The telomeric Cdc13 protein interacts directly withthe telomerase subunit Est1 to bring it to telomericDNA ends in vitroYun Wu and Virginia A. Zakian1

Department of Molecular Biology, Princeton University, Princeton, NJ 08544

Edited by Neal F. Lue, Weill Cornell Medical College, New York, NY, and accepted by the Editorial Board September 1, 2011 (received for review April 21, 2011)

In Saccharomyces cerevisiae, a Cdc13–Est1 interaction is proposed to mediate recruitment of telomerase to DNA ends. Here we provideunique in vitro evidence for this model by demonstrating a direct interaction between purified Cdc13 and Est1. The Cdc13–Est1interaction is specific and requires the in vivo defined Cdc13 recruitment domain. Moreover, in the absence of this interaction, Est1is excluded from telomeric single-stranded (ss)DNA. The apparent association constand (Kd) between Est1 and a Cdc13-telomeric ssDNAcomplex was ∼250 nM. In G2 phase cells, where telomerase is active, Cdc13 and Est1 were sufficiently abundant (∼420 and ∼110 copiesper cell, respectively) to support complex formation. Interaction between Cdc13 and Est1 was unchanged by three telomerase-deficientmutations, Cdc13E252K (cdc13-2), Est1K444E (est1-60), and Cdc13S249,255D, indicating that their telomerase null phenotypes are not dueto loss of the Cdc13–Est1 interaction. These data recapitulate in vitro the first step in telomerase recruitment to telomeric ssDNA andsuggest that this step is necessary to recruit telomerase to DNA ends.

Telomeres, the specialized nucle-oprotein structures that cap theends of linear chromosomes, areessential for genome integrity

and hence cell viability because they pro-tect chromosome ends from fusions anddegradation. They also counter the pro-gressive loss of terminal sequences due tothe inability of the conventional DNApolymerase to replicate the very ends oflinear DNAmolecules. In most eukaryotes,telomeres are maintained by the enzymetelomerase, a conserved reverse tran-scriptase that extends telomeric DNA us-ing an integral RNA component as thetemplate. In the absence of telomeraseactivity, most cells undergo progressivetelomere shortening, eventually stop di-viding, and senesce (1). In the buddingyeast Saccharomyces cerevisiae, EST2 andTLC1 encode the telomerase reversetranscriptase subunit (2, 3) and RNAtemplate (4), respectively. Additionally,EST1 and EST3, each of which encodes atelomerase accessory factor, and CDC13,which encodes the sequence-specific telo-meric single-stranded (ss)DNA bindingprotein (5–7), are needed for telomeremaintenance in vivo (8). Deletion or mu-tation of any of these genes leads to anever-shorter telomeres phenotype (1, 8)that is shared with est2Δ and tlc1Δmutants (4).The replication of telomeres is tightly

controlled during the cell cycle. Aftersemiconservative replication of telomericDNA, which takes place in late S phase,telomeres are processed to generate tran-sient, long, single-stranded G tails (9).Telomerase action also takes place in lateS/G2 phase (10, 11), coincident with anincrease in telomere association of theessential telomerase components Cdc13,Est1, Est2, and Est3 (12–14). Among theessential components of telomerase, only

Cdc13 is not part of the telomerase holo-enzyme (15) but rather arrives at telomereends presumably via its high affinity andselectivity for TG-rich ssDNA (5, 6).Cdc13 telomere association is indepen-dent of and presumably before all othertelomerase components, as the chromatinimmunoprecipitation (ChIP) signal forCdc13 at telomere ends or at a de novotelomere addition site can be detected ina number of telomerase null strains [tlc1Δ(13, 16), cdc13-2 (13, 16), or est1-60 (17)],and Cdc13 levels at telomeres do not in-crease with increased telomere associationof Est1 or Est2 (18). On the other hand,the late S/G2 phase assembly of the telo-merase holoenzyme at telomere ends isstrongly dependent on Est1, a cell cycle-controlled recruiter and activator oftelomerase (16).Even though telomerase action is re-

stricted to late S/G2 phase in vivo (10, 11),telomerase activity can be detected in ex-tracts from both G1 and G2/M phase cellsin vitro (10). In addition, despite theircritical importance in vivo, Est1, Est3, andCdc13 are dispensable for telomerase ac-tivity in vitro (19). Moreover, Cdc13 andits functional counterpart in humans andSchizosaccharomyces pombe, POT1, inhibittelomerase activity in vitro (20–22). Giventhe high affinity of Cdc13 for telomericssDNA, Cdc13 may hamper telomeraseaction in vivo by competing with telomer-ase for binding to telomeric ssDNA. Thecurrent model to explain these data is that,in vivo, Est1 brings telomerase to Cdc13-occupied ssDNA. This hypothesis can ex-plain the discrepancy between the in vivoand in vitro requirements for Cdc13 andEst1. Moreover, it is consistent with cur-rent models indicating that telomeraseaction is regulated at the level of telomereaccess (18).

Genetic evidence strongly implies thattelomerase recruitment to telomeres isachieved by a specific interaction betweenCdc13 and Est1. In vivo interactionbetween Cdc13 and Est1 has been dem-onstrated by yeast two-hybrid and coim-munoprecipitation analyses of over-expressed proteins (23–25). Fusion ofCdc13 and Est2 bypasses the need forEST1 to maintain stable telomeres (26).The in vivo defined Cdc13 recruitmentdomain (RD) is localized to amino acids211–331 (27). Furthermore, a “charge-swap” mutant of Cdc13, cdc13-2(Cdc13E252K), a mutation within the RD,confers a telomerase-null phenotype on itsown (6, 8) but is suppressed by a charge-swap allele of Est1, est1-60 (Est1K444E)(27). These results suggest that interactionbetween Cdc13 and Est1 is supported bythe electrostatic attraction of a specificLys-Glu pair (27). Consistent with this in-terpretation, Est1 binding is low in cdc13-2cells at both telomeres (16) and double-strand breaks (17). However, inconsistentwith the aforementioned hypothesis, the

This paper results from the Arthur M. Sackler Colloquium ofthe National Academy of Sciences, “Telomerase and Retro-transposons: Reverse Transcriptases that Shaped Genomes”held September 29–30, 2010, at the Arnold and Mabel Beck-man Center of the National Academies of Sciences and Engi-neering in Irvine, CA. The complete program and audio filesof most presentations are available on the NAS Web site atwww.nasonline.org/telomerase_and_retrotransposons.

Author contributions: Y.W. and V.A.Z. designed research;Y.W. performed research; Y.W. analyzed data; and Y.W.and V.A.Z. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. N.F.L. is a guesteditor invited by the Editorial Board.1To whom correspondence should be addressed. E-mail:vzakian@princeton.edu.

This article contains supporting information online atwww.pnas.org/lookup/suppl/doi:10.1073/pnas.1100281108/-/DCSupplemental.

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in vivo biochemistry data showed that Est1interacts equally well with both wild-type(WT) Cdc13 and Cdc13E252K (23), andEst1K444E binds telomeres as well as WTEst1 (28).The yeast TEL1 and MEC1 checkpoint

kinases, the homologs of ATM and ATR,respectively, are also involved in regulat-ing telomerase action. Deletion of TEL1or MEC1 leads to stably short (29) or nearWT-length (30) telomeres, respectively,whereas the tel1Δ mec1Δ double mutant isunable to maintain telomeres (30). Fusionof Cdc13 to Est2 bypasses the need forTEL1 and MEC1 for telomere mainte-nance, suggesting that TEL1 and MEC1function in telomerase recruitment (7).Indeed, TEL1 is required for efficient Est1and Est2 telomere association (31) andpreferential elongation of at least someshort telomeres (18, 32, 33). Cdc13 isthought to be a Tel1/Mec1 target as bothkinases can phosphorylate N-terminalfragments of Cdc13 in vitro. Moreover,simultaneous mutation of two of the Tel1/Mec1 sites in Cdc13, Ser-249, and Ser-255,to alanine, leads to cellular senescence,a phenotype that can be rescued by ex-pressing a Cdc13–Est1 fusion (34). Thesefindings suggest that telomerase recruit-ment is controlled by Tel1- or Mec1-dependent phosphorylation of Cdc13 inthe RD, with phosphorylated Cdc13 beingmore favorable for interaction with Est1.However, contrary to the expectations ofthis model, Ser255 phosphorylation is un-detectable in Cdc13 purified from yeast,and simultaneous mutation of all of theSQ sites in Cdc13 to SA, where SQ is theTel1 consensus sequence, does not leadto telomere shortening (25).In this report, we took in vitro approaches

to examine the Cdc13–Est1 interaction,

a step central to telomerase recruitmentand regulation in vivo. We provide uniqueevidence for a direct interaction betweenCdc13 and Est1 and show that this in-teraction can support the first step in telo-merase recruitment to DNA ends in vitro.However, mutant proteins that are de-fective in telomerase recruitment in vivo,Cdc13E252K, Est1K444E, and Cdc13S249,255D,had WT levels of Cdc13–Est1 interactionsin vitro. We also determined the in vivoconcentrations of Cdc13 and Est1 in bothG1 (when telomerase is not active) and G2(when it is). Only in G2 phase cells are theconcentrations of the two proteins suffi-ciently high to support productive complexformation. Our results confirm and extendthe current models on the molecularmechanisms that are needed to recruit te-lomerase to yeast telomeres.

ResultsPurification and Characterization of Recom-binant Cdc13 and Est1. The DNA bindingactivity of Cdc13 has been thoroughlycharacterized by several research groupsusing recombinant proteins purified fromEscherichia coli or insect cells (5, 6, 35).The DNA binding domain of Cdc13,which maps to residues 497–694 (36),binds to telomeric ssDNA with high af-finity and sequence specificity (36, 37).However, proteins obtained from a heter-ologous host will likely be devoid ofposttranslational modifications that areimportant for their function and regula-tion. We therefore overexpressed and pu-rified full-length Cdc13 (hereafter calledCdc13FL) and the DNA binding domain,Cdc13DBD (amino acids 445–694) from itsnative host, S. cerevisiae to near homoge-neity (Fig. S1A). Purification was per-formed in the presence of phosphatase

inhibitors to preserve phosphorylation,and the affinity tag was removed by pro-tease cleavage after purification. Wealso purified the N-terminal 455-aa(Cdc13N ter) polypeptide containing thepredicted Est1 RD from E. coli (Fig. S1A).For all proteins used in this study, weconfirmed protein identity and integrity byliquid chromatography-mass spectrometry(LC-MS) and MS/MS analysis.Using a filter binding assay, we showed

that purified Cdc13FL and Cdc13DBD ex-hibited indistinguishable DNA bindingactivity for an 11-mer minimal consensussequence (TEL11) across various salt andmagnesium conditions at affinities com-parable to previously reported values forrecombinant Cdc13FL purified from E. coli(6) or insect cells (35, 36, 38) (Fig. S1 Band C). In contrast, Cdc13N ter did notbind the TEL11 substrate or a 43-merlonger substrate to any appreciable extentunder the same conditions (Fig. S1 Band D). Our results confirm previouslypublished findings that the Cdc13 DNAbinding domain (DBD) alone is sufficientfor high-affinity telomeric ssDNA binding(35–37).We also purified full-length, C-terminally

strep-tagged Est1 from S. cerevisiae (Fig.S2A). The sequence of Est1 from aYPH499 strain differed at eight amino acidpositions from that from the Saccharomy-ces Genome Database (S288c strain) (Fig.S2B). We purified and carried out experi-ments with both the YPH499 and S288cversions of Est1 and obtained identicalresults. For simplicity, we show only theresults obtained with the YPH Est1.Consistent with findings by others (28,

39), our purified Est1 bound TG-ssDNA,and its affinity for TG-ssDNA increased asthe length of the ssDNA increased (Fig.

Fig. 1. Cdc13 interacts directly with Est1 to form a 1:1 stoichiometric complex that brings Est1 to telomeric ssDNA. (A) Magnetic beads pull-down experimentbetween purified strep-tagged Est1 and untagged Cdc13FL. Each experiment contained 0.5 μM of Est1 (lanes 1–8) and varying amounts of Cdc13FL (lanes 1–9).Upper, beads fractions (“beads”); Lower, 20% of the input materials shown as a control (“20% input”). Positions of Cdc13FL, Est1, and BSA are indicated on theright. (B) Beads-associated Cdc13FL in the presence of 0.5 μM (solid squares) or 1 μM (open squares) Est1 was quantified against Cdc13FL concentrations in theinput. In this and subsequent figures, error bars represent 1 SD from at least three independent experiments.

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S2C). Furthermore, the strep-tagged Est1supported WT-length telomeres in vivowhen expressed from a CEN plasmid andits native promoter (Fig. S2D). On elec-trophoretic mobility shift assay (EMSA),the strep-tagged Est1-ssDNA complex re-mained in the well. This behavior is likelydue to the high pI value of the protein(9.59), as ssDNA bound by a myc9-strep–tagged Est1 (pI 7.66) purified using thesame strategy migrated into the gel (Fig.S3A). Furthermore, in a glycerol gradientexperiment, strep-tagged Est1 sedimentedin a manner expected for a largely mono-meric protein (Fig. S3B). Therefore, strep-tagged Est1 was not aggregated in thepresence or absence of ssDNA. We con-clude that our purified Cdc13 (and itsderivatives) and Est1 are functional bypreviously established criteria with theexception that we did not observe a G4DNA promoting activity of Est1 reportedby a previous paper (40).

Cdc13 and Est1 Interact Directly to Form a 1:1Complex in Vitro. In vivo studies suggest thatCdc13 and Est1 interact and this inter-action is important to recruit telomerase totelomeres (17, 23, 26, 27). To determinewhether this interaction is direct, we testedpurified untagged Cdc13FL and C-termi-nally tagged Est1 for their ability to in-teract in vitro, using magnetic beads pull-down experiments (Fig. 1A). Varyingamounts of Cdc13FL in the presence orabsence of Est1 were mixed with strepta-vidin-coated magnetic beads that capturethe C-terminal affinity tag of Est1 (Fig. 1,lane 1). Cdc13FL alone was not pulleddown by the beads (Fig. 1, lane 9). How-ever, in the presence of Est1, Cdc13FL wasbead associated, and the amount of bead-associated Cdc13FL increased as the con-centration of input Cdc13FL increased(Fig. 1, lanes 2–8). The nonspecific con-trol, BSA, was not pulled down by thebeads either in the presence or in the ab-sence of Est1.The amount of Cdc13FL brought down

by the beads saturated at ∼0.56 μM (Fig.1B, solid squares), which was the approx-imate concentration of Est1 in the re-action. To establish the stoichiometricrelationship between these two proteins,we increased Est1 concentration by two-fold and observed that the saturatingamount of Cdc13FL on the beads alsoincreased to ∼1.1 μM (Fig. 1B, opensquares). Thus, Cdc13FL and Est1 forma complex at an apparent 1:1 ratio.

Cdc13–Est1 Interaction Is Specific. To furthercharacterize the Cdc13–Est1 interaction,we subjected Cdc13N ter and Cdc13DBD tothe same magnetic beads pull-down ex-periment used for Cdc13FL (Fig. 2B andFig. S4). Neither Cdc13N ter nor Cdc13DBD

were bead associated in the absence of

Est1 (Fig. 2B, lanes 3 and 4). However,in the presence of Est1, Cdc13N ter waspulled down by the beads (Fig. 2B, lane 6)with similar efficiency to Cdc13FL (Fig. 2B,lane 5), whereas Cdc13DBD was not beadassociated (Fig. 2B, lane 7). This in vitroresult confirms the in vivo findings (17, 27)that the Est1 RD of Cdc13 resides in itsN terminus.The N terminus of Cdc13 contains two

additional oligosaccharide-oligonucleotidebinding (OB) folds in addition to thegenetically defined RD (41). The firstOB fold (OB1) is required for Cdc13dimerization (41, 42) as well as Pol1 (thelargest subunit of DNA polymerase α)interaction (23, 41). We therefore char-acterized Cdc13–Est1 interaction with adimerization-defective mutant of Cdc13,L91R (41, 42) (Fig. S1E and Fig. 2C, lanes4 and 5). Est1 interaction was not com-promised in the Cdc13L91R mutant, in-dicating that Cdc13 dimerization is not

a perquisite for its interaction with Est1.We also tested two smaller fragments ofCdc13, Cdc131–340 (Fig. 2C, OB1-RD,lanes 6 and 7) and Cdc13190–340 (Fig. 2C,RD, lanes 8 and 9) for Est1 interaction:Both variants were beads associated in thepresence of Est1. This result suggests thatthe RD is necessary and sufficient for Est1interaction.To determine whether the interaction

between Cdc13 and Est1 is specific, wecompared it with the interaction betweenEst1 and RPA, the major cellular sequencenonspecific ssDNA binding protein (Fig.2D). Because the largest subunit of RPA(Rfa1) migrates at the same position asBSA, we omitted BSA from the reaction.In separate control experiments, weshowed that BSA was not bead associatedeither in the presence or in the absence ofEst1 (Fig. 2D, lanes 4 and 7), whereasCdc13FL was bead associated at an ∼1:1ratio of Cdc13 to Est1 (Fig. 2D, lane 5;

Fig. 2. Cdc13 interacts with Est1 via its N-terminal RD and the interaction is specific. (A) Schematic il-lustration of Cdc13 fragments used. (B) A total of 0.5 μM strep-tagged Est1 was incubated with 0.5 μMuntagged Cdc13FL, Cdc13Nter, or Cdc13DBD in a magnetic beads pull-down experiment. Upper, beadsfractions (“beads”); Lower, 20% of the input material as controls (“20% input”). (C) A total of 1 μM ofstrep-tagged Est1 was incubated with 1 μM untagged Cdc13 fragments in the beads pull-down assay.The beads fractions and 15% of the input material are shown as “beads” and “15% input”, respectively.An Est1 degradation product is indicated by an asterisk (*). (D) A total of 1 μM of strep-tagged Est1 orEst3 was incubated with 1 μM untagged Cdc13FL, RPA, or BSA. Experiments were performed as describedin Materials and Methods, except BSA was substituted with 20 μg/mL aprotinin. The beads fractions and15% of the input material are shown as “beads” and “15% input”, respectively. The same gels stained bySyproRuby for better detection are provided in Fig. S4.

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∼102% of the input levels). Reproducibly,a low level of Rfa1, the largest subunitof RPA, was bead associated in the pres-ence of Est1 (Fig. 2D, lane 6; ∼18% of theinput levels), suggesting a weak interactionbetween Est1 and RPA. Using a moresensitive stain, SyproRuby, we could alsodetect Rfa2 in the beads fraction (Fig.S4D), suggesting that Est1 interacts withthe functional heterotrimeric complexof RPA.As another test for specificity, we ex-

amined the ability of Cdc13FL to interactwith Est3, another telomerase subunitthat, like Est1, is essential for telomeraseaction in vivo (8, 15). When E. coli-expressed strep-tagged Est3 was used toreplace Est1 in the beads pull-down ex-periment (Fig. 2D, lanes 8 and 9), Cdc13FL

was not bead associated (Fig. 2D, lane 9).Using the same assay, purified Est3 in-teracted with Est1 (14), demonstratingthat our purified Est3 is capable of form-ing specific interactions. Therefore, theinteraction between Cdc13 and Est1is specific.

Cdc13–Est1 Interaction Is Critical for Recruit-ing Est1 to Telomeric ssDNA. We confirmedour observations on the specific Cdc13–Est1 interaction from the beads pull-downexperiments with an EMSA (Fig. 3A). Inthis and subsequent EMSA experiments,we first formed a complex betweenCdc13FL and a 15-mer TG-ssDNA

(TEL15) and then added purified Est1.We monitored formation of the Est1-Cdc13FL-TEL15 tertiary complex by fol-lowing the position of radioactively la-beled TEL15 (Fig. 3A, Left, lanes 14–20).As reported previously (28, 39), Est1 didnot bind efficiently to short oligonucleo-tides (28, 39) (Kd for Est1-TEL15 was2.14 ± 0.31 μM) (Fig. 3A, Left, lanes 1–6and Fig. 3A, Right, triangles). However,Est1 efficiently shifted the Cdc13FL-TEL15 ssDNA complex, forming a tertiarycomplex in the well with a Kd of 252 ±19 nM, ∼8.5 times lower than that forTEL15 alone (Fig. 3A, Right, squares).Thus, not only do Cdc13 and Est1 interactdirectly with each other, but also this in-teraction promotes Est1 association withtelomeric ssDNA.To determine whether Est1 interaction

with Cdc13-TEL15 is physiologically rele-vant, we asked whether this associationrequires the Cdc13-RD. In contrast to theefficient tertiary complex formation ob-served with Cdc13FL-TEL15, Est1 did notform a tertiary complex with Cdc13DBD-complexed TEL15 (Fig. 3A, Left, lanes7–13, and Fig. 3A, Right, open circles).Moreover, when Cdc13N ter was added tothe Est1-Cdc13FL-TEL15 tertiary com-plex, the tertiary complex dissociated toyield the Cdc13FL-TEL15 complex in aCdc13N ter concentration-dependentmanner (Fig. 3B, Right, solid squares).Cdc13N ter did not effectively dissociate a

preformed Est1-TEL15 complex (Fig. 3B,Right, open circles). Both of these resultsindicate that the Cdc13–Est1 interaction isessential for Est1 recruitment to Cdc13-bound ssDNA. Because Cdc13 is boundto telomere ends in vivo throughout thecell cycle (13), our results imply that it isthe specific interaction between Cdc13and Est1, not DNA binding by Est1, thatbrings Est1 to telomere ends.

Est1 Has Similar Affinity for WT and MutantCdc13 in Vitro. The EST4/cdc13-2 telomer-ase-defective allele is a point mutation inCDC13 that changes Glu-252 to Lys (8).Purified Cdc13E252K binds telomericssDNA as well as WT Cdc13 (6). How-ever, genetic experiments suggest thatCdc13E252K is defective in telomerase re-cruitment (6, 26, 27). In a cdc13-2 back-ground, Est1 is still telomere associated,but the levels of association are signifi-cantly reduced (16). The EST1 (K444E)allele est1-60 also confers a telomerase-null phenotype but suppresses the cdc13-2phenotype in an allele-specific manner(17, 27). The charge-swap nature of thesemutations led to the hypothesis that thetelomerase null phenotypes of the twosingle mutants are due to their loss of theCdc13–Est1 interaction.To test whether the Est1 interaction is

compromised by the cdc13-2 mutation, wepurified full-length Cdc13E252K and sub-jected it to the magnetic beads pull-down

Fig. 3. Cdc13–Est1 interaction is important for Est1 recruitment to the telomeric ssDNA end. (A) A total of 50 nM of TEL15, alone or complexed with Cdc13DBD

(150 nM) or Cdc13FL (100 nM), was incubated with varying amounts of Est1 in an EMSA. Left, a representative gel; Right, quantification. (B) Cdc13N ter out-competes Est1 for binding to the Cdc13FL–TEL15 complex. A total of 50 nM TEL15 was complexed with 100 nM Cdc13FL without (lane 1) or with (lanes 2–8) 500nM Est1 to form a tertiary complex. After 5 min, varying amounts of Cdc13N ter were added to the reaction to disrupt the cocomplex. Left, a representative gel;Right, quantification.

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experiment (Fig. 4A, “EK”). In this assay,Cdc13E252K interacted with Est1 as wellas WT Cdc13FL. In the EMSA experimentwhere affinity was quantified, Est1 had a Kdof 268 ± 26 nM for Cdc13E252K, which isnot significantly different from that for WTCdc13 (Fig. 4B). In addition, we observedno interaction difference between Est1 withWT Cdc13 or Cdc13E252K when the ex-periments were carried out at 200 mMNaCl, a condition in which the Est1–Cdc13interaction was weakened (Fig. S5). Like-wise, purified Est1K444E (encoded by theest1-60 allele) interacted with WT andCdc13E252K equally well in bead pull-downexperiments (Fig. 4C), with an observedKd of 241 ± 38 and 245 ± 30 for WTand Cdc13E252K-complexed TEL15, respec-tively, in the EMSA experiment (Fig. 4D).It has also been proposed that the

Cdc13–Est1 interaction is controlled byTel1/Mec1 phosphorylation of residuesS249 and S255 in the Cdc13 RD. Simul-taneous mutation of both residues to un-phosphorylatable alanine leads to an estphenotype (34). However, exhaustive massspectrometry analysis did not detectphosphorylation of either residue in puri-fied Cdc13FL. Also, the Cdc13N ter purified

from E. coli is not expected to be phos-phorylated. Thus, Cdc13 and Est1 can in-teract in the absence of phosphorylationwithin the Cdc13 RD.To determine whether phosphorylation

would enhance the Cdc13–Est1 interactionwe detect in vitro, we purified full-lengthCdc13S249,255D in which the two serineresidues are replaced by phosphomimeticamino acids. The mutant protein wassubjected to the magnetic beads pull-down experiment with Est1 (Fig. 4A,“DD”). The Cdc13S249,255D mutant inter-acted with Est1 as effectively as the WTCdc13. In the EMSA experiment where theinteraction was quantitatively measured(Fig. 4B), Est1 had a Kd of 269 ± 29 nM forthe Cdc13S249,255D mutant, which was notsignificantly different from that for WTCdc13. Therefore, we conclude that the invivo defects of the Cdc13E252K (cdc13-2),Est1K444E (est1-60), and Cdc13S249,255D

mutants do not reflect a change in Cdc13–Est1 interaction in vitro as measured by theassays used in this paper.

Cdc13 and Est1 Abundance Is Sufficient toSupport Complex Formation in Vivo. Cdc13and Est1 are both low-abundance proteins

in vivo: In asynchronous cells, Cdc13 andEst1 are present at, respectively, ∼320(43) and ∼70 copies per cell (14). Using aquantitative Western blot approach (14),we determined Cdc13 and Est1 concen-trations (Fig. 5). We used strains express-ing either Cdc13-myc9 or Est1-myc9 fromtheir endogenous promoters and nativeloci. The tagged proteins are fully func-tional as they support WT-length telo-meres (7, 12). In asynchronous cultures,our methods yielded values of 324 ± 66copies of Cdc13 per cell and 67.6 ± 23.7copies of Est1 per cell, in excellentagreement with previous reports (14, 43).We then determined Cdc13 and Est1 lev-els in α-factor–arrested cells (late G1)when telomerase is not active and noco-dazole-arrested (G2) cultures, when it is(10, 11). Cdc13 and Est1 were present at288.9 ± 23.2 and 20.3 ± 6.9 copies percell in G1 phase and 417.8 ± 67.2 and109.1 ± 25.8 copies per cell in G2 phasecells, respectively. The cell cycle-de-pendent increase in G2 was statisticallysignificant for both proteins (P = 0.03 and0.003 for Cdc13 and Est1, respectively).We previously concluded that the increasein Cdc13 levels detected (but not quanti-

Fig. 4. Est1 interacts with Cdc13 mutants at strengths indistinguishable from that of the WT protein. (A) A total of 0.5 μM Strep-tagged Est1 was incubatedwith 0.5 μM untagged Cdc13WT (WT), Cdc13E252K (EK), or Cdc13S249,255D (DD) in a magnetic beads pull-down assay. Upper, beads fractions (beads); Lower, 20%of the input materials are shown as control (20% input). (B) A total of 50 nM of TEL15, complexed with 100 nM of FL-Cdc13-WT, EK, or DD, was subjected tococomplex formation with Est1 in EMSA experiments. Upper, a representative gel; Lower, quantification. (C) A total of 0.5 μM Strep-tagged Est1K444E (KE) wasincubated with 0.5 μM untagged Cdc13WT (WT) or Cdc13E252K (EK) in a magnetic beads pull-down assay. Upper, beads fractions (beads); Lower, 20% of theinput material is shown as controls (20% input). (D) A total of 50 nM of TEL15 complexed with 100 nM of Cdc13WT or Cdc13E252K (EK) was subjected to co-complex formation with Est1K444E (KE) in EMSA experiments. Left, a representative gel; Right, quantification.

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fied) by Western blotting is consistentwith the increase in cell mass and DNAcontent as cells progress through the cellcycle, whereas the increase in Est1 abun-dance represents cell cycle-dependent up-regulation (13) via proteosome-dependentdegradation (44). The volume of theyeast nucleus expands quickly in G1 phasebut remains largely unchanged from lateG1 to S phase, at ∼2.91 fL (45). Using thisvalue for the volume of nuclei in bothconditions, the in vivo concentrations forCdc13 were 164.8 ± 13.2 nM in G1 and238.4 ± 38.3 nM in G2, whereas Est1concentration was 11.6 ± 4 nM in G1 and60.3 ± 12.6 nM in G2. These estimatessuggest that there are no more than ∼8(4.5 nM) and ∼48 (27.6 nM) Cdc13–Est1complexes in, respectively, G1 and G2phase cells. All of these concentrations arebelow the Kd for Cdc13–Est1 interactioncalculated in vitro (Fig. 1). Thus, unlessthere are additional interactions thatstabilize the Cdc13–Est1 interactionson telomeric DNA, in vivo, most of theCdc13 and Est1 is expected to exist as freeproteins, rather than in complex form.

DiscussionHere we used full-length Cdc13 and Est1proteins purified from their native host,S. cerevisiae, to provide unique in vitroevidence that Cdc13 and Est1 interact di-rectly to form a 1:1 complex (Fig. 1)through the genetically defined RD ofCdc13 (Fig. 2C). This interaction, whichwas essential for Est1 recruitment to te-lomeric ssDNA in vitro (Fig. 3), mimicsthe in vivo roles of Cdc13 and Est1 ascomediators for telomerase recruitment(26). Unexpectedly, the Cdc13–Est1 in-teraction was unchanged by charge-swapmutations in either Cdc13 (cdc13-2) orEst1 (est1-60) or by simultaneous phos-phomimetic mutations of the potentialTel1/Mec1 phosphorylation sites on Cdc13(cdc13-S249,255D) (Fig. 4). Additionally,we show that the in vivo concentrations ofCdc13 and Est1 in G2 phase cells aresufficiently high to support complex for-mation in vivo (Fig. 5).Genetic experiments strongly suggest

that a Cdc13–Est1 interaction is critical torecruit telomerase to telomeres in vivo(17, 26, 27). Previously, a weak Cdc13–Est1 interaction was demonstrated by

yeast two-hybrid and in vivo coimmuno-precipitation experiments (23–25). How-ever, it is not clear from these earlierexperiments whether the Cdc13–Est1 in-teraction is direct or bridged by anothercellular component, and there was noquantitative information on the strength ofthis interaction. Our in vitro results es-tablish that these two proteins interactdirectly, and the Est1 recruitment domainof Cdc13 resides within a small regionpredicted from in vivo experiments (17,27). The apparent Kd for the Cdc13–Est1interaction was ∼250 nM, which fallswithin the range of that for other transientinteractions between yeast nuclear pro-teins, such as the replication machinerycomponents PCNA and Polη (∼100 nM)(46), the Pho80-Pho85 kinase and its in-hibitor Pho81 (∼120 nM) (47), Pho80-Pho85 and its substrate Pho4 (400–800nM) (48), and Cdc13 OB1 and a 36-aafragment from Pol1 (3.8 μM) (41).The abundance of Cdc13 andEst1 in vivo

was extremely low, with Cdc13 levels closeto the Kd value but the level of Est1 farbelow it (Fig. 5). On the basis of the de-termined in vivo concentrations and the Kdfor the Cdc13–Est1 complex, we estimatethat there are no more than 8 and 48Cdc13–Est1 complexes in G1 andG2 phasecells, respectively. G tails in G1 phase areshort, 12–14 nt in length (49), and cantherefore accommodate only a singleCdc13 molecule. On the basis of theseconsiderations, most (∼89%) of the ∼300Cdc13 molecules will not be telomere as-sociated in G1 phase cells [78% if Cdc13forms a dimer (41, 42) on ssDNA]. Thus,even though the number of Cdc13–Est1complexes is low in G1 phase cells, thenumber of telomere-associated complexesis even lower, ≤2 complexes per cell.However, G tails in S/G2 phase are50–100 nt in length (9, 50), and thereforeeach of these G tails can bind moreCdc13. Assuming that four to six Cdc13molecules (or two to three dimers) arebound to each of the 64 telomeres in ahaploid G2-phase cell, ∼75% of the Cdc13molecules could be telomere associatedat the time in the cell cycle when telomer-ase is active. Thus, given the number ofCdc13–Est1 complexes that are predictedto be telomere associated, up to 57% of thetelomeres in a G2-phase cell can poten-tially be elongated by telomerase.Although we estimate that more than

half of the telomeres in G2-phase cellscould be Cdc13–Est1 associated, in vivodata demonstrate that telomere extensionoccurs at a much lower frequency, withonly ∼7% of WT-length telomeres elon-gated in a given cell cycle (51). Therefore,productive Cdc13–Est1 interactions mustoccur at a much lower efficiency in vivo.There are multiple possible explanationsfor this discrepancy. For example, if the

Fig. 5. Cell cycle-specific in vivo concentration of Cdc13 and Est1. (A) A representative gel of quanti-tative Western blot analysis. Total cell lysates were extracted from asynchronous, α-factor–arrested, ornocodazole-arrested cultures expressing Est1-myc9 and an untagged strain. Lysates equivalent to 2 × 107

cells were loaded alongside a myc9-tagged standard protein (10, 6, 4, 2, 1, and 0.5 fmol) mixed withextract from 2 × 107 cells from the untagged control for anti-myc Western blot analysis. Right, Flowcytometry profiles are shown. (B) Quantification of in vivo concentrations of Cdc13 and Est1 fromasynchronous, G1-phase, and G2-phase cells. Concentrations in nanomoles are shown in parentheses.

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interaction between Cdc13 and Est1 issufficiently transient, the complex coulddissociate from telomeres before pro-ductive telomerase assembly or action, aprocess that could be hastened by the Pif1DNA helicase, which removes active telo-merase from DNA ends in vivo andin vitro (52). Productive Cdc13–Est1complex formation is likely also reducedby the presence of other proteins thatcompete with Est1 for Cdc13 binding, suchas Stn1 (53, 54) and Pol1 (DNA Pol α)(23). These considerations can explain whyin vivo Cdc13–Est1 interactions are de-tected by coimmunoprecipitation onlywhen the proteins are overexpressed (23,24). Taken together, these results suggestthat the Cdc13–Est1 interaction is neces-sary but insufficient to support stabletelomere association of Est1 (and the te-lomerase holoenzyme). Other interactionssuch as Est1–ssDNA (28, 39), Est1–Est2(28), or Est1–TLC1 (28, 55) may preventdissociation of Est1 and/or the telomeraseholoenzyme from telomeres. In vivo datasupport this interpretation as in the ab-sence or destabilization of TLC1 or Est2,Est1 telomere association is greatly re-duced (13, 16, 17), even though theCdc13–Est1 interaction should still occur.We examined two Cdc13 mutants,

Cdc13E252K (cdc13-2) and Cdc13S249,255D,that show reduced Est1 recruitment invivo, defects that are generally attributedto their presumed inability to interactwith Est1 (27, 34). Surprisingly, we sawno significant change in Est1 affinity forthese two mutants (Fig. 4). We also didnot observe a change in the strength of theWT–Cdc13 interaction for Est1K444E (est1-60). These findings are consistent with invivo results showing that Est1 interactswith WT and Cdc13E252K equally well bythe criteria of yeast two hybrid and coim-munoprecipitation analyses (23), andEst1K444E associates as well as WT Est1with telomeres (28). These data provideadditional support for our view that theCdc13–Est1 interaction is not sufficient tosupport stable telomerase assembly at te-lomeres. We propose that, although thecharge-swap or phosphorylation mutantssupport WT levels of Cdc13–Est1 in-teraction, the resulting Cdc13–Est1 com-plex is defective. For example, the Cdc13-Est1-K444E and Cdc13-E252K-Est1 com-plexes might be improperly oriented sothat Est1 cannot interact stably with Est2,TLC1, or other telomerase subunits.These defects could lead to Est1 dissoci-ation from the telomere and thus loss ofEst2 binding and telomerase activity.Both Cdc13 and Est1 proteins are

reported to bind telomeric ssDNA specif-ically (6, 28, 39, 40, 56), and our results areconsistent with these previous findings(Fig. 1–3). One might question why telo-merase recruitment depends on a Cdc13–

Est1 interaction when Est1 itself bindstelomeric ssDNA. In vivo, Cdc13 telomereassociation is independent of other telo-merase components (16, 17). The affinityof Est1 for telomeric DNA is not nearlyas high as that of Cdc13 (6, 37), and thepresence of large quantities of nuclearRNA may compete with ssDNA for Est1binding, because DNA binding and RNAbinding by Est1 are mutually exclusive(28). Therefore, in the absence of an Est1-interacting partner that confers highaffinity for telomeric ssDNA, Est1 is ex-pected to be excluded from telomericssDNA. Indeed, when ssDNA was boundby Cdc13DBD, a protein that does notinteract with Est1, Est1 was unable tobind telomeric ssDNA (Fig. 3). Taken to-gether, we propose that it is the Cdc13interacting ability of Est1, not its ssDNAbinding activity, that is essential fortelomerase recruitment.It is intriguing that we observed a weak

but reproducible interaction betweenRPA and Est1 (Fig. 2), given that certainmutations in RPA result in telomereshortening in both S. cerevisiae (57) andS. pombe (58, 59). A previous report sug-gested that interaction between Est1 andRPA helps telomerase recruitment (60).However, short telomeres that are pre-ferred substrates for telomerase have verylow levels of RPA binding, and eventhis binding can be explained as being dueto semiconservative DNA replication (61).At this time, it is not possible to assesswhether the RPA–Est1 interaction hasfunctional significance.In summary, our results provide the

biochemical basis of Cdc13–Est1 in-teraction and insights into the molecularmechanism of telomere recruitment of te-lomerase. Our work recapitulates the firststep of telomerase recruitment in vitro.How the Cdc13–Est1 interaction is regu-lated in vivo and the necessary componentsfor stable assembly of telomerase at thetelomere are areas for future investigation.

Materials and MethodsStrains. Est1, Cdc13FL, both WT and mutants, andall Cdc13 fragments except Cdc13N ter werepurified from yeast strain BCY123 carrying anarc1-K86R mutation. RPA was purified from yeastBJ2168. Cdc13N ter (amino acids 1–455) and Est3were purified from E. coli Rosetta2(DE3) (Nova-gen). Complementation test and quantitativeWestern blot analyses were carried out in aYPH499 background. Genotypes of the yeaststrains are listed in Table S1.

Protein Purification. Cdc13FL, WT and mutants, andall Cdc13 fragments except Cdc13N ter were clonedinto a pYES2 vector fused to a carboxyl-terminaltag consisting of a Gly8 linker, an HRV 3C cleavagesite, 5× streptag II (Novagen), and a HAT tag(Clontech). Protein overexpression was inducedwith 2% galactose at 30 °C for 12 h. Cdc13N ter

was cloned into pGEX6P-1 fused to an amino-terminal GST tag. Fresh E. coli transformants

were grown at 18 °C, and protein overexpressionof Cdc13N ter was induced with 0.2 mM iso-propylthiogalactose for 16 h.

Cdc13FL, Cdc13OB1-RD, and Cdc13RD were puri-fied over 0.1% polyethyleneimine precipitation,50% ammonium sulfate precipitation, streptactinagarose (Novagen), and Talon Metal Affinityresin (Clontech). The affinity tags were removedby HRV 3C protease (Novagen) digestion at 4 °C.Untagged Cdc13FL or Cdc13OB1-RD was concen-trated and buffer exchanged to TDEG/100 buffer(25 mM Tris·Cl, pH 7.5, 0.1 mM DTT, 0.1 mM EDTA,10% glycerol, 100 mM NaCl) on an Amicon Ultra-4 [molecular weight cut off (MWCO) 30 kDa]concentrator that retained Cdc13 but not theprotease and the tag. The affinity tag on Cdc13RD

was removed on column and the untaggedCdc13RD was separated from the resin and con-centrated, and buffer was exchanged to TDEG/100buffer on an Amicon Ultra-4 (MWCO 10 kDa).Cdc13DBD was purified as described except theammonium sulfate precipitation step was omittedand a GST-tagged HRV 3C was used to cleave thetag. The protease was then removed by an AmiconUltra-4 (MWCO 50 kDa). Untagged Cdc13DBD in thefiltrate was then concentrated and buffer ex-changed to TDEG/100 buffer on an Amicon Ultra-4(MWCO 10 kDa) concentrator. Cdc13N ter was pu-rified using glutathione Sepharose (GE Health-care), and the tag was removed on column by GST-HRV 3C protease digestion. Cdc13N ter was sepa-rated from resins and concentrated buffer ex-changed to TDEG/100 buffer on an Amicon Ultra-4(MWCO 50 kDa) concentrator. Concentrations ofCdc13 fragments were determined using an ex-tinction coefficient at 280 nm calculated on thebasis of the amino acid composition.

Est1andEst3werepurifiedasdescribed(14)exceptthat the affinity tag of Est3 was not removedpostpurification. RPA was purified as described (62).

All proteins from the final step of purificationwere subjected to sequential in-gel endoproteinasedigestion. Peptides were eluted, desalted, andthen subjected to reversed-phase nano–LC-MSand MS/MS coupled to an LTQ-Orbitrap hybrid massspectrometer (Thermo) to confirm protein identityand integrity (PrincetonMass Spectrometry Facility).

Oligonucleotide DNA Substrates. DNA oligonu-cleotides were purchased from Integrated DNATechnologies (Table S2). DNA concentrations weredetermined using extinction coefficients providedby the vendor.

Magnetic Bead Pulldown. Experiments were carriedout as described in ref. 14 at indicated concen-trations. The gels were first stained with SyproRuby (Invitrogen) and analyzed using a Storm 860system (Molecular Dynamics). The relative amountsof bead-associated Cdc13 or RPA were measuredby comparing Cdc13/Est1 or RPA/Est1 ratio inthe input gel with proteins of known quantity.After quantification, the gels were restained withCoomassie brilliant blue for visualization.

EMSA. Unless otherwise indicated, reactions wereperformed in 20 mM Tris·HCl, pH 8, 100 mM NaCl,5 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, 5%glycerol, and 0.005% bromophenol blue at roomtemperature for 20 min before loading onto a0.8% agarose gel in 1× TBE. Gels were run at 120V for 2 h at 4 °C, dried on a DE81 paper (Millipore),and visualized and quantified using a Storm860 system.

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Quantitative Western Blot Analysis. Early log-phaseculture (OD 0.1–0.2) expressing myc9-taggedCdc13 (YTSF32) or Est1 (YCTT373) was arrestedin G1 or G2 by 10 ng/mL α-factor or 15 μg/mLnocodazole, respectively, at 24 °C for 3 h. Cell cycleprofiles were determined by flow cytometryanalysis, cells counted on a Coulter Z2 cell counter

(Beckman), and quantitative Western blots wereperformed as described (14).

ACKNOWLEDGMENTS. We thank T. Cech and hislaboratory, A. Mazin, M. Bochman, K. McDonald,and C. Webb for careful reading of this manu-script; B. Garcia, S. Kyin, and D. Perlman for

help with mass spectrometry; and C. DeCostefor help with flow cytometry. This work wassupported by US National Institutes of HealthGrant GM43265 (to V.A.Z.) and fellowshipDRG-1943-07 from the Damon Runyon–Robert Black Cancer Research Foundation(to Y.W.).

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