The Est1 Subunit of Saccharomyces cerevisiae Telomerase Makes Multiple Contributions to Telomere Length Maintenance

Corresponding author: Victoria Lundblad, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030., lundblad{at}bcm.tmc.edu (E-mail)

Communicating editor: L. PILLUS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The telomerase-associated Est1 protein of Saccharomyces cerevisiae mediates enzyme access by bridging the interaction between the catalytic core of telomerase and the telomere-binding protein Cdc13. In addition to recruiting telomerase, Est1 may act as a positive regulator of telomerase once the enzyme has been brought to the telomere, as previously suggested by the inability of a Cdc13-Est2 fusion protein to promote extensive telomere elongation in an est1- strain. We report here three classes of mutant Est1 proteins that retain association with the telomerase enzyme but confer different in vivo consequences. Class 1 mutants display a telomere replication defect but are capable of promoting extensive telomere elongation in the presence of a Cdc13-Est2 fusion protein, consistent with a defect in telomerase recruitment. Class 2 mutants fail to elongate telomeres even in the presence of the Cdc13-Est2 fusion, which is the phenotype predicted for a defect in the proposed second regulatory function of EST1. A third class of mutants impairs an activity of Est1 that is potentially required for the Ku-mediated pathway of telomere length maintenance. The isolation of mutations that perturb separate functions of Est1 demonstrates that a telomerase holoenzyme subunit can contribute multiple regulatory roles to telomere length maintenance.

TELOMERES, the ends of chromosomes, are composed of duplex G-rich repeat sequences that terminate in single-strand 3' extensions. Maintenance of both the length and the terminal structure of telomeres is crucial for genomic integrity and, consequently, for long-term cell viability. The telomerase enzyme is a key factor involved in telomere length maintenance (EVANS and LUNDBLAD 2000 ; MCEACHERN et al.. 2000 ; DUBRANA et al.. 2001 ). The catalytic core of this enzyme consists of a reverse transcriptase protein, as well as an RNA subunit that templates the sequence of nucleotides added to the G-rich strand of the telomere (O'REILLY et al.. 1999 ; COLLINS 2000 ). In the budding yeast Saccharomyces cerevisiae, the template RNA and the catalytic subunit are encoded by TLC1 and EST2, respectively (SINGER and GOTTSCHLING 1994 ; COUNTER et al.. 1997 ; LINGNER et al.. 1997A ). Two additional proteins, Est1 and Est3, comprise subunits of the S. cerevisiae telomerase holoenzyme that each contribute essential in vivo regulatory functions to telomere replication (LUNDBLAD and SZOSTAK 1989 ; STEINER et al.. 1996 ; MORRIS and LUNDBLAD 1997 ; EVANS and LUNDBLAD 1999 ; HUGHES et al.. 2000 ; ZHOU et al.. 2000 ).

Telomere length maintenance requires not only a functional telomerase enzyme, but also a proficient pathway for mediating enzyme access to the end of the chromosome (for review, see EVANS and LUNDBLAD 2000 ). In S. cerevisiae, recruitment of telomerase to the chromosome terminus depends on the single-strand end-binding protein Cdc13 and the telomerase-associated Est1 protein (NUGENT et al.. 1996 ; EVANS and LUNDBLAD 1999 ; QI and ZAKIAN 2000 ; PENNOCK et al.. 2001 ). The recruitment function of Cdc13 is reduced by missense mutations at amino acid (aa) 252, which can be suppressed by missense mutations at residue 444 of the Est1 telomerase subunit (NUGENT et al.. 1996 ; PENNOCK et al.. 2001 ). This reciprocal suppression depends on oppositely charged residues on Cdc13 and Est1, indicating that a direct interaction between these two proteins is necessary for telomere replication (PENNOCK et al.. 2001 ). The recruitment model is further supported by the analysis of a series of fusion proteins that fuse various subunits of telomerase to the DNA-binding domain of Cdc13; in the presence of any of these telomerase fusion proteins, the recruitment activity of either Est1 or Cdc13 now becomes dispensable (EVANS and LUNDBLAD 1999 ; HUGHES et al.. 2000 ). Collectively these results support a model whereby Est1 mediates telomerase access as the bridging component between the telomerase catalytic machinery and the telomere end-binding Cdc13 protein. Est1 may perform an additional activity that promotes telomere replication, in a step that occurs subsequent to recruitment of the enzyme to the telomere. This proposed second function stems from the observation that although telomeres can be maintained in the absence of Est1 by fusing the catalytic subunit of telomerase directly to Cdc13, the extensive telomere elongation conferred by this Cdc13-Est2 fusion protein occurs only in the presence of the Est1 telomerase subunit (EVANS and LUNDBLAD 1999 ).

A recent study has further investigated the roles of Est1, Est2, and Cdc13 in telomere replication, by monitoring the association of each of these proteins with chromosome termini, using chromatin immunoprecipitation (ChIP) with a subtelomeric probe that detects sequences located between 350 and 600 bp from the end of the chromosome (TAGGART et al.. 2002 ). As expected, the Est2 protein shows greatly enhanced association during late S phase, consistent with the timing of telomerase action (MARCAND et al.. 2000 ). Surprisingly, Est2 also exhibits a strong association with telomeric chromatin during G₁ phase. This has led to the proposal that the primary function of Est1 and Cdc13 is to activate an inactive, but telomere-bound, Est2-TLC1 complex (TAGGART et al.. 2002 ) rather than to recruit the Est2-TLC1 complex to telomeres. One limitation of these ChIP studies, however, is that association of the Est2-TLC1 complex with the extreme chromosome end (where telomerase must be ultimately recruited) vs. association with subtelomeric sites cannot be differentiated by this technique. This caveat suggests an interesting modification of the recruitment model, which is that the Est2-TLC1 complex resides as a component of subtelomeric chromatin until it is recruited from these subtelomeric sites to the ends of chromosomes during late S phase. In fact, TAGGART et al. (2002) observe that, in a cdc13-2 (E252K) mutant strain, Est2 association is unaffected during G₁ phase, but Est2 association is lost during late S phase, consistent with the model that Cdc13 is responsible for its recruitment to single-stranded chromosome termini.

The Est1 protein also potentially contributes to telomere replication through nucleic acid interactions. Partially purified recombinant Est1 protein from Escherichia coli binds both single-strand RNA and DNA in vitro (VIRTA-PEARLMAN et al.. 1996 ). The interaction between Est1 and telomeric DNA is both sequence and structure specific: Est1 binds single-strand telomeric DNA substrates (albeit with a low affinity) with a dependence on a free 3' end for binding (VIRTA-PEARLMAN et al.. 1996 ). This suggests that Est1, like Cdc13, binds the chromosome terminus in vivo, an activity that may be required for Est1 to perform its proposed second function. The Est1 protein also appears to associate with telomerase through a direct interaction with the TLC1 subunit. Est1 retains association with a TLC1-containing complex even in the absence of the Est2 and Est3 subunits (HUGHES et al.. 2000 ; ZHOU et al.. 2000 ), and a specific region of TLC1 has been identified that determines association of Est1 with the telomerase complex (LIVENGOOD et al. 2002 ). Est1 has been proposed to interact with TLC1 through an RNA-binding domain that possesses weak similarity to an RNA recognition motif (RRM; ZHOU et al.. 2000 ), although deletion of this proposed RRM does not abolish the association of Est1 with a TLC1-containing complex (this work). This therefore leaves open the question of the specific residues responsible for RNA binding by the Est1 protein.

To correlate the biochemical activities of Est1—interaction with Cdc13, telomeric DNA, and the telomerase holoenzyme—with the proposed roles based on in vivo analysis, we have subjected the Est1 protein to a detailed site-directed mutational analysis. Analysis of 30 est1 mutants, which introduced missense mutations into 74 residues of the 699-aa Est1 protein, has yielded several distinct classes of separation-of-function mutations. Three classes of mutations encode Est1 proteins that retain association with an active telomerase complex, but confer different in vivo phenotypic consequences for telomere replication. The first (and largest) class exhibits genetic properties indicating that this set of mutant proteins is defective in a step involved in enzyme recruitment. A second class of mutations fulfills criteria predicted for a defect in the putative second function: these mutants fail to elongate telomeres in the presence of the Cdc13-Est2 fusion, despite retaining association with the telomerase enzyme. A third set of mutants exhibits extremely short telomeres but no associated senescence phenotype; epistasis analysis suggests that this class defines an activity of EST1 that is potentially required for the Ku-mediated pathway of telomere length maintenance. We also report a cluster of mutations in the N-terminal region of Est1 that substantially reduce the association with the telomerase RNP and thus potentially define a domain required for stabilizing the Est1-TLC1 association. The identification of distinct classes of separation-of-function mutants, which associate with an active telomerase enzyme but appear to perturb different aspects of Est1 function, supports the proposal that Est1 has multiple roles in telomere replication.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains:
Isogenic strains TVL409, TVL415, TVL418, and TLV474 were derived from the protease-deficient strain AVL78 and were used for all experiments in Fig 2; 4A; 5, A–C; and 6. TVL409 [MATa est1-::kan^R leu2 trp1 ura3-52 prb prc pep4-3/pVL232 (CEN URA3 EST1)] and TVL474 [MATa myc₃-EST2 est1-::kan^R leu2 trp1 ura3-52 prb prc pep4-3/pVL232 (CEN URA3 EST1)] were constructed from AVL78 and TVL291 (HUGHES et al.. 2000 ), respectively, by one-step gene disruption to delete aa 36–664 of EST1. TVL415 (MATa ProA-EST2 leu2 trp1 ura3-52 prb prc pep4-3) was derived by replacing the endogenous EST2 gene with ProA-EST2 (pKF409; FRIEDMAN and CECH 1999 ). TVL418 [MATa ProA-EST2 tlc1-::LEU2 leu2 trp1 ura3-52 prb prc pep4-3 /pSD120 (CEN URA3 TLC1)] was constructed from TLV415 by one-step gene disruption to remove nucleotides (nt) 285–1008 of TLC1.

View larger version (63K): In this window In a new window Download PPT slide   Figure 1. Sequence alignment of the S. cerevisiae and S. carlsbergensis Est1 proteins. (A) Clustered charged-to-alanine scanning mutations are indicated by asterisks and a numerical designation of the allele number. The area delineated by the brackets corresponds to the 130-aa domain discussed in the text. (B) Five single mutations were constructed in a conserved 17-aa block. (C) The positions of the five classes of mutations (see Table 1 and text) are shown.

View larger version (96K): In this window In a new window Download PPT slide   Figure 2. Growth analysis of est1 mutants in a YKU70 background. Plasmids bearing mutant est1 allele derivatives of pVL1355 were transformed into strain TVL409 [est1-/pVL232 (EST1 URA3 CEN)], followed by eviction of the wild-type copy of the EST1 gene. Mutant alleles were tested in parallel with wild-type EST1 (pVL499), parental EST1-myc18 (pVL1355), and vector (YCplac22) controls. Two independent isolates of each mutant were propagated by serial streakout. Growth at the 75-generation timepoint following loss of the wild-type EST1 gene is shown, except for the bottom right, which shows the growth phenotype for est1-6 and est1-54 mutant strains after 100 generations.

Two isogenic haploid strains, TVL471 [MAT yku70-::kan^R est1-::HIS3 ura3-52 ade2-101 lys2-801 leu2-1 trp1-1 his3-200/pVL232 (CEN URA3 EST1)] and TVL304 [MATa yku80-::kan^R ura3-52 ade2-101 lys2-801 leu2-1 trp1-1 his3-200/pVL867 (CEN URA3 YKU80)], were used for the experiments shown in Fig 3 and Fig 5D; both strains are related to the TVL120 strain background used in previous publications from this laboratory (LUNDBLAD and SZOSTAK 1989 ; LENDVAY et al.. 1996 ). The est1- cdc13-/pVL438 (CEN URA3 CDC13) strain was generated by tetrad dissection of DVL336 (est1-::HIS3/EST1 cdc13-::LYS2/CDC13 ura3-52/ura3-52 ade2-101/ade2-101 lys2-801/lys2-801 leu2-1/leu2-1 trp1-1/trp1-1 his3-200/his3-200/pVL438).

View larger version (110K): In this window In a new window Download PPT slide   Figure 3. Growth analysis of est1 mutants in a yku70- background. Plasmids bearing est1 mutant derivatives of pVL1355 were introduced into strain TVL471 [yku70- est1-/pVL232 (EST1 URA3 CEN)]. Tenfold serial dilutions were plated at 30° onto media selecting for the presence of both pVL232 and the pVL1355 derivatives (-Trp -Ura) and on media that selected for eviction of the covering pVL232 plasmid (-Trp 5-FOA). Analysis of the mutant strains was done in parallel with wild-type EST1 (pVL499), parental EST1-myc18 (pVL1355), and vector (YCplac22) controls.

View larger version (130K): In this window In a new window Download PPT slide   Figure 4. Analysis of telomere length of est1 mutant strains. (A) Plasmids bearing est1 mutant derivatives of pVL1355 were introduced into strain TVL409 [est1-/pVL232 (EST1 URA3 CEN)]. A Southern blot of genomic DNA, prepared 60 generations of growth following loss of the covering EST1 plasmid, was probed to detect yeast telomeric sequences, as previously described. Telomere length analysis for the est1 mutations was performed in parallel with untagged EST1 (pVL499), EST1-myc18 (pVL1355), and vector (YCplac22) controls. (B) The Est1-42 and Est1-19 mutant proteins fail to promote telomere elongation in the presence of the Cdc13-Est2 fusion. Plasmids expressing the est1 mutations (derivatives of pVL1355) were cotransformed with a plasmid expressing the Cdc13-Est2 fusion (pVL1107) into an est1- cdc13-/pVL438 (CEN URA3 CDC13) strain. DNA was prepared 50 generations following eviction of the wild-type CDC13 plasmid.

View larger version (73K): In this window In a new window Download PPT slide   Figure 5. Association of telomerase with mutant Est1 proteins. (A) Plasmid-borne est1 mutants (derivatives of pVL1355) were transformed into strain TVL409 [est1-/pVL232 (EST1 URA3 CEN)], and cultures were grown immediately following loss of the covering wild-type EST1 plasmid. Extracts prepared from asynchronous log-phase cultures expressing C-terminal myc18-tagged mutant Est1 proteins were immunoprecipitated with anti-myc antibody, in parallel with strains expressing wild-type Est1-myc18 and untagged Est1 proteins. A representative subset of immunoprecipitates, which were analyzed by Northern blotting to detect the telomerase RNA (TLC1) and an unrelated control RNA (U1) in the crude extract (C) and immunoprecipitate (IP), are shown. (B) Immunoprecipitates were assayed for telomerase activity in the presence of labeled TTP and the chain-terminating nucleotide ddGTP, which concentrates the telomerase signal into a +2 product, as indicated. (C) Immunoprecipitates were analyzed by Western blotting with an anti-myc antibody to detect the mutant Est1-myc18 protein; a subset of the mutants is shown here. (D) Plasmids overexpressing HA3-est1 mutations on a 2µ plasmid from the strong ADH promoter (derivatives of pVL1008) were introduced into a yku80-/pVL986 (KU80 URA3 CEN) haploid strain, and the wild-type YKU80 plasmid was evicted by plating on media containing 5-FOA. Colonies that had lost the covering YKU80 plasmid were resuspended in water and plated at equivalent cell counts at 10-fold serial dilutions at 30°.

Construction of EST1 mutations:
Two epitope-tagged versions of the Est1 protein were used in this work. Missense mutations were initially constructed in pVL1007 (derived from pRS415), with HA₃-EST1 expressed from the native EST1 promoter; this HA₃-EST1 construct has been shown previously to be fully functional when integrated into the genome (HUGHES et al.. 2000 ). Mutations were created using oligo-directed single-strand mutagenesis (KUNKEL et al.. 1987 ) and partially sequenced to confirm introduction of mutation. Initial genetic and biochemical analyses were performed with this panel of plasmids; however, pVL1007 was subsequently shown to not fully complement an est1- strain, presumably due to variations in plasmid copy number specific to this vector (data not shown). In addition, the HA₃ tag did not achieve high-efficiency recovery during immunoprecipitations (typically 1–2% of the telomerase RNA). Therefore, a second parental plasmid (pVL1355, derived from YCplac22) was constructed, which introduced an myc₁₈ epitope onto the C terminus of Est1, with EST1-myc₁₈ expressed from the native EST1 promoter. This plasmid fully complemented an est1- strain as assessed by several different assays (see Fig 2 Fig 3 Fig 4 for a comparison between EST1 and EST1-myc₁₈). Furthermore, the presence of the myc₁₈ epitope greatly increased Est1 IP efficiency (typically 10–30% of TLC1). The 19 mutations that showed a potential defect when analyzed in the pVL1007 construct were subcloned into pVL1355, and the subcloned 1.6-kb region was completely sequenced for each mutant allele to rule out the possibility of potential additional unlinked mutations. All of the in vivo and in vitro analyses presented in this article, unless noted otherwise, were performed with the pVL1355-derived constructs. Nine mutant alleles (est1-32, est1-33, est1-35, est1-37, est1-40, est1-43, est1-44, est1-58, and est1-59) that showed no defect (or only very modest effects) on telomere length, when analyzed in the slightly less functional pVL1007 backbone, were not subcloned into pVL1355 for further analysis. The est1-19 deletion mutation was generated by oligo-directed single-strand mutagenesis in pVL198, resulting in the deletion of aa 499–518 (T. LENDVAY and V. LUNDBLAD, unpublished data), and was subsequently cloned into pVL1355.

For the overexpression studies presented in Fig 5D, mutations were subcloned into pVL1008 (derived from the 2µ pRS424 plasmid) with the HA₃-EST1 gene expressed by the strong ADH promoter. Construction of plasmids expressing the Cdc13-Est2 (pVL1107) and HA₃-Cdc13-Est2 (pVL1115) fusion proteins has been previously described (EVANS and LUNDBLAD 1999 ).

Cloning the Saccharomyces carlsbergensis EST1 gene:
Genomic DNA prepared from a strain of S. carlsbergensis (ATCC no. 2345) was digested with EcoRI and probed at low stringency with the S. cerevisiae EST1 gene, which detected a faint hybridizing 2.0-kb band. Preparative amounts of EcoRI-digested S. carlsbergensis DNA in the 1.9- to 2.1-kb size range were gel purified and cloned into pBluescript SK⁺. Approximately 1000 E. coli transformants containing this size-fractionated library were screened by low-stringency hybridization, and a single clone containing a 2.0-kb insert (pVL259) that encoded a gene with homology to S. cerevisiae EST1 was recovered. The S. carlsbergensis EST1 sequence has been deposited in GenBank under the accession no. AF411028.

Genetic characterization of mutants:
Multiple (typically two to three) yeast isolates from each transformation were analyzed for growth, as described below, and telomere length, as described previously (LENDVAY et al.. 1996 ). For analysis of growth phenotypes in a YKU70 strain background, plasmids harboring est1-myc₁₈ mutations were introduced into TVL409, followed by eviction of the wild-type EST1 plasmid by plating onto media containing 5-fluoroorotic acid (5-FOA). Each mutant was analyzed at 30° by serial streakouts and assessed for senescence at the third and fourth streakouts. For analysis of growth phenotypes in a yku70- strain background, plasmids expressing each est1-myc₁₈ mutant were transformed into strain TVL471 at 30°. Transformants were inoculated into a 2-ml culture that maintained selection for both EST1 plasmids, plated at 10-fold serial dilutions onto -Trp -Ura and -Trp 5-FOA plates, and incubated for 2–3 days at 30°. For analysis of overexpression growth phenotypes, plasmids overexpressing the est1 mutations (on a 2µ plasmid and expressed as N-terminally tagged HA₃ proteins from the strong ADH promoter) were introduced into TVL304. Following eviction of the covering YKU80 plasmid, colonies were resuspended in 100 µl of water and plated at equivalent cell counts at 10-fold serial dilutions onto -Trp plates that were subsequently incubated at 30°.

Biochemical characterization of mutants:
Cultures (initiated from est1- strains expressing plasmid-borne est1-myc₁₈ mutations immediately following the loss of the wild-type EST1 covering plasmid) were grown at 30° in selective media and harvested at an OD₆₀₀ of 0.6–1.0. Extracts were prepared as described previously (HUGHES et al. 2000 ) to yield 500–600 µl of extract at protein concentrations of 5–7 mg/ml. Immunoprecipitations were performed essentially as described previously (HUGHES et al.. 2000 ): 500 µl of extract was incubated with 2 µl 9E10 anti-myc antibody (BabCo) and 40 µl of protein A/G agarose (Calbiochem, San Diego), and the beads were washed 3 times in TMG plus 200 mM NaCl and once in TMG plus 50 mM NaCl. The IP pellet was resuspended in final wash buffer and aliquoted for subsequent biochemical analyses. For Northerns, TLC1 and U1 were detected as described previously (LINGNER et al.. 1997A ). The amount of extract loaded in the IP lane represented 30 times the amount loaded in the crude lane, and the efficiency of RNA recovery was quantitated by PhosphorImager analysis. For Western analysis, Est1-myc₁₈ proteins were visualized using the 9E10 anti-myc antibody, horseradish peroxidase-conjugated goat anti-mouse antibody and ECL-Plus reagent (Amersham, Arlington Heights, IL). The amount of protein loaded into each lane varied by less than twofold, as measured by Bradford assay. Chain-terminating telomerase assay reactions were performed as described previously (HUGHES et al.. 2000 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Alignment of two Est1 homologs identifies conserved charged residues as targets for mutagenesis:
For the majority of the mutant alleles analyzed in this article, selection of amino acids for mutation relied on a comparison of the S. cerevisiae Est1 protein sequence with that from a related budding yeast species, Saccharomyces carlsbergensis. A genomic 2.0-kb fragment containing most of the S. carlsbergensis EST1 gene was recovered by reduced-stringency hybridization (see MATERIALS AND METHODS for details). This incomplete clone, which lacks N-terminal sequences, encodes an open reading frame that exhibits 59% aa identity to the S. cerevisiae Est1 protein over 551 amino acids (corresponding to aa 149–699 of the S. cerevisiae protein). The highest degree of sequence identity (75%) lies within the 130-aa region that harbors the DNA- and RNA-binding activities (VIRTA-PEARLMAN et al.. 1996 ).

Regions of conservation between the two Est1 homologs were used to dictate the choice of residues to mutate, on the basis of a clustered charged-to-alanine scanning mutagenesis strategy. In this method, small groups of charged aa (which are likely to be located on the solvent-accessible surface of the protein) are mutated to alanine (BASS et al.. 1991 ; BENNETT et al.. 1991 ; GIBBS and ZOLLER 1991 ). The principles of the clustered charged-to-alanine scan make it a potentially effective method of systematically surveying a significant portion of the protein surface in a relatively unbiased and nondestructive manner. In our analysis, the algorithm used to define a "charged cluster" was loosened to allow inclusion of two to four charged aa in a window of seven residues. The residues mutated in the N-terminal region, where sequence information on the S. carlsbergensis homolog was absent, were chosen solely on the basis of charged cluster criteria. A total of 23 clustered mutations were constructed (Fig 1A).

In addition, seven additional mutations were examined that were not constructed on the basis of the charged cluster criteria (Fig 1B). These single aa missense mutations were targeted to a completely conserved block of residues, from aa 498 to 514 (located within the 130-aa domain required for DNA and RNA binding). Since basic and aromatic groups have been implicated in direct nucleic acid interaction (DRAPER 1999 ; JONES et al.. 2001 ), phenylalanine and arginine residues in this block were mutated to generate est1-50 (R499A), est1-51 (F501A), and est1-53 (F506A). Two additional mutations in this region, est1-6 (F511S) and est1-7 (D513I), had previously been shown to affect telomere replication in vivo (VIRTA-PEARLMAN et al.. 1996 ). Alanine substitutions at residues 511 and 513 (est1-54 and est1-55, respectively) were constructed for this study to preserve the congruity of the alanine mutagenesis approach. Collectively, the clustered mutagenesis and the single mutational changes introduced missense mutations into 74 of the 699 residues in the Est1 protein. Of the 30 mutations constructed for this analysis, 14 resided in the N-terminal portion of the protein, 14 in the central 130-aa domain, and 2 in the C-terminal region (Fig 1).

Missense mutations in the central 130-aa region of Est1 have a severe telomere replication defect:
The 30 clustered and single mutant alleles were subjected to a battery of in vivo phenotypic assays to determine effects on growth and telomere length maintenance. The panel was first tested for whether any mutations exhibited a senescence phenotype comparable to that of an est1- null mutation. Plasmids harboring the est1 mutations (in single copy and expressed by the native EST1 promoter) were transformed into a haploid est1- strain that harbored a covering EST1 plasmid. Following eviction of the wild-type EST1 plasmid, the collection of mutant strains was analyzed for a senescence phenotype (see MATERIALS AND METHODS for details). The results are summarized in Table 1, and data for a subset of mutant strains propagated for 75 or 100 generations are shown in Fig 2. Strains bearing six mutations, which all resided within the central 130-aa region of the protein, displayed a detectable senescence phenotype, indicating that EST1 function was severely impaired. Four mutant strains (bearing est1-7, est1-47, est1-48, or est1-52 alleles) displayed a senescence phenotype indistinguishable from that of an est1- null strain, and two additional mutant strains (containing the est1-6 and est1-55 mutations) exhibited a moderate senescence phenotype (Fig 2).

A more stringent assay, capable of detecting partial defects in the telomerase-mediated pathway for telomere replication, was also used to examine the panel of 30 mutants. We have shown previously that a strain bearing null mutations in both telomerase and the Ku heterodimer exhibits greatly accelerated senescence, compared to the slower senescence displayed by a telomerase-defective strain (NUGENT et al.. 1998 ). Furthermore, in the absence of Ku, even modest disruptions in telomerase function (which would not otherwise exhibit a senescence phenotype) confer a growth defect (EVANS 2000 ). Therefore, a yku70- or yku80- strain can provide a sensitized background for detecting intermediate reductions in the activity of a telomerase subunit. We introduced each of the 30 est1 mutations into a haploid est1- yku70-/pEST1 CEN URA3 strain and examined the strains for growth following eviction of the wild-type EST1 plasmid (Fig 3 and data not shown). As expected, the 6 est1 mutants described above that displayed moderate to severe senescence in a YKU70 background exhibited a further reduction in viability when combined with a yku70- mutation. Five additional mutant strains with lesions that also mapped to the 130-aa central domain exhibited either severe (est1-46, est1-49, and est1-54) or partial (est1-50 and est1-51) reduction in viability in the yku70- background. Three additional mutants located within this region (est1-53, est1-56, and est1-57) had little or no defect in either growth assay (Fig 2 and Fig 3). Thus, of the 14 missense mutations that were introduced into this 130-aa domain, 11 conferred a notable growth defect by either or both of these two assays. Only a single mutation (est1-42) that mapped outside the 130-aa region displayed a reduction in EST1 function that was sufficient to confer growth defect: although the est1-42 strain did not exhibit a senescence phenotype in a YKU70 background (Fig 2), a proliferative defect was observed in the est1-42 yku70- strain (Fig 3).

The 30 mutations were also tested for their effects on telomere length. Plasmids expressing the est1 alleles were introduced into an est1-/pEST1 CEN URA3 strain and telomere length was analyzed 60 generations following loss of the covering EST1 plasmid. Mutations in the 130-aa domain displayed the most severe reductions in telomere length (many were comparable to an est1- strain), while mutations outside of this region showed more modest reductions in telomere length (Fig 4A). All of the 12 mutations described above that exhibited growth defects in either the YKU70 or the yku70- strain backgrounds also displayed telomere lengths that were significantly shortened.

An unexpected conclusion from this initial phenotypic analysis was that 18 of the 30 missense mutations—a strikingly large number—showed little or no effect on telomere length, with a corresponding lack of a discernable effect in either of the two growth assays. Eleven mutant strains exhibited telomere length comparable to that of wild type, and 7 additional mutations resulted in only modest effects on telomere length (Fig 4A and data not shown; summarized in Table 1 as class 4 and class 5 mutations). This may be a consequence of the emphasis placed on charged residues for mutagenesis; a similar rationale may also explain why mutant phenotypes were restricted primarily to the highly basic 130-aa domain.

Est1 proteins with missense mutations in the central 130-amino-acid region still retain association with telomerase:
One explanation for an in vivo defect in telomere replication could be a reduced association of a mutant Est1 protein with the telomerase holoenzyme complex. To test this, each mutant Est1 protein was examined for the ability to co-immunoprecipitate the TLC1 telomerase RNA subunit (Fig 5A and data not shown); a subset was also tested for association with enzyme activity (Fig 5B). Notably, 13 of the 14 mutations introduced into the 130-aa central domain retained roughly wild-type levels of association with telomerase (Fig 5; Table 1), indicating that the telomere replication defect displayed by these mutant proteins was not due to a gross inability to associate with the enzyme complex. The remaining mutant in this region, Est1-48, appeared to be reduced for telomerase association (Fig 5A and Fig B); however, both protein levels and the degree of association with TLC1 varied significantly from experiment to experiment (data not shown). The biochemical nature of this variability is unknown, but may reflect an unstable protein that is particularly susceptible to proteolysis in cell-free extracts (Fig 5C); the est1-48 mutation was therefore excluded from further analysis. The majority of the remaining mutant Est1 proteins exhibited a degree of interaction with telomerase roughly comparable to that of the wild-type Est1 protein; several notable exceptions are discussed in later sections.

The panel of est1 mutants was further examined by an additional in vivo assay that also potentially monitored association with the telomerase complex, by asking whether overexpression of these mutant proteins could influence telomere replication. We have previously shown that increasing the levels of wild-type Est1 protein results in a slight increase in telomere length, whereas overexpression of the Est1-6 and Est1-7 mutant proteins in a wild-type strain results in a modest telomere length decline (VIRTA-PEARLMAN et al.. 1996 ). This dominant negative effect can be further exaggerated by overexpressing these mutant proteins in a yku80- EST1 strain: in this sensitizing strain background, overexpression of either est1-6 or est1-7 results in a clear growth defect (EVANS 2000 ). To examine whether this phenotype extended to other mutations, the remaining 28 est1 missense mutant proteins were similarly overexpressed in a yku80- EST1 strain. Collectively, 8 of the 14 mutations in the 130-aa domain conferred an overexpression dominant negative phenotype by this assay, whereas no mutations outside this region exhibited such a phenotype (Fig 5D and data not shown; summarized in Table 1). These 8 mutations also reduced telomere length when these mutant proteins were overexpressed in a YKU80 strain (VIRTA-PEARLMAN et al.. 1996 and data not shown), suggesting that overexpression of mutant forms of Est1 that are able to interact with telomerase components may titrate out a limiting factor (or factors) required for telomere replication. This same set of 8 mutations exhibited severe synthetic lethality in the assay shown in Fig 3, which examined each est1 mutant allele (expressed by its native promoter) in combination with a yku70- mutation. On the basis of both telomere length defects and impaired growth in the two yku-dependent assays shown in Fig 3 and Fig 5D, these 8 mutants have been placed into a single class, referred to as class 1 in Table 1.

Identification of a class of mutations defective for the proposed second function of EST1:
We have previously observed that in an est1- strain, the Cdc13-Est2 fusion fails to promote the extensive telomere elongation that occurs in the presence of Est1 (EVANS and LUNDBLAD 1999 ). This suggests that Est1 has a second role in telomere replication, following recruitment of the enzyme to the telomere. To identify alleles that may be defective for this putative second function, est1 missense mutations were screened for those that failed to promote telomere elongation in the presence of the Cdc13-Est2 fusion (Fig 4B). The majority of the est1 mutations were capable of promoting extensive telomere elongation, comparable to that conferred by the parental EST1-myc₁₈ gene. Mutations in class 1 promoted either extensive (est1-46, est1-47, est1-49, est1-54, and est1-55) or intermediate (est1-52) telomere elongation in the presence of the Cdc13-Est2 fusion (Fig 4B). Therefore, the severe telomere replication defects displayed by these mutants can be relieved by the presence of the Cdc13-Est2 fusion, fulfilling the prediction for a defect in telomerase recruitment. Included in this class is the est1-47 mutation that we had previously argued, on the basis of other criteria, was impaired for enzyme recruitment (EVANS and LUNDBLAD 1999 ).

In contrast, two alleles, est1-42 and est1-19, failed to promote extensive telomere elongation by the Cdc13-Est2 fusion. (The est1-19 mutation, which removes amino acids 499–518, is described in more detail in a later section in this article.) These two mutant alleles are therefore defective in this proposed second function, as defined by this genetic test, and thus constitute a second class of mutant alleles, referred to as class 2 in Table 1. Protein levels in these two mutant strains were not noticeably impaired (Fig 5C), although a moderate reduction in the association with telomerase was observed (see Table 1). However, other mutant proteins (Est1-38, Est1-39, and Est1-41, discussed in a later section of this article) displayed an even greater reduction in the ability to associate with telomerase, but were still able to promote extensive telomere elongation in the presence of the Cdc13-Est2 fusion protein (Table 1; Fig 4B). This argues that the failure of the Est1-42 or Est1-19 proteins to promote telomere elongation in the presence of the Cdc13-Est2 fusion could not be attributed to a reduced interaction with the telomerase enzyme.

A third class of mutations defines an activity of EST1 that is potentially required for the Ku-mediated pathway of telomere length maintenance:
In the analysis described above, there was generally a good correlation between telomere length and growth: mutants with the shortest telomeres also displayed the most severe growth defects. However, two mutants (est1-50 and est1-51) were striking exceptions to this observation. Telomere length in these two strains was as short as that of an est1- null strain (Fig 4A), and yet neither of these strains exhibited even a weak senescence phenotype (Fig 2). This lack of correlation between telomere length and a growth phenotype argued that the inability to maintain telomere length in these two strains was not due to a defect in the same activity that is lost in class 1 mutants. Furthermore, both est1-50 and est1-51 promoted telomere elongation in the presence of the Cdc13-Est2 fusion (Fig 4B), indicating that they are proficient for the proposed second function of EST1. Thus, these two alleles appear to define a functionally distinct class of est1 mutations, perturbing an activity of EST1 different from that altered in the other two classes of mutations discussed in the previous sections; these alleles are designated as class 3 mutants (Table 1).

In addition, est1-50 and est1-51 exhibited only a slightly impaired growth defect when placed in combination with a yku70- mutation, in contrast to all other mutations that conferred short or very short telomeres (Fig 3). Furthermore, increased expression of Est1-50 and Est1-51 did not affect the growth of a yku80- strain (Fig 5D). This epistasis behavior is reminiscent of that of a mutation in another subunit in telomerase: the tlc1-48 allele impairs an activity of TLC1 that has been proposed to interact, either directly or indirectly, with the Ku heterodimer to facilitate telomere length maintenance (PETERSON et al. 2001 ). Thus, this third class of est1 mutations may be similarly altered for an activity that is required for this pathway.

Mutations in the N terminus of Est1 result in reduced association with telomerase:
Previous work demonstrated that deletion of the 130-aa central region completely abolished in vitro RNA-binding activity (VIRTA-PEARLMAN et al.. 1996 ), arguing that essential determinant(s) for interaction between Est1 and TLC1 reside in this region of the protein. Surprisingly, none of the missense mutations in this region significantly altered the ability of the Est1 protein to associate with the telomerase RNP (Fig 5). However, it is possible that multiple weak interactions in this region each contribute to RNA binding and that disrupting only a subset of interactions does not globally affect the association between Est1 and TLC1.

In contrast, three mutations that mapped to the N-terminal region of the Est1 protein displayed a markedly reduced association with telomerase (class 4 in Table 1). All three proteins were greatly reduced for the ability to co-immunoprecipitate a TLC1-containing complex: Est1-38 (<2% of wild type, which is at the detection limit for these experiments), Est1-39 (3% of wild type), and Est1-41 (2–8% of wild type; Fig 5A; Table 1). These three mutants were similarly reduced for the presence of telomerase activity in the immunoprecipitates (Fig 5B). This reduced association with telomerase was not a result of decreased protein levels or reduced IP efficiency (Fig 5C and data not shown). In addition, this defect was not specific for the C-terminal myc₁₈-tagged versions, as IP experiments with these three mutant proteins bearing an HA₃ epitope at the N terminus were similarly reduced for association with telomerase (data not shown).

However, despite the significant reduction in telomerase association observed in these co-immunoprecipitation experiments, est1-38, est1-39, and est1-41 mutant strains did not exhibit any detectable defects in telomere replication. Telomere length was only slightly reduced in the est1-38 and est1-39 alleles and unaffected in the est1-41 mutant strain (Fig 4A), and none of these mutant strains exhibited any growth defects in the assays shown in Fig 2, Fig 3, and Fig 5D. These three mutants also displayed only minor reductions in the ability to promote telomere elongation in the presence of the Cdc13-Est2 fusion (Fig 4B). Potential explanations for the lack of correlation with an in vivo telomere replication defect are considered in DISCUSSION.

The Est1 protein fails to associate with telomerase in the absence of TLC1:
Previous biochemical analysis has shown that the Est1 and Est2 proteins co-immunoprecipitate (HUGHES 1998 ), as expected for subunits of the telomerase holoenzyme complex. However, these experiments did not examine the dependency of the Est1-Est2 association on the telomerase RNA. To answer this question, we analyzed the association of these two proteins (present as Est1-myc₁₈ and ProA-Est2) in isogenic TLC1 and tlc1- strains. The ProA-Est2 protein was immunoprecipitated, and the IP complex was assayed for the presence of Est1-myc₁₈ by Western analysis. Although the Est1-myc₁₈ protein could readily be detected in a ProA-Est2 IP in the presence of TLC1, it failed to co-immunoprecipitate with ProA-Est2 in tlc1- cells (Fig 6A). This was not due to a gross destabilization of either Est1 or Est2 in the absence of TLC1 (data not shown; see also Fig 6B and HUGHES et al.. 2000 ). These results demonstrate that Est1 and Est2 do not form a stable complex in the absence of TLC1.

View larger version (52K): In this window In a new window Download PPT slide   Figure 6. Est1 and Est2 do not co-immunoprecipitate in the absence of the telomerase RNA. (A) Plasmids expressing either the tagged Est1-myc18 protein or the untagged Est1 protein were introduced into strains TVL415 (ProA-EST2) and TVL418 (tlc1- ProA-EST2/TLC1 URA3 CEN). The wild-type TLC1 plasmid was evicted from strain TVL418, and cultures were initiated immediately following the loss of the TLC1 plasmid. Immunoprecipitates from extracts were analyzed by Western blotting with an anti-myc antibody to detect the presence of the Est1-myc18 protein (top). Northern blot analysis on extracts from samples (prior to immunoprecipitation) demonstrates the absence of the TLC1 RNA in tlc1- strains (bottom). Duplicate lanes represent two separate isolates analyzed in parallel. (B) Extract prepared from strain TVL415 expressing EST1-myc18 on a plasmid was divided into two equal samples and both were incubated with IgG Sepharose to immunoprecipitate the ProA-Est2 protein; one sample was additionally treated with RNaseA (20 µl for 4 hr). Aliquots of crude extract and IP pellet were analyzed by Western blotting with an anti-myc antibody to detect the presence of the Est1-myc18 protein (top). Northern blot analysis of the samples demonstrates the absence of detectable TLC1 RNA in RNaseA-treated preparations (bottom).

One possible mechanism to explain the above observation is that TLC1 functions as a "scaffold" onto which Est1 and Est2 bind, with Est1 and Est2 bound to distinct sites on the TLC1 RNA and held in proximity like beads on a string. In this model, the bridge between the two proteins is lost in the absence of the telomerase RNA. However, the recent finding that TLC1 may be required for assembly of the telomerase enzyme adds a layer of complexity to this model. The TLC1 RNA has binding sites for Sm proteins, suggesting that telomerase is assembled through a multistep pathway (SETO et al. 1999 ). Therefore, a caveat of the experiment presented in Fig 6A is that in a tlc1- strain, a telomerase complex may not be assembled. To address this, extracts were prepared from TLC1 cells and incubated in either the presence or the absence of RNaseA during the course of the immunoprecipitation; this method allowed for assembly of the telomerase complex prior to removal of the telomerase RNA. Fig 6B shows that the amount of Est1-myc₁₈ protein present in the ProA-Est2 IP was substantially reduced upon RNaseA treatment. This was not due to destabilization of the Est1 protein in the absence of TLC1, as Est1 levels were unchanged in RNaseA-treated vs. untreated extracts. Thus, the presence of TLC1 is required for a stable association between Est1 and Est2 proteins. These results, in combination with prior observations that Est1 and Est2 independently associate with TLC1 (HUGHES et al.. 2000 ; ZHOU et al.. 2000 ) and recent observations demonstrating that Est1 and Est2 interact with separate regions of TLC1 (LIVENGOOD et al. 2002 ), suggest that Est1 and Est2 do not directly interact, but rather are held in close association largely through their interactions with the telomerase RNA.

A previously proposed RNA recognition motif in Est1 is not required for association with telomerase:
The above observation indicates that the association of Est1 with the catalytic core is through a direct interaction with the telomerase RNA subunit. During the course of this analysis, the Futcher lab reported the presence of a potential RRM within the 130-aa central domain of Est1 and proposed that this sequence motif was required for TLC1 binding (ZHOU et al.. 2000 ). This conclusion was based on the apparent failure of a mutant Est1 protein, containing three missense mutations in the conserved RNP-1 subdomain of the proposed RRM (R499G, I504A, and F506A), to associate with telomerase. However, in our studies, mutant proteins with alterations in two of these aa residues [est1-50 (R499A) and est1-53 (F506A)] retained full association with telomerase (Fig 5). This group also reported that Est1-6 and Est1-7, which contained single missense mutations in residues immediately adjacent to RNP-1, displayed a greatly reduced association with the enzyme. In contrast, we did not observe a defect in strains carrying these same mutations: Est1-6-myc₁₈ and Est1-7-myc₁₈ proteins associated with a TLC1-containing complex at levels of 35 and 45%, respectively (data not shown). This difference was not due to the location of the epitope tag used for immunoprecipitation of the Est1 proteins, since we also observed that LexA-HA₃-Est1-6 and LexA-HA₃-Est1-7 proteins (constructed by ZHOU et al.. 2000 for their studies) retained association with the telomerase RNP at levels of 38 and 50%, respectively (data not shown).

To address this discrepancy further, we assessed the ability of an Est1 mutant protein deleted for the entire putative RNP-1 motif and adjacent residues (est1-19, deleted from aa 499 to 518) to associate with telomerase. Deletion of these 20 amino acids resulted in a complete telomere replication defect, comparable to that of an est1- null strain (Fig 2 Fig 3 Fig 4). However, the Est1-19 mutant protein retained substantial association with a TLC1-containing complex (Fig 5A) and with a catalytically active enzyme (Fig 5B). Thus, removal of the putative RNA-binding motif does not abolish the ability of the mutant Est1 protein to interact with the telomerase RNA, arguing against a significant role for this region of the Est1 protein in the association with telomerase.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Est1 protein is an essential regulatory component of the telomerase holoenzyme. Previous work has shown that a primary function of this subunit is to recruit telomerase to chromosome termini, in collaboration with the telomere binding protein Cdc13 (NUGENT et al.. 1996 ; EVANS and LUNDBLAD 1999 ; QI and ZAKIAN 2000 ; PENNOCK et al.. 2001 ). Additional studies have suggested that Est1 may contribute to telomere elongation in a step that occurs subsequent to telomerase recruitment (EVANS and LUNDBLAD 1999 ). To further elucidate the role of Est1 in telomere replication, we have identified and characterized an extensive set of new alleles of the EST1 gene. Alanine scanning mutagenesis, directed by regions of high homology between the S. cerevisiae and S. carlsbergensis Est1 proteins, was used to identify amino acids required for Est1 function. A number of different assays assessed the telomere replication proficiency of these alleles, and the results of these tests placed the mutations into five classes, summarized in Table 1 and schematically presented in Fig 1C. The first three classes of mutant proteins, which retained association with the telomerase complex, were distinguished by unique in vivo properties, whereas class 4 mutants resulted in a substantial reduction in association with the holoenzyme. Class 5 mutants showed little or no alteration in the above phenotypic assays, indicating that this set of missense mutations had no significant impact on EST1 function. The identification of separation-of-function mutations with distinct properties indicates that the Est1 protein makes multiple contributions to telomere length maintenance.

Identification of est1 mutants defective in the telomerase recruitment function:
Although class 1 mutants display a severe telomere replication defect, the inability to elongate telomeres is bypassed by the introduction of a Cdc13-Est2 fusion protein. This argues that the defect in this class of mutants occurs either at a step prior to or at the recruitment step. The behavior of these mutants is consistent with previous work demonstrating that Est1 mediates the association between the catalytic core of telomerase and the telomere (NUGENT et al.. 1996 ; EVANS and LUNDBLAD 1999 ; QI and ZAKIAN 2000 ). This activity appears to depend on a direct interaction between Est1 and Cdc13, as indicated by the reciprocal suppression observed between cdc13-2 and est1-60 mutations (PENNOCK et al.. 2001 ). Notably, class 1 mutations, which span residues 455–514, map immediately adjacent to the est1-60 mutation at residue 444. Thus, determinants that dictate the interaction with Cdc13 may reside within an 70-aa region of the Est1 protein. Similarly, a 15-kD domain of Cdc13 is sufficient for telomerase recruitment (PENNOCK et al.. 2001 ), suggesting that these two proteins may interact through small modular protein interaction domains.

This 70-aa region also overlaps with the 130-aa stretch of Est1 that has been previously implicated in nucleic acid binding. This region, spanning from aa 435 to aa 565, was defined by screening relatively large deletions for the ability to bind telomeric DNA in vitro (VIRTA-PEARLMAN et al.. 1996 ), an approach that presumably did not precisely delineate the boundaries of the nucleic acid binding activity. In fact, two of the class 1 mutant proteins, Est1-6 and Est1-7, which are altered at residues 511 and 513, respectively, still bind telomeric DNA in vitro, consistent with the premise that additional biochemical properties may reside within this region of the protein.

Identification of mutations that disrupt a step that occurs subsequent to telomerase recruitment:
Two est1 mutant strains display a telomere replication defect that cannot be bypassed by the presence of the Cdc13-Est2 fusion. The behavior of these two alleles recapitulates the phenotype of an est1- strain carrying the Cdc13-Est2 fusion—even though the requirement for the Est1 recruitment function is alleviated, telomeres are not fully elongated (EVANS and LUNDBLAD 1999 ). This argues that Est1 contributes a second activity to telomere maintenance, which is supported by the identification of two mutations, est1-42 and est1-19, that display the same characteristics.

Although the est1-42 mutant strain is completely defective for the ability to promote Cdc13-Est2-dependent telomere elongation, this strain exhibits only a partial defect in telomere replication in an otherwise wild-type background (i.e., one that lacks the Cdc13-Est2 fusion): telomeres are short, although not as short as those of an est1- null strain (Fig 4A), and an est1-42 strain does not exhibit a senescence phenotype (Fig 2). The phenotype of the est1-42 mutant strain argues that this second activity contributes to telomere length maintenance, but this contribution is not as critical as the Est1-mediated telomerase recruitment function. This is consistent with our prior observations showing that a Cdc13-Est2 fusion, which alleviates the requirement for the recruitment function of Est1, maintains viability of an est1- strain (EVANS and LUNDBLAD 1999 ). If the second function made an equal contribution, the est1-/Cdc13-Est2 strain should have instead displayed a full telomere replication defect.

The est1-19 mutant also falls into this class on the basis of the inability to elongate telomeres in the presence of the Est2-Cdc13 fusion. However, this strain exhibits a more severe phenotype than does the est1-42 mutant strain. We suggest that est1-19 may not be a complete separation-of-function allele—in other words, both the proposed second function and other Est1-mediated activities may be impaired by this mutation (which is consistent with the fact that this deletion removes residues altered in the class 1 mutants).

Since the defining phenotype for this proposed second function is the inability of the catalytic core to extensively elongate telomeres when telomerase is tethered to the telomere as a Cdc13-Est2 fusion, the proposed second function of Est1 may mediate enzyme catalysis in some way. However, in the absence of a biochemical defect for the est1-42 mutant, models for this activity remain speculative at this point. For example, the est1-42 mutation may impair either DNA or RNA binding (on the basis of the assumption that additional determinants that contribute to nucleic acid binding may lie outside of the 130-aa domain). Thus, the second function of Est1 may be to promote accessibility of the 3' terminus to the active site of telomerase by virtue of its DNA-binding activity. Alternatively, the est1-42 mutant may exert its effects through an interaction with the RNA subunit, influencing parameters such as an inability to effectively position the templating RNA. Either model predicts that the telomerase holoenzyme should display altered enzymatic properties in the absence of the Est1 subunit, such as reduced ability of the primer to interact with the enzyme active site. Although the Est1 protein is dispensable for catalytic activity in vitro (COHN and BLACKBURN 1995 ; LINGNER et al.. 1997B ), these in vitro assays have been performed in primer excess and thus would not have detected more subtle changes in enzyme properties. Further tests of the proposed second function for Est1 will require a reexamination of the potential Est1-dependent effects on the ability of telomerase to extend a primer.

Identification of mutations that define a potential third role for EST1 in telomere length maintenance:
Two mutant strains (est1-50 and est1-51) do not manifest a senescence phenotype, even though telomeres appear to be as short as those of an est1- null strain, and thus these two strains fall into a third mutant class. However, an alternative possibility is that these two mutations are hypomorphic class 1 alleles, such that telomere length in these two strains is not quite reduced to that of a null mutation (but the slight difference cannot be detected on the Southern blot shown in Fig 4). Thus, there might be sufficient activity in the est1-50 and est1-51 mutants to maintain telomeres just barely above the length that would confer an obvious growth defect. Furthermore, est1-50 and est1-51, which disrupt aa 499 and 501, respectively, overlap with a class 1 mutation [est1-49 (R499A E500A)].

However, the phenotype of the est1-50 and est1-51 mutations in a yku80- background clearly distinguishes these two alleles from class 1 mutations. Class 1 alleles confer a complete synthetic growth defect when combined with a yku70- mutation. In contrast, the two class 3 mutations show only a minor growth defect in the yku70- epistasis test, despite the fact that these two class 3 mutant strains have shorter telomeres than do many of the class 1 alleles (for example, compare telomere length of the est1-50 and est1-51 strains with the more moderate telomere length defects of est1-46, est1-55, and est1-7 strains).

Recent work from the Gottschling laboratory has shown that a 48-nt stem-loop structure in the TLC1 component of telomerase influences telomere length through the same pathway as Ku (PETERSON et al. 2001 ). This conclusion was based in part on the behavior of a tlc1-48 mutant strain (lacking the stem loop), which conferred short telomeres but was not synthetic when placed in combination with a yku- strain. Furthermore, overexpression of the stem-loop structure did not exacerbate the telomere phenotype of a yku- strain. The similar behavior of the est1-50 and est1-51 mutations prompts our suggestion that Est1 may also be involved in this same pathway. Additional work will be required to determine whether this is due to a direct role or whether this effect is exerted indirectly through the RNA subunit.

Residues comprising a putative RRM motif are not required for TLC1 binding:
One goal of this mutational analysis was to determine if mutations in the 130-aa domain, previously shown to be required for in vitro RNA binding, were defective for association in vivo with the holoenzyme. Strikingly, none of the mutants in this central region significantly affect the ability of the mutant Est1 protein to associate with the telomerase RNA, as all Est1 mutants in this region co-immunoprecipitate with the telomerase RNA at levels comparable to that of the wild-type Est1 protein. It is possible that multiple weak interactions in this region may contribute to RNA binding, so that disruption of one or two of the contacts is not sufficient to alter the association between Est1 and TLC1.

The results presented in this article also contradict the results presented by the Futcher laboratory regarding the functionality of the putative RNP-1 motif (ZHOU et al.. 2000 ). Zhou et al. have reported that mutations in this proposed motif abolish the ability to interact with telomerase, even when overexpressed. In contrast, in our immunoprecipitation experiments, we observed association of these same mutant proteins with the telomerase complex at levels roughly comparable to that of the wild-type Est1 protein. Furthermore, deletion of the entire RNP-1-like sequence does not abolish the interaction between Est1 and telomerase. The reason for the discrepancy between the data presented here and in Zhou et al. is not clear, but must be attributable to differences in either the strain background or the experimental procedure. Typically, an HA₃-Est1 protein, even when overexpressed, will immunoprecipitate only 2–4% of the TLC1 telomerase RNA; perhaps Futcher and colleagues were close to the detection limit in their experiments, so that even a two- to threefold reduction would appear as an inability to associate with TLC1.

An N-terminal region is required for interaction with telomerase:
Although mutations in the 130-aa domain that had lost association with TLC1 were not recovered, three mutants that map to the N terminus (est1-38, est1-39, and est1-41) show substantially reduced association with telomerase. Unexpectedly, these mutants do not display discernable defects in telomere replication. It is possible that the immunoprecipitation results reflect an in vivo decrease in the association of Est1 with telomerase in these three mutant strains. If so, this would suggest that substantial reductions in the ability of a mutant Est1 protein to associate with the telomerase enzyme have little negative consequences on telomere replication. Alternatively, the substantial reduced association with telomerase may be an in vitro artifact of the stringent immunoprecipitation conditions employed in these experiments. Thus, a partially weakened association between the mutant Est1 protein and telomerase might not result in a detectable in vivo phenotype, but might translate to a significant reduction during immunoprecipitation conditions (possibly due to loss of additional associated proteins). Notably, these three mutants cluster in a region spanning 115 aa near the amino terminus of the protein. Additional mutagenesis is in progress to test whether this region represents a direct Est1-TLC1-binding interface.

Future perspective:
This study, as well as several recent investigations (PRESCOTT and BLACKBURN 1997 ; FRIEDMAN and CECH 1999 ; PENG et al. 2001 ; PETERSON et al. 2001 ), has begun to reveal the complex contributions that telomerase holoenzyme subunits make to telomere length regulation. Recently, human homologs of many of the proteins required for yeast telomere replication have been identified. Information gained from the detailed molecular analyses of the yeast holoenzyme will therefore likely provide insights into similar regulatory mechanisms governing human telomerase.

	ACKNOWLEDGMENTS

We thank Alex Rhode and Kathleen Becherer for isolating the S. carlsbergensis EST1 homolog, Tom Lendvay for constructing the est1-19 mutation, and Danna Morris for assistance in planning the site-directed mutagenesis. We also thank Kathy Friedman and Bruce Futcher for generously providing plasmids and members of the Lundblad laboratory, Thomas Cech, and several anonymous reviewers for helpful discussions and comments on the manuscript. This work was supported by a U.S. Army Medical Research and Materiel Command Breast Cancer Research Pre-doctoral Fellowship to S.K.E. and National Institutes of Health grant AG11728 grant to V.L.

Manuscript received November 30, 2001; Accepted for publication August 26, 2002.

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