band shift experiments, performed as described in the legend to Fig. 2, revealed that all six Cdc13 mutant proteins were severely defective in DNA binding, with Cdc13-23-2HA-6His, Cdc13-30-2HA-6His, and Cdc13-280-2HA-6His being more affected than Cdc13-69-2HA-6His, Cdc13-243-2HA-6His, and Cdc13-273-2HA-6His, which still showed some binding. The specificity of the DNA-protein interaction was attested to by visualizing a supershift upon addition of a monoclonal anti-HA antibody to the reaction mixture. Unlabeled arrows indicate the position of the DNA-protein and DNA-protein-antibody complexes. (B) Western blot analysis of crude extracts from cells expressing Cdc13-2HA-6His proteins under the control of the inducible GAL1 promoter, using a monoclonal anti-HA antibody, revealed that basically all of the Cdc13 mutant proteins were produced to the same extent within the cell, with the exception of Cdc13-243-2HA-6His, whose levels were lower than those of the other mutant proteins. (C) The cdc13-69i strain, harboring the telomere-shortening cdc13-69 allele integrated at the CDC13 locus, exhibited telomeres shortened to the same extent as those in a cdc13-1 YCp-cdc13-69 strain grown at 34°C (Fig. 1A, lane 12) or in a cdc13 YCp-cdc13-69 strain (Fig. 4A, lane 15).

Stn1 exerts a negative effect on Cdc13-mediated telomerase loading. We have previously proposed that Stn1 might negatively regulate Cdc13 activity (17). In view of the potential positive modulation of Cdc13 activity by yKu70, we speculated that Stn1 might feed negative signals into the Cdc13-regulating machinery. In our previous studies on Stn1 (17), it had not been demonstrated that a loss of Stn1 function led to deregulation of telomerase recruitment. We now demonstrate that the telomeric defect of stn1-13, a mutant which exhibits a severe growth defect at the restrictive temperature of 37°C and telomere elongation at any temperature between 25 and 37°C (17), results from a deregulation in telomerase recruitment and/or activity (Fig. 6A).

View larger version (58K): [in this window] [in a new window]   FIG. 6.   Stn1 behaves as an inhibitor of telomerase recruitment. (A) A "young" stn1-13 mutant strain (in which the stn1-13 mutation had been introduced at the STN1 locus only for a week so as to obtain cells with telomeres of nearly wild-type size) was disrupted for TLC1 and grown at 30°C for approximately 50 generations. In this stn1-13 tlc1::LEU2 strain, telomeres remained short (lane 4), of the same size as in a wild-type strain disrupted for TLC1 (lane 2), and shorter than in wild-type TLC1+ cells (lane 1), in contrast with telomeres in stn1-13 TLC1+ cells which during the same period of growth had become very long (lane 3). (B) Overexpression of STN1 diminishes the cdc13-276-associated telomere elongation and aggravates the cdc13-273-associated telomere shortening. A cdc13-1 YCp-cdc13-273 mutant was transformed with YEp-STN1, a multicopy plasmid overexpressing STN1 under the control of its natural promoter (lane 3), or with vector alone (lane 2). Both strains were grown at 32°C (to inactivate Cdc13-1), and the size of their telomeres were measured after approximately 100 generations. Short telomeres conferred by the Cdc13-273 protein (lane 2; compare to the telomere size in a cdc13-1 strain grown at 25°C [lane 1]) were further shortened following STN1 overexpression (lane 3). In addition, abnormal telomere lengthening conferred by the Cdc13-276 mutant protein (lane 4) was partially relieved when STN1 was overexpressed (lane 5).

View larger version (31K): [in this window] [in a new window]   FIG. 7.   Expression of Stn1-Cdc13 fusion proteins confirm that Stn1 negatively regulates Cdc13. (A) Expression of a cdc13-231-STN1 hybrid gene from a single-copy centromeric plasmid under the control of the CDC13 promoter in a cdc13 strain grown for approximately 100 generations totally suppressed the long telomere phenotype conferred by Cdc13-231 (compare lanes 2 and 3). (B) Expression of a CDC13-stn1-63 hybrid gene in an stn1 strain under the same conditions as those described above totally suppressed the telomere lengthening phenotype conferred by Stn1-63 (compare lanes 2 and 3), which resulted in the acquisition of telomeres of wild-type size (lane 1).

Sequence analysis of the cdc13 alleles. DNA sequencing revealed that all six sequenced cdc13 alleles (cdc13-7, cdc13-69, cdc13-109, cdc13-243, cdc13-273, and cdc13-276) contained multiple point mutations. In addition, a mutation in lysine 706 of Cdc13-276 introduced a termination codon that resulted in the truncation of the last 218 amino acids, while a frameshift in cdc13-7 sequence at lysine 702 led to the introduction of a premature stop codon at amino acid 721. We have not been able so far to identify the mutations responsible for the phenotypes of the corresponding mutants.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The survival of an organism relies on the proper duplication, segregation, and stability of its genome. Telomeres play an important role in maintaining chromosome structure because, for instance, chromosomes that lose a telomere are themselves eliminated from the cell (50). In yeast, mutations in telomerase components or regulators produce a gradual erosion of chromosomes that eventually leads to death by senescence due to chromosome instability (29, 34). Cdc13 has been previously implicated, together with Est1, as the main regulator of telomerase access to telomeric ends (10, 30, 45, 48, 61).

The present study extends our knowledge on the role of Cdc13 in telomerase control through the analysis of novel cdc13 alleles and their functional relationships with mutations in other telomeric proteins. Our data suggest that the yeast Ku proteins, previously implicated in DNA repair and telomere maintenance in yeast and humans (4, 7, 16, 24, 27, 37, 39), promote telomerase recruitment by Cdc13. Our results also demonstrate that regulation by yKu70 or yKu80 is opposite of that by Stn1, since we found that Stn1 negatively regulates Cdc13-mediated telomerase recruitment. The present study also provides telomere-shortening cdc13 alleles that do not confer senescence and may be useful in some biochemical or genetic assays.

Stn1 inhibits telomerase recruitment via Cdc13. Our observation that STN1 overexpression produces telomere shortening (Fig. 6B) is reminiscent of the effects of overexpressing RIF1 or RIF2 (59). A striking parallel between these two situations is that Rif1 and Rif2 physically associate with Rap1, a master regulator of telomere length which binds double-stranded telomeric DNA (23, 36, 59), while Stn1 physically associates with Cdc13, a master regulator of telomere length which binds single-stranded telomeric DNA (5, 17, 26, 30, 45). However, a noticeable difference between the two situations was that STN1 overexpression did not affect telomere length in wild-type cells (17; the present data), whereas overexpression of RIF1 and RIF2 did (59). We propose that overproduction of Stn1 can affect telomere length only when Cdc13 function is already compromised, as explained below.

yKu70 promotes the recruitment of telomerase by Cdc13. Disrupting either YKU70 or YKU80 had a larger suppressing effect on telomere elongation conferred by the Cdc13-276 or Cdc13-109 mutant proteins than disrupting RAD50 or TEL1 (Fig. 3). Although at first glance tel1 may appear to have an effect similar to that of yku70 or yku80 in diminishing the cdc13-109-associated telomere elongation, careful examination of the Southern blot revealed a difference between the two, which was confirmed in the cdc13-1 YCp-cdc13-276 strain. It could be argued that such experiments are difficult to interpret because, for instance, all of the telomere-shortening mutations considered here are in proteins known to be involved each in several pathways, including telomere maintenance. Moreover, since the two mutations present in a given strain affect telomere length in opposite directions, it is difficult to determine whether the resulting average telomere length corresponds to equilibrium between the two opposing effects or rather reflects actual genetic interaction between the two mutations. For these reasons, we were very cautious in interpreting these epistasis experiments. In the end, a noticeable result is that the yku70 and yku80 mutations totally suppressed the cdc13-109-induced telomere elongation (Fig. 3A), while conferring wild-type length telomeres to strains bearing the cdc13-276 mutation (Fig. 3B). The effects of rad50 and tel1 were smaller than those of yku70 and yku80 in either cdc13 strain.

A model for the control of Cdc13-mediated telomerase recruitment by Stn1 and by the yKu70 and yKu80. Based on the experiments presented here and the data available in the literature (principally, references 4, 10, 14, 17, 18, 25, 26, 29, 30, 40, 44-48, 55, 57, and 61), we propose an improved model for the control of telomerase recruitment by Cdc13 that involves the existence of a balance between the effects of yKu70 and Stn1 on Cdc13 (Fig. 8). The top panel of Fig. 8 depicts the situation in wild-type cells, while the bottom panels propose two hypotheses to account for the situation encountered in the cdc13-109i mutant strain.

View larger version (25K): [in this window] [in a new window]   FIG. 8.   (Top) A speculative model for yKu70 and Stn1 as positive and negative regulators, respectively, of Cdc13-Est1-mediated telomerase loading in wild-type cells. Stn1, which physically associates with Cdc13, a single-strand telomeric DNA-binding protein, acts as an inhibitor of Cdc13-mediated telomerase recruitment (telomerase contains the catalytic subunit, Est2, and TLC1, the RNA template; Est1, a regulator of telomerase which binds single-stranded telomeric DNA, also physically associates with TLC1). Stn1 release from the Cdc13-Stn1 complex might represent the signal allowing interactions between Cdc13 and Est1-telomerase. According to this model, interactions between Cdc13 and the DNA repair yKu proteins, yKu70 and yKu80 (yKu70 physically associates with yKu80, which itself binds double-strand telomeric DNA), might promote efficient association of Est1-telomerase with the telomere end, thus allowing telomeric DNA addition. Re-binding of Stn1 to Cdc13 might compete with Cdc13-yKu70 or -yKu80 and Cdc13-Est1 interactions, therefore breaking interactions between Est1-telomerase and telomeric DNA and terminating the telomere replication process. (Bottom) Two hypotheses are proposed to explain the deregulation of telomere length conferred by the telomere-elongating cdc13 alleles described in the present study (cdc13-109, which has been used in most of the experiments presented here, has been chosen for illustration). For both hypotheses, the situation has been envisioned either in the presence of the cdc13-109i mutation alone (cdc13-109 YKU70 cells) or in the simultaneous presence of the cdc13-109i and the yku70 mutations (cdc13-109 yku70 cells). Full ovals represent a higher than normal physical association between the deregulated Cdc13-109 mutant protein and yKu70 (hypothesis 1, left) or between Cdc13-109 and Est1 (hypothesis 2, right). In all of the configurations represented here, Stn1 is in the off position, physically apart from Cdc13, the position that presumably allows telomerase recruitment by Cdc13. In hypothesis 1 (left), constitutive interactions between Cdc13 and either yKu70 or yKu80 provokes recruitment of telomerase at higher than normal levels, thus leading to telomere lengthening (top), while the absence of yKu70 presumably results in inefficient recruitment of Est1-telomerase, thus suppressing cdc13-109-induced telomere lengthening (bottom). In hypothesis 2 (right), constitutive interactions between Cdc13 and Est1 also provoke recruitment of telomerase at higher-than-normal levels and leads to telomere lengthening (top), but this time the absence of yKu70 presumably generates an abnormal single-stranded telomeric DNA extension which competes for Cdc13-109-Est1 interactions and results in inefficient recruitment of Est1-telomerase and suppression of the cdc13-109-induced telomere lengthening (bottom). See the text for further explanations.