Replication Proteins Influence the Maintenance of Telomere Length and Telomerase Protein Stability

We investigated the effects of fission yeast replication geneson telomere length maintenance and identified 20 mutant allelesthat confer lengthening or shortening of telomeres. The telomereelongation was telomerase dependent in the replication mutantsanalyzed. Furthermore, the telomerase catalytic subunit, Trt1,and the principal initiation and lagging-strand synthesis DNApolymerase, Pol ${alpha}$ , were reciprocally coimmunoprecipitated, indicatingthese proteins physically coexist as a complex in vivo. In apol ${alpha}$ mutant that exhibited abnormal telomere lengthening andslightly reduced telomere position effect, the cellular levelof the Trt1 protein was significantly lower and the coimmunoprecipitationof Trt1 and Pol ${alpha}$ was severely compromised compared to those inthe wild-type pol ${alpha}$ cells. Interestingly, ectopic expression ofwild-type pol ${alpha}$ in this pol ${alpha}$ mutant restored the cellular Trt1protein to the wild-type level and shortened the telomeres tonear-wild-type length. These results suggest that there is aclose physical relationship between the replication and telomerasecomplexes. Thus, mutation of a component of the replicationcomplex can affect the telomeric complex in maintaining bothtelomere length equilibrium and telomerase protein stability.

INTRODUCTION

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Introduction

The chromosome ends of almost all eukaryotes are capped by telomeresto ensure the integrity of chromosomes and to maintain the overallgenome stability. Telomeres consist of simple DNA repeats, whichassociate with proteins to protect chromosomes from end-to-endfusion, degradation, and inappropriate recombination. TelomericDNA consists of tandem arrays of short repeats rich in G residueson the strand that runs towards the 3' end of the chromosome.The telomeric tracts of all organisms are of heterogeneous length,ranging from 28 bases in some ciliates to over 50 kb in mice.However, in a given cell type, the telomeric DNA length is keptwithin a certain size range. A common structural feature oftelomeres from different organisms is a single-stranded G-richoverhang. This G-rich overhang structure and the proteins thatbind to it are thought to play an important role in mediatingchromosome integrity, since in its absence, chromosome lossand fusion rates are elevated (14, 32, 43, 49, 67, 72, 75, 77).

Telomeres are also essential for the complete replication ofeukaryotic chromosomes. DNA synthesis by DNA polymerases hasa 5'-to-3' polarity (38). At the replication fork, the leading-strandsynthesis is thought to initiate once and then proceeds continuously.The lagging-strand synthesis is discontinuous throughout andrequires repeated initiations by polymerase ${alpha}$ (Pol ${alpha}$ ) and primase(9, 12, 73). Due to the polarity of DNA synthesis, the telomericG-rich strand is synthesized by the leading-strand replicationmachinery, and the C-rich strand is synthesized by the lagging-strandreplication machinery. Removal of the terminal RNA primer bynucleases leaves an 8- to 12-nucleotide gap at the 5' end ofthe newly replicated DNA that cannot be refilled by conventionalDNA replication, resulting in an end replication dilemma (44,74). The end replication problem is potentially resolved bytelomerase, an unusual reverse transcriptase. The catalyticsubunit of telomerase (Trt1) contains an integral RNA moleculewith a small template domain that is utilized by telomeraseas template to add telomeric repeats onto the 3' end of thetelomere (57). Although telomerase activity is required to maintainthe steady-state length of telomeric DNA repeats at the chromosomeend, other proteins have been shown to affect telomere lengthmaintenance. In Saccharomyces cerevisiae (budding yeast) andSchizosaccharomyces pombe (fission yeast), proteins that bindeither directly to telomeric sequences or to telomeric sequence-bindingproteins have been shown to positively or negatively influencetelomere length (10, 11, 27, 43, 57). In addition, mutationsin several checkpoint genes in fission or budding yeast andin Caenorhabditis elegans have been shown to affect telomerelength (3, 22, 42, 48, 51, 54, 66).

Mutations of components of budding yeast DNA replication machinerysuch as POL1 (Pol ${alpha}$ ) or the large subunit of replication factorC (CDC44/RFC1) confer telomere elongation, whereas a rad27 deletionconfers destabilized telomere length (1, 15, 60). Moreover,in a POL1 mutant (pol1-17), the telomeric position effect (TPE)is reduced concomitantly with telomere elongation (2). Recentdata have shown that fission yeast Pol ${alpha}$ mediates recruitmentof Swi6 to heterochromatin, including that of telomeres to establishsilencing (4). By using a novel in vivo assay, it has been shownthat telomerase-mediated telomere addition requires the activitiesof Pol ${alpha}$ , Pol ${delta}$ , and primase in budding yeast (23). In budding yeast,Cdc13p plays an important role in protecting and maintainingtelomeric DNA (31, 56). Budding yeast Cdc13p interacts withthe catalytic subunit of DNA Pol ${alpha}$ (Pol1p) by two-hybrid analysiscriteria. Point mutations in either CDC13 or POL1 not only reducethe interaction but also affect telomere length (64). Aphidicolin,an inhibitor of Pol ${alpha}$ and Pol ${delta}$ , causes abnormal lengthening ofthe G- and C-strand heterogeneity of telomeres of the ciliateEuplotes (28), and Euplotes telomerase has been shown to physicallyassociate with primase, a component of the lagging-strand machinery(65). Together, these studies suggest that telomere replicationby telomerase requires coordination of C- and G-strand syntheses.

Fission yeast is evolutionarily distant from budding yeast.The chromosome organizations of fission yeast, such as centromerestructure (18, 19), the replication origin structure (24), andchromosome segregation (5), are more similar to those of mammaliancells than are those of budding yeast. Much less is known aboutfission yeast telomeres than about their budding yeast counterparts.Fission yeast telomeres consist of about 300 bp of repeat units,which includes a consensus sequence of 5'-TTACAG_1-8-3' on thestrand that runs towards the 3' end (25, 68). To investigatewhether telomere length maintenance in fission yeast is similarto that in the evolutionarily distant budding yeast, we haveanalyzed the telomere length of a large panel of replicationmutants. We report here that telomere length is altered in allof the replication mutants analyzed. Using the principal initiationand lagging-strand synthesis DNA polymerase (Pol ${alpha}$ ) as a representativeof replication proteins, we show here that Pol ${alpha}$ and the telomerasecatalytic subunit, Trt1, coexist in a complex in vivo. Analysisof the telomere length and the Trt1 protein level in a pol ${alpha}$ mutantreveals a close physical association between the replicationcomplex and the telomerase complex in S. pombe. Mutations incomponents of the replication complex, such as Pol ${alpha}$ , could affectthe telomeric complex in maintaining telomere length and telomeraseprotein stability.

MATERIALS AND METHODS

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Materials and Methods

Fission yeast strains and growth conditions. All genetic operations were performed as described previously(34). To construct the spp2-9 trt1 ${Delta}$ double mutant strain, theCF248 diploid strain (h⁺/h^- leu1-32/leu1-32 ura4-D18/ura4-D18his3-D1/his3-D1 ade6-M210/ade6-M216 trt1⁺/trt1::his3⁺) was sporulated,and the spores were germinated on Edinburgh minimal medium (EMM)(50) lacking histidine to select for trt1::his3⁺. This strainwas crossed with an h^- spp2-9 strain (69), which had grown for100 generations, to generate the spp2-9 trt1 ${Delta}$ mutant. The spp2-9trt1 ${Delta}$ double mutant was verified by temperature sensitivity andits ability to grow on media lacking uracil and histidine. Tetradswere dissected and germinated on YES (yeast extract plus supplements).The pol ${alpha}$ ts13 strain [pKAN1-C-myc9trt1⁺ (kanMX Cmyc9trt1⁺)] (hereafterthe pol ${alpha}$ ts13 myc-trt1⁺ strain) was made by crossing the h⁺ pol ${alpha}$ ts13strain (13), which had been grown for 100 generations, and theCF830 (h^- leu1-32 ade6-M210 ura4-D18 his3-D1 trt1::his3⁺ [pKAN1-C-myc9trt1⁺(kanMX Cmyc9trt1⁺)] strain (36). The pol ${alpha}$ ts13 myc-trt1⁺ mutantstrain was verified by Western blotting with anti-myc antibody,temperature sensitivity, and Southern blot analysis of telomerelength. The pol ${alpha}$ ts13 ura-tel strain was constructed by crossingthe h^- pol ${alpha}$ ts13 strain, which had been grown for 100 generationsand the FY1872 strain (h⁹⁰ leu1-32 ade6-210 his3D1 ura4DS/Eade6OTRsph ura4-tel) and was verified by PCR with primers 5'-TGAGGGGATGAAAAATCCCATTG-3'and 5'-TTCGACAACAGGATTAC-GACCAG-3' directed against the ura4⁺gene.The pol ${alpha}$ ts13 strain [pKAN1-Cmyc9trt1⁺ (kanMX Cmyc9trt1⁺)] [pART-pol ${alpha}$ ⁺(ura4⁺ pol ${alpha}$ ⁺)] (hereafter the pol ${alpha}$ ts13 myc-trt1⁺ + pol ${alpha}$ ⁺ strain)was constructed by transforming the pol ${alpha}$ ts13 myc-trt1⁺ strainwith pART-pol ${alpha}$ ⁺, followed by selection for expression of pol ${alpha}$ ⁺by shifting the temperature to 36°C to rescue the temperaturesensitivity of the pol ${alpha}$ ts13 myc-trt1⁺ strain. The CF830 (pol ${alpha}$ ⁺myc-trt1⁺), pol ${alpha}$ ts13 myc-trt1⁺, and pol ${alpha}$ ts13 myc-trt1⁺ + pol ${alpha}$ ⁺strains were grown in yeast extract with G418 (0.1 mg/ml) at25°C to maintain the episomal plasmid pKAN1-C-myc9trt1⁺;all other strains were grown in YES at the indicated temperatures.

Detection of telomeres by Southern blotting. Genomic DNA was isolated by a glass bead-phenol protocol andanalyzed on agarose gels. Agarose gels were stained with ethidiumbromide (EtBr) and photographed to ensure that equal amountsof DNA were loaded. A 2.4-kb ApaI fragment of unique S. pombegenomic DNA (22) was also used as a loading control. Detectionof telomeres by Southern blotting was carried out as describedpreviously (22).

G-strand overhang assay. To detect G-strand overhangs, a nondenaturing hybridizationassay was used (72). Oligonucleotide probes 5'-GGGTTACAGGTTACAGGGTTAC-3'(G-specific oligonucleotide) and 5'-GTAACCCTGTAACCTGTAACCC-3'(C-specific oligonucleotide) were designed based on reference20. The C- and G-strand-specific probes were end labeled with[ ${gamma}$ -³²P]ATP (3,000 Ci/mmol; Amersham) by T4 polynucleotide kinaseto identical specific activities (9,160 cpm/pmol for the C-strandprobe and 9,220 cpm/pmol for the G-strand probe). Labeled probeswere purified on MicroSpin G-25 columns (Amersham PharmaciaBiotech). Four microliters of labeled probe (8 nM) was usedfor each reaction to hybridize to 5 µg of ApaI-restrictedgenomic DNA (from cells grown for 100 generations). The labeledprobes were added to a final volume of 25 µl and thenincubated for 12 to 15 h at 50°C. Hybridized samples wereseparated on a 1.5% agarose gel, stained with EtBr to ensureequal loading, dried, and autoradiographed.

Preparation of protein extracts from S. pombe. A total of 3 x 10⁸ cells were harvested, washed once in ice-coldstop buffer (150 mM NaCl, 50 mM NaF, 10 mM EDTA [pH 8.0], 1mM NaN₃), and then resuspended in 25 µl of lysis buffer(150 mM HEPES [pH 7.9], 300 mM KCl, 1 mM EDTA, 10% glycerol),with the addition of 1 tablet of Roche Complete Protease InhibitorCocktail per 25 ml of buffer, and lysed by vortexing with glassbeads. Soluble protein extracts were prepared by centrifugationat 4°C for 10 min, and the protein concentration was determinedby the Bradford assay.

Immunoprecipitation and Western blotting. Five hundred micrograms to 1 mg of total protein was solubilzedin 1 ml of lysis buffer containing 0.2% RNase inhibitor RNasin(Promega) and 0.1% NP-40. Anti-c-myc (9E10) antibody was preadsorbedonto protein G plus/protein A agarose beads (Oncogene Research).Affinity-purified anti-Pol ${alpha}$ antibody (61) was conjugated withCNBr-activated Sepharose beads according to the manufacturer'sinstructions. Ten microliters of a 50% slurry of antibody-labeledbeads was added to the protein extract, and this mixture wasincubated for 2 to 3 h at 4°C with end-over-end rotation.Immunocomplexes were washed four times with lysis buffer andresuspended in 30 µl of sodium dodecyl sulfate (SDS)-samplebuffer. Ten microliters was analyzed on SDS-polyacrylamide gelelectrophoresis (PAGE) gels (8% polyacrylamide) and then transferredto polyvinylidene difluoride membranes. The membrane was blockedin 5% dry milk powder in phosphate-buffered saline (PBS) and0.2% Tween 20. Anti-Pol ${alpha}$ antibody at a 1:4,000 dilution, anti-mycantibody at a 1:500 dilution, and antitubulin antibody at a1:5,000 dilution were used as the primary antibodies. A rabbitanti-chicken secondary antibody at a 1:1,000 dilution was usedfor detecting anti-Pol ${alpha}$ immunoglobulin Y (IgY), and a goat anti-mousesecondary antibody at a 1:3,000 dilution was used for detectinganti-myc. Proteins were detected by the ECL enhanced chemiluminescencesystem (New England Nuclear). Five milligrams of EtBr per mlwas added to the myc-trt1 cell extracts prior to immunoprecipitation.

Cell synchronization. The myc-trt1⁺ strain was grown at 25°C to a cell densityof 6 x 10⁶ cells per ml before hydroxyurea (HU) was added toa final concentration of 12 mM. The cells were incubated inHU for 4 h and then released into media without HU. Cell samplesof 50 ml were taken every 15 min. One milliliter of the cellsample was fixed with ethanol. A total of 3 x 10⁶ fixed cellswere stained with propidium iodide for flow cytometry analysis(62). The rest of the cells (49 ml) were used for protein extractpreparation.

Telomere position effect. The pol ${alpha}$ ⁺ (ura4-tel) (FY1872), pol ${alpha}$ ts13 (ura4-tel), and pol ${alpha}$ ts13(ura4^-) strains were grown to 5 x 10⁶ cells per ml at 25°C.Serial dilutions were applied as spots to PM + adenine-histidine-leucine-uracil(AHLU) (complete), PM + AHL (ura^-), and PM + AHLU + 5-fluorooroticacid (5-FOA) plates and grown at 25°C for 4 days or untilcolonies appeared.

Ectopic expression of pol ${alpha}$ ⁺. Full-length pol ${alpha}$ ⁺ was expressed from pART-pol ${alpha}$ ⁺ in the pol ${alpha}$ ts13myc-trt1⁺ strain. The myc-trt1⁺ (CF830), pol ${alpha}$ ts13 myc-trt1, andpol ${alpha}$ ts13 myc-trt1⁺ + pol ${alpha}$ ⁺strains were grown at 25°C to 6x 10⁶ cells per ml, and the cultures were then divided intotwo halves: one-half was shifted to 36°C to select for cellssustained by pART-pol ${alpha}$ ⁺, while the other half continued culturingat 25°C. Cell samples were taken at 6 and 15 h after shiftingthe temperature to 36°C for protein extract and DNA preparation.Western blotting and telomere length analyses were performedas described above.

RESULTS

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Results

Telomere length in fission yeast replication mutants is deregulated. We analyzed the telomere length of 20 mutant alleles of replicationgenes that are involved in DNA replication. Since there is atypical phenotypic lag of telomere length regulation (22, 45),each mutant strain was grown for $~$ 100 generations at its respectivesemipermissive temperature in liquid media. Many of the mutantstrains analyzed in the experiments had been propagated formany generations prior to this study, and consequently theyhad already reached their steady-state telomere lengths before100 generations of subculture. We chose to routinely analyzestrains after 100 generations of subculture and compared themto their respective isogenic parental strains. We first analyzedmutants with mutation of primase and pol ${alpha}$ that are essentialfor initiation of Okazaki fragments. All five mutants with mutationof the primase catalytic subunit, spp1 (33), exhibited an averageof 400- to 600-bp extensions of telomeres compared to theirparental wild-type strains (Fig. 1A, compare lanes 2 to 6 tolane 1). Out of these spp1 mutants, the spp1-9 mutant had thelongest telomeres (Fig. 1A, lane 3). The two pol ${alpha}$ mutants (13)displayed differences in their telomere length after subculturefor 100 generations at 25°C. The pol ${alpha}$ ts11 mutant had telomeresabout 270 bp longer than those of the wild type (Fig. 1A, lane7), while the pol ${alpha}$ ts13 mutant had a majority of telomeres 550bp longer than those of the wild type (Fig. 1A, compare lanes1 and 8). Longer exposure of the gel revealed that a minor populationof the telomeres in the pol ${alpha}$ ts13 strain were $~$ 1,600 bp longerthan wild-type telomeres (appearing as a weak smear in Fig.1A, lane 8). Of the three spp2 mutants, the mutant containingthe gene encoding the primase coupling subunit, spp2-9 (69),exhibited the longest telomeres: $~$ 300 bp longer than those ofthe wild type (Fig. 1B, lane 4). These results show that theextent of telomere lengthening in primase and pol ${alpha}$ mutants isallele dependent.

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FIG. 1. Analysis of telomere length in pol ${alpha}$ and primase mutants. pol ${alpha}$ and spp1 and spp2 (genes coding for S. pombe primase subunits 1 and 2, respectively) mutants were subcultured for $~$ 100 generations at their respective semipermissive temperature (pol ${alpha}$ and spp1 mutants, 25°C; spp2 mutants, 33°C) before harvest. Genomic DNA was restricted with ApaI and separated on 1.2% agarose gels, transferred to Hybond N⁺ filters, and probed with a 1.9-kb ApaI fragment of pEN42 (55). The positions of DNA size markers and their sizes in base pairs are given on the left. Brackets indicate the weak smear seen in the pol ${alpha}$ ts13 mutant. TR, terminal telomeric repeats; TAR, telomere-associated repeat sequences. (A) Lanes: 1, KG2 (wild type); 2, spp1-4; 3, spp1-9; 4, spp1-14; 5, spp1-19; 6, spp1-21; 7, pol ${alpha}$ ts11; 8, pol ${alpha}$ ts13. (B) Lanes: 1, KG2 (wild type); 2, spp2-7; 3, spp2-8; 4, spp2-9.

To test whether the aberrant lengthening of telomeres also occursin replication mutants that are required for elongation of Okazakifragments and leading strand synthesis, we analyzed the telomerelengths of five pol ${delta}$ mutants (30, 58). All five pol ${delta}$ mutants hadon average 100- to 150-bp-longer telomeres than their respectiveisogenic wild-type strains (Fig. 2A, lanes 3 to 7). Furthermore,mutants with defects in two small subunits of pol ${delta}$ , cdc1 andcdc27 (46), exhibited telomere lengths about 180 bp longer thanthose of the wild type (Fig. 2B, lanes 2 and 3). Similar topol ${alpha}$ and pol ${delta}$ mutants, a mutant with a ligase mutation, cdc17-K42,had telomeres about 160 bp longer than those of the wild-typestrain (Fig. 2B, lane 4). Although the exact role of Pol ${varepsilon}$ inreplication is not yet clear, similar to a recent observationin S. cerevisiae (59), a pol ${varepsilon}$ mutant, the cdc20-M10 strain, exhibitedshorter telomeres than the wild-type strain (Fig. 2B, lane 5).A strain with deletion of rad2, encoding a nuclease requiredfor Okazaki fragment maturation (73), also had shorter telomeresthan those of the wild-type strain (Fig. 2B, lane 6). Thus,mutations of genes encoding fission yeast replication proteinsinduce deregulation of telomere length.

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FIG. 2. Analysis of telomere length in replication mutants. Mutants with mutation of pol ${delta}$ subunits (cdc1 and cdc27), ligase (cdc17), pol ${varepsilon}$ (cdc20), and rad2 were grown at their respective semipermissive temperatures (indicated in parentheses after each mutant) for $~$ 100 generations. DNA was analyzed as described in the legend to Fig. 1. (A) Lanes: 1, 972 (wild type); 2, SP808 (wild type); 3, pol ${delta}$ ts1 (25°C); 4, pol ${delta}$ ts2 (25°C); 5, pol ${delta}$ ts3 (30°C); 6, cdc6-23 (25°C); 7, cdc6-121 (30°C); 8, SP808. (B) Lanes: 1, 972 (wild type); 2, cdc1-7 (25°C); 3, cdc27-K3 (30°C); 4, cdc17-K42 (30°C); 5, cdc20-M10 (33°C); 6, rad2.d (30°C); 7, 972 (wild type).

Since the two primase subunit spp1-9 and spp2-9 mutants andthe pol ${alpha}$ and pol ${alpha}$ ts13 mutants exhibited substantially longer telomeresthan the wild type, we further analyzed whether the telomericextension in these mutant strains is a G- or C-strand extensionas described in Materials and Methods. With C- or G-strand-specificprobes of identical radioactive specific activities, the resultssuggest that the telomere elongation seen in these replicationmutants is an extension of the G strand (data not shown).

Mutation at a pol ${alpha}$ mutant allele, pol ${alpha}$ ts13, affects the silencing at telomere loci. It is known that mutation of genes that causes a telomere lengthphenotype could also affect the TPE. In S. cerevisiae, the pol1-17allele causes elongation of telomeres and reduces the TPE (2).We therefore investigated the possible effect of the pol ${alpha}$ ts13,spp1-9, and spp2-9 mutations on silencing at the telomere locus.The pol ${alpha}$ ts13 ura4-tel strain with the ura4⁺ gene placed at atelomere locus was constructed as described in Materials andMethods. The pol ${alpha}$ ts13 ura4-tel mutant was able to grow on medialacking uracil, indicating a slight reduction of silencing atthe telomere locus (Fig. 3). This result corroborates a recentreport that showed an interaction between Pol ${alpha}$ and Swi6, an elementrequired for silencing. In a thermosensitive pol ${alpha}$ mutant (theswi7-H4 strain), the interaction was impaired and the silencingwas defective (4). In contrast, the spp1-9 and spp2-9 mutationsdo not affect the TPE (data not shown).

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FIG. 3. The telomere position effect is slightly reduced in the pol ${alpha}$ ts13 mutant. Shown is the organization of the telomere region on chromosome II where the ura4⁺ marker is inserted between the telomere repeats (TR) and the telomere-associated repeat sequences (TAS). Serial dilutions of the pol ${alpha}$ ⁺ ura4-tel, pol ${alpha}$ ts13 ura4-tel, and pol ${alpha}$ ts13 ura4^- mutants were applied as spots on complete, ura^- and FOA plates.

The telomere elongation in the pol ${alpha}$ and primase mutants is telomerase dependent. We then investigated whether the telomere length extension occurringin these replication mutants is mediated by telomerase. Attemptswere made to generate pol ${alpha}$ ts13 (13) or primase subunit spp1-9(33) and spp2-9 (69) double mutants in the telomerase catalyticsubunit (trt1 ${Delta}$ ) deletion background (16, 52, 53) as describedin Materials and Methods. However, both random sporulation andcrossing followed by tetrad analysis failed to generate spp1-9trt1 ${Delta}$ or pol ${alpha}$ ts13 trt1 ${Delta}$ double mutants. Mating of the trt1 ${Delta}$ strainwith either the spp1-9 or pol ${alpha}$ ts13 mutant strain resulted inone to two spores per tetrad, with none of them being the desireddouble mutant. Hence, both the spp1-9 and pol ${alpha}$ ts13 strains havea strong synthetic interaction with deletion of trt1⁺. In contrast,the spp2-9 trt1 ${Delta}$ double mutant was successfully generated. Themutant spp2-9 trt1 ${Delta}$ double mutant was cultured in liquid mediumat the semipermissive temperature (33°C) up to 100 generations,and its telomere length was compared to the telomere lengthsof the wild type and the spp2-9 single mutant. The telomerelength of the spp2-9 single mutant after subculture for 100generations was about 300 bp longer than that of the wild type(Fig. 4, compare lane 2 to lane 1). After growing for 30, 50,70, and 100 generations, the spp2-9 trt1 ${Delta}$ mutant exhibited progressivelyshorter telomere lengths (Fig. 4, lanes 3 through 6). After100 generations of growth, the telomere length was even shorterthan that of the parental wild-type strain (Fig. 4, comparelane 6 to lanes 1 and 7). This result strongly suggests thatthe telomere lengthening seen in the spp2-9 mutant requiresthe action of telomerase. By extending the interpretation ofour results, the telomere lengthening in the pol ${alpha}$ and primasemutants may be also mediated by telomerase.

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FIG. 4. Telomere shortening in an spp2-9 trt1::his3⁺ double mutant is telomerase dependent. The spp2-9 mutant cells (grown for $~$ 100 generations at the semipermissive temperature [33°C) were crossed with a haploid trt1::his3⁺ strain as described in Materials and Methods. An spp2-9 trt1::his3⁺ colony was grown in liquid medium until the total number of progeny reached 5 x 10⁸ cells. Assuming cell death to be insignificant, this is equivalent to $~$ 30 cell divisions since the meiotic segregation. Numbers in parentheses in lanes 3 through 6 correspond to the number of cell divisions after the meiotic segregation. Lanes: 1, 972 strain (wild type); 2, spp2-9 (100 generations); 3, spp2-9 trt1::his3⁺, 30 generations; 4, 50 generations; 5, 70 generations; 6, 100 generations; 7, 972 strain (wild type).

Pol ${alpha}$ and Trt1 physically interact in vivo. Studies of S. cerevisiae and Euplotes have suggested a coordinationof replication of the two strands at the telomere (23, 28, 47,65). Our results indicate that Trt1 is responsible for the aberranttelomere extension in the pol ${alpha}$ and primase mutants (Fig. 4).Furthermore, pol ${alpha}$ ts13 and trt1 ${Delta}$ strains showed a strong syntheticphenotype. These data bring to mind a physical interaction betweenPol ${alpha}$ and telomerase. To test this possibility, coimmunoprecipitationexperiments were performed with a myc-tagged trt1⁺ strain ectopicallyexpressed at a moderate level from its own promoter in a trt1 ${Delta}$ background (CF830) (36), and a wild-type nontagged trt1 strainas a negative control. An $~$ 130-kDa myc-tagged Trt1 protein, butno protein of the size of Pol ${alpha}$ , was detected when cell extractsprepared from the myc-trt1⁺ strain were probed with the anti-mycmonoclonal antibody 9E10 (Fig. 5A, lane 1). When the same cellextract was probed with polyclonal anti-Pol ${alpha}$ antibody (61), threeprotein species of Pol ${alpha}$ were detected—a major band of 180kDa and minor amounts of 165 and 155 kDa, which are degradedforms of Pol ${alpha}$ (61): however, no protein band of the same sizeas myc-tagged Trt1 was detected (Fig. 5A, lane 2). These resultsindicate that the anti-myc and anti-Pol ${alpha}$ antibodies do not cross-reactwith Pol ${alpha}$ and myc-Trt1, respectively.

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FIG. 5. Coimmunoprecipitation of myc-Trt1 and Pol ${alpha}$ . (A) Antibodies against Pol ${alpha}$ and c-myc do not cross-react. Protein extracts from the myc-trt1⁺ strain CF830 were Western blotted with anti-c-myc antibody (lane 1) and anti-Pol ${alpha}$ antibody (lane 2). (B) Coimmunoprecipitation of myc-Trt1 and Pol ${alpha}$ . Protein extracts from the myc-trt1⁺ strain and the nontagged wild-type strain (WT) were immunoprecipitated with anti-Pol ${alpha}$ antibody or anti-c-myc antibody, followed by transfer to membranes. Membranes were probed with either anti-c-myc antibody (lanes 1 through 4) or anti-Pol ${alpha}$ antibody (lanes 5 through 8). (C) Coimmunoprecipitation of Trt1 and Pol ${alpha}$ is not due to association with nucleic acid. EtBr (+EtBr) was added to the myc-trt1⁺ extract at 5 mg/ml prior to immunoprecipitation (lanes 3 and 4). EtBr-treated extracts and nontreated extracts (-EtBr) were immunoprecipitated with anti-Pol ${alpha}$ antibody or anti-c-myc antibody. The membrane was probed with anti-c-myc antibody.

When immunoprecipitating Pol ${alpha}$ with anti-Pol ${alpha}$ from myc-trt1⁺ cellextracts and probing with anti-myc antibodies, a myc-Trt1 proteinwas detected (Fig. 5B, lane 1). As a control, myc-Trt1 was detectedfrom the same cell extracts when immunoprecipitated and probedwith anti-myc antibodies (Fig. 5B, lane 2). No myc-Trt1 wasdetected in immunoprecipitates made from the nontagged wild-typestrain (Fig. 5B, lane 3 and 4). Reciprocally, Pol ${alpha}$ was detectedin the anti-myc immunoprecipitates from the myc-trt1⁺ strainwhen probed with anti-Pol ${alpha}$ antibody (Fig. 5B, lane 6). To verifythat this was indeed Pol ${alpha}$ , the anti-Pol ${alpha}$ immunoprecipitate fromthe same cell extract was probed with anti-Pol ${alpha}$ antibody. Itexhibited proteins of the size of Pol ${alpha}$ (Fig. 5B, lane 5). Anti-Pol ${alpha}$ -antibodiesdetected Pol ${alpha}$ in anti-Pol ${alpha}$ immunoprecipitates from lysates ofthe nontagged wild-type trt1 strain (Fig. 5B, lane 7), whereasno Pol ${alpha}$ could be detected in the anti-myc immunoprecipitatesfrom the same cell extract (Fig. 5B, lane 8). Thus, Trt1 andPol ${alpha}$ can be reciprocally coimmunoprecipitated by these antibodies.

To ascertain that the coimmunoprecipitation of Trt1 and Pol ${alpha}$ was not caused by a random nonspecific coassociation with DNAor RNA fragments in the cell extract, EtBr was added to themyc-trt1⁺ cell extract prior to immunoprecipitation. myc-Trt1was detected in the anti-Pol ${alpha}$ -immunoprecipitates from cell extractswith or without EtBr added (Fig. 5C, lanes 2 and 4). Thus, thecoimmunoprecipitation of Trt1 and Pol ${alpha}$ was not due to nonspecificrandom association of these proteins with nucleic acids in thecell extract. Together, these results strongly suggest thattelomerase and DNA Pol ${alpha}$ physically interact in vivo.

We then investigated whether the association of Trt1 and Pol ${alpha}$ is cell cycle dependent. The myc-trt1⁺ strain was arrested inearly S phase with HU at 25°C for 4 h and then releasedinto fresh medium to continue growing at 25°C. After releasefrom the HU arrest, the cells took approximately 60 min to recoverand progress through S phase. Cell samples were removed at 0,60, 75, and 120 min after release from the HU block for flowcytometry analysis (Fig. 6A) and for preparation of proteinextracts for coimmunoprecipitation experiments (Fig. 6B and C).Cells arrested in HU for 4 h at 25°C displayed a 1CDNA profile (Fig. 6A, t = 0 min). The cells gradually shiftedtowards a greater-than-1C fluorescence-activated cell sorter(FACS) profile, and after 120 min, cells had a 2C DNA profile,indicating that cells had completed S phase and entered theG₂ phase (t = 120 min). Since S. pombe has a very abbreviatedG₁ phase, no G₁-phase cells are observed in the 120-min cellsample. Throughout S phase and in G₂ phase, myc-Trt1 was detectablein the anti-Pol ${alpha}$ immunoprecipitates (Fig. 6B, lane 2) and theanti-myc immunoprecipitates (Fig. 6B, lane 3). Pol ${alpha}$ was effectivelydetected by the anti-Pol ${alpha}$ antibody in the anti-Pol ${alpha}$ immunoprecipitate(Fig. 6C, lane 2). Furthermore, Pol ${alpha}$ coimmunoprecipitated withmyc-Trt1 as cells progressed through S phase and entered theG₂ phase, although the level was low at t = 0 min (Fig. 6C,lane 3, t = 0, 60, 75, and 120 min). These experiments indicatethat Trt1 and Pol ${alpha}$ interact throughout S and G₂ phases of thecell cycle.

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FIG. 6. myc-Trt1 and Pol ${alpha}$ interact in the S and G₂ phases. The myc-trt1⁺ strain was incubated with HU for 4 h at 25°C, and the cells were then released from the HU block. (A) Flow cytometry analysis. Cell samples were removed every 15 min for FACS analysis and protein extract preparation. After 4 h of HU arrest, immediately before release, a cell sample was taken (t = 0). The cells at t = 0 have a 1C content. After 60 min (t = 60), the peak has started to move towards 2C content. After 75 min (t = 75), the cells have a 1.5C content. Finally, after 120 min, the cells have a 2C content corresponding to G₂-phase cells. (B) myc-Trt1 in Pol ${alpha}$ immunoprecipitates. Cell extracts were prepared from samples released from the HU block at the times indicated and subjected to Western blotting with anti-c-myc antibody (lane 1), immunoprecipitated with anti-Pol ${alpha}$ or anti-c-myc antibody, and probed with anti-c-myc antibody (lanes 2 and 3). (C) Pol ${alpha}$ in anti-myc immunoprecipitates. The study was performed as described for panel B, but the membrane was probed with anti-Pol ${alpha}$ antibody.

The interaction between Pol ${alpha}$ and Trt1 is compromised in a pol ${alpha}$ mutant exhibiting long telomeres. To test the status of interaction between Trt1 and Pol ${alpha}$ in apol ${alpha}$ mutant (the pol ${alpha}$ ts13 strain) that exhibited abnormally longtelomeres (Fig. 1A, lane 8), we constructed a strain containingthe pol ${alpha}$ ts13 allele and myc-trt1⁺ and analyzed the interactionof these two proteins. As expected, myc-Trt1 was detected inanti-myc immunoprecipitates (Fig. 7A, lane 1) and also coimmunoprecipitatedwith Pol ${alpha}$ (Fig. 7A, lane 2), and Pol ${alpha}$ was detected in anti-Pol ${alpha}$ immunoprecipitates from cell lysates of the myc-trt1⁺ (pol ${alpha}$ ⁺)strain (Fig. 7A, lane 3). In contrast, although myc-Trt1 wasimmunoprecipitated by identical amounts of anti-myc antibodyfrom myc-trt1⁺ (pol ${alpha}$ ⁺) (Fig. 7A, lane 1) and pol ${alpha}$ ts13 myc-trt1⁺cell extracts (Fig. 7B, lane 1), no detectable myc-Trt1 wasfound in the anti-Pol ${alpha}$ immunoprecipitates from the pol ${alpha}$ ts13 myc-trt1⁺cell extract (Fig. 7B, lane 2). This is not due to the failureof anti-Pol ${alpha}$ antibody to immunoprecipitate the mutant Pol ${alpha}$ protein,because mutant Pol ${alpha}$ protein was readily detectable in the anti-Pol ${alpha}$ immunoprecipitates from pol ${alpha}$ ts13 myc-trt1⁺ cell extract (Fig.7B, lane 3).

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FIG. 7. The interaction between myc-Trt1 and Pol ${alpha}$ is compromised in a pol ${alpha}$ ts13 background. The myc-trt1⁺ (pol ${alpha}$ ⁺) strain and the pol ${alpha}$ ts13 myc-trt1⁺ mutant strain were grown at 25°C. Cells were harvested, and protein extracts were prepared. Asterisks indicate three unrelated bands, which were used as loading controls. (A) Coimmunoprecipitation of myc-Trt1 from myc-trt1⁺ (pol ${alpha}$ ⁺) cells. Extracts from the myc-trt1⁺ cells were immunoprecipitated (IP) with anti-c-myc antibody (lane 1) or anti-Pol ${alpha}$ antibody (lanes 2 and 3) and probed with anti-c-myc antibody. (B) Coimmunoprecipitation of myc-Trt1 from pol ${alpha}$ ts13 myc-trt1⁺ cells. Extracts from pol ${alpha}$ ts13 myc-trt1⁺ cells were immunoprecipitated with anti-c-myc antibody (lane 1) or anti-Pol ${alpha}$ antibody (lanes 2 and 3) and probed with anti-c-myc antibody. (C) Comparison of the myc-Trt1 and Pol ${alpha}$ protein levels and their efficiency at coimmunoprecipitation in myc-trt1⁺ (pol ${alpha}$ ⁺) and pol ${alpha}$ ts13 myc-trt1⁺ cells. Cell extracts were diluted to 1, 0.5, 0.25, and 0.1 mg of total protein extract of myc-trt1⁺ (pol ${alpha}$ ⁺) and pol ${alpha}$ ts13 myc-trt1⁺ cells. One percent of each diluted extract was loaded onto SDS gels followed by Western blotting with anti-Pol ${alpha}$ antibody to determine the level of Pol ${alpha}$ proteins or anti-myc antibody to determine the level of myc-Trt1 protein. Pol ${alpha}$ proteins were immunoprecipitated with anti-Pol ${alpha}$ antibody from the same diluted extracts as in the three top lanes: myc-trt1⁺ (pol ${alpha}$ ⁺) and pol ${alpha}$ ts13 myc-trt1⁺. Ten microliters of anti-Pol ${alpha}$ immunoprecipitates was loaded onto SDS gel followed by Western blotting with anti-Pol ${alpha}$ antibody to determine the levels of Pol ${alpha}$ protein and with anti-myc antibody for coimmunoprecipitation of Pol ${alpha}$ and myc-Trt1.

To investigate why the coimmunoprecipitation of myc-Trt1 wascompromised in the pol ${alpha}$ ts13 myc-trt1⁺ mutant, we tested whetherprotein levels of Pol ${alpha}$ or myc-Trt1 were altered in the pol ${alpha}$ ts13myc-trt1⁺ mutant. Cell extracts of 0.1 to 1 mg of protein fromthe myc-trt1⁺ (pol ${alpha}$ ⁺) and pol ${alpha}$ ts13 myc-trt1⁺ strains were Westernblotted to determine the levels of the Pol ${alpha}$ and myc-Trt1 proteins(Fig. 7C, top two panels). Comparable levels of Pol ${alpha}$ proteinwere present in both myc-trt1⁺ (pol ${alpha}$ ⁺) cells and the pol ${alpha}$ ts13myc-trt1⁺ mutant (Fig. 7C, first panel). Surprisingly, the levelof myc-Trt1 protein was severely reduced in the pol ${alpha}$ ts13 myc-trt1⁺mutant (Fig. 7C, second panel). The level of myc-Trt1 in pol ${alpha}$ ts13myc-trt1⁺ mutant cell extract was about 10% of the myc-Trt1level in the myc-trt1⁺ (pol ${alpha}$ ⁺) cell extract: compare the myc-Trt1protein level in 0.1 mg of myc-trt1⁺ (pol ${alpha}$ ⁺) cell extract withthe myc-Trt1 protein level in 1 mg of pol ${alpha}$ ts13 myc-trt1⁺ cellextract. We then analyzed the interaction of myc-Trt1 and Pol ${alpha}$ in these cell extracts. With comparable levels of Pol ${alpha}$ proteinimmunoprecipitated from myc-trt1⁺ (pol ${alpha}$ ⁺) and pol ${alpha}$ ts13 myc-trt1⁺cells, myc-Trt1 was detectable in the anti-Pol ${alpha}$ immunoprecipitatesfrom myc-trt1⁺ (pol ${alpha}$ ⁺) cell extracts, but not in pol ${alpha}$ ts13 myc-trt1⁺extracts. Furthermore, with comparable levels of myc-Trt1 presentedin 0.1 mg of myc-trt1⁺ (pol ${alpha}$ ⁺) extract and in 1 mg of pol ${alpha}$ ts13myc-trt1⁺ cell extract, myc-Trt1 protein was again detectableonly in the anti-Pol ${alpha}$ -immunoprecipitates from 0.1 mg of myc-trt1⁺(pol ${alpha}$ ⁺) cell extract, but not from 1 mg of pol ${alpha}$ ts13 myc-trt1⁺cell extract (Fig. 7C, panels 4 and 5). Moreover, although 1mg of pol ${alpha}$ ts13 myc-trt1⁺ cell extract had a much higher levelof Pol ${alpha}$ protein than 0.1 mg of myc-trt1⁺ (pol ${alpha}$ ⁺) cell extract(Fig. 7C, panel 1), myc-Trt1 was only detectable in the anti-Pol ${alpha}$ -immunoprecipitatesfrom myc-trt1⁺ (pol ${alpha}$ ⁺) cell extract and not in those from pol ${alpha}$ ts13myc-trt1⁺ extract. These results suggest that the pol ${alpha}$ ts13 mutationnot only causes an abnormal telomere lengthening, but also severelyreduces the cellular Trt1 protein level and compromises theability of Trt1 and Pol ${alpha}$ to physically coexist as a complex invivo.

Ectopic expression of pol ${alpha}$ ⁺ restores myc-Trt1 level and shortens telomeres in the pol ${alpha}$ ts13 mutant. As shown above in Fig. 7C, the myc-Trt1 protein level in thepol ${alpha}$ ts13 myc-trt1⁺ mutant strain was significantly lower thanthat in the myc-trt1 (pol ${alpha}$ ⁺) strain. It is possible that themutant Pol ${alpha}$ in the pol ${alpha}$ ts13 strain might affect the stabilityof Trt1 in the Pol ${alpha}$ -Trt1-containing protein complex, causingTrt1 to become unstable. To test this hypothesis, we transformedpART-pol ${alpha}$ ⁺ into the pol ${alpha}$ ts13 myc-trt1⁺ strain (hereafter the pol ${alpha}$ ts13myc-trt1⁺ + pol ${alpha}$ ⁺ strain) to express the wild-type pol ${alpha}$ gene.The myc-trt1⁺(pol ${alpha}$ ⁺), pol ${alpha}$ ts13 myc-trt1⁺, and pol ${alpha}$ ts13 myc-trt1⁺+ pol ${alpha}$ ⁺ strains were cultured at 25°C to early log phaseand then shifted to 36°C for 6 h to select for cells sustainedby the ectopic expression of pol ${alpha}$ ⁺. At 25°C, when there isno selection for pART-pol ${alpha}$ ⁺, most cells have probably lost theplasmid, and the myc-Trt1 level is comparable to that of pol ${alpha}$ ts13myc-trt1⁺ strain (Fig. 8A, lane 2 and 3). The pol ${alpha}$ ts13 myc-trt1⁺strain without the ectopic expression of pol ${alpha}$ ⁺ is viable at 36°Cfor 6 h, but then starts to gradually lose viability, whereasthe pol ${alpha}$ ts13 myc-trt1⁺ strain transformed with pART-pol ${alpha}$ ⁺ is viable.After 6 h at 36°C, the myc-Trt1 level is increased in thepol ${alpha}$ ts13 myc-trt1⁺ + pol ${alpha}$ ⁺ strain compared to that in the pol ${alpha}$ ts13myc-trt1⁺ strain (Fig. 8A, compare lanes 5 and 6). After 15h at 36°C, the level of myc-Trt1 in the pol ${alpha}$ ts13 myc-trt1⁺+ pol ${alpha}$ ⁺ strain is comparable to that of the myc-trt1⁺ (pol ${alpha}$ ⁺)strain expressing wild-type Pol ${alpha}$ (Fig. 8B, compare lanes 1 and2), with equal amounts of protein extracts analyzed, as shownby the tubulin levels used as the control. These experimentsindicate that ectopic expression of pol ${alpha}$ ⁺ is able to restorethe myc-Trt1 level in the pol ${alpha}$ ts13 myc-trt1⁺ strain, suggestingthat Pol ${alpha}$ affects the physical status of Trt1.

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FIG. 8. Ectopic expression of pol ${alpha}$ ⁺ in the pol ${alpha}$ ts13 mutant can restore the myc-Trt1 protein and telomere length to near-wild-type level. (A) Ectopic expression of pol ${alpha}$ ⁺ increases the cellular myc-Trt1 protein in pol ${alpha}$ ts13 cells. Prior to the experiment, the pol ${alpha}$ s13 myc-trt1⁺ + pol ${alpha}$ ⁺ (pol ${alpha}$ ts13 + pol ${alpha}$ ⁺) transformants had been grown at 36°C to select for expression of pol ${alpha}$ ⁺. The myc-trt1⁺ (pol ${alpha}$ ⁺), pol ${alpha}$ ts13 myc-trt1⁺ (pol ${alpha}$ ts13), and pol ${alpha}$ s13 myc-trt1⁺ + pol ${alpha}$ ⁺ (pol ${alpha}$ ts13 + pol ${alpha}$ ⁺) strains were grown at 25°C to a cell density of 6 x 10⁶ cells per ml (lanes 1 through 3) and then shifted to 36°C and continued to culture for 6 h (lanes 4 through 6). (B) Ectopic expression of pol ${alpha}$ ⁺ restores myc-Trt1 to the wild-type level. The myc-trt1⁺ (pol ${alpha}$ ⁺) and pol ${alpha}$ s13 myc-trt1⁺ + pol ${alpha}$ ⁺ (pol ${alpha}$ ts13 + pol ${alpha}$ ⁺) cells were grown at 36°C for 15 h, and cell extracts were analyzed as described for panel A. (C) Ectopic expression of pol ${alpha}$ ⁺ reduces telomere length in the pol ${alpha}$ ts13 mutant. DNA was isolated from the same cell extracts as in panels A and B for analysis of the telomere lengths by Southern blotting. Lanes: 1, KG2 (wild type); 2, myc-trt1⁺ (pol ${alpha}$ ⁺) (25°C); 3, pol ${alpha}$ ts13 myc-trt1⁺ (pol ${alpha}$ ts13) (25°C); 4, pol ${alpha}$ s13 myc-trt1⁺ + pol ${alpha}$ ⁺ (pol ${alpha}$ ts13 + pol ${alpha}$ ⁺) (first selected at 36°C and then shifted to 25°C); 5, myc-trt1⁺ (pol ${alpha}$ ⁺) (36°C, 6 h); 6, pol ${alpha}$ ts13 myc-trt1⁺ (pol ${alpha}$ ts13) (36°C, 6 h); 7, pol ${alpha}$ s13 myc-trt1⁺ + pol ${alpha}$ ⁺ (pol ${alpha}$ ts13 + pol ${alpha}$ ⁺) (36°C, 6 h); 8, myc-trt1⁺ (pol ${alpha}$ ⁺) (36°C, 15 h); 9, pol ${alpha}$ s13 myc-trt1⁺ + pol ${alpha}$ ⁺ (pol ${alpha}$ ts13 + pol ${alpha}$ ⁺) (36°C, 15 h).

To test whether ectopic expression of pol ${alpha}$ ⁺ also affects thetelomere length in the pol ${alpha}$ ts13 myc-trt1⁺ strain, genomic DNAwas prepared from the same strains shown in Fig. 8A and B, andthe telomere lengths were analyzed by Southern blotting. Interestingly,ectopic expression of a functional pol ${alpha}$ ⁺ gene rapidly restoredthe telomere length to near-wild-type length in the pol ${alpha}$ ts13myctrt1⁺ mutant (Fig. 8C). These results indicate that Pol ${alpha}$ playsa significant role in maintaining the Trt1 protein level andalso the telomere length of cells, supporting the finding ofa close physical association between Pol ${alpha}$ and Trt1 in vivo.

DISCUSSION

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Discussion

In this study, we analyzed telomeres of a large panel of fissionyeast replication mutants and found telomere length abnormalitiesin all mutants analyzed (Fig. 1 and 2). Analysis of one of thereplication mutants, the primase spp2-9 mutant, shows that theabnormal telomere lengthening is telomerase dependent (Fig.4), inferring that this may also be the case for other replicationmutants. Importantly, we show that Pol ${alpha}$ associates with Trt1in a complex in vivo and that the status of Pol ${alpha}$ influences telomerelength as well as the cellular level of the Trt1 protein (Fig.5, 7, and 8). Below, we discuss these findings and their possibleeffects on the cell biology.

Do replication proteins of fission yeast and budding yeast affect telomere length differently? In this study, we analyzed 20 replication mutants for theirtelomere length (Fig. 1 and 2). Among these mutants analyzed,mutation of ligase (cdc17-K42) induces abnormal telomere lengthening(Fig. 2B, lane 4). In contrast to the fission yeast ligase mutant(cdc17-K42), an S. cerevisiae mutant with a ligase mutation(cdc9) has telomeres of wild-type length (2). In addition, deletionof fission yeast rad2 (S. pombe homolog of Fen1/RAD27), whichis essential for maturation of Okazaki fragments (9, 73), inducestelomere shortening (Fig. 2B, lane 6). In budding yeast, deletionof RAD27 (the homolog of S. pombe rad2⁺) causes destabilizationof telomeres but not a shortening of telomeres (60). The differencesseen in telomere length regulation of ligase and of rad2⁺/RAD27mutants between the two yeasts might simply be due to allelespecificity. On the other hand, they might reflect a differencein the effect of replication proteins on telomere length regulationbetween the two yeasts.

In budding yeast, telomerase-mediated telomere addition requiresPol ${alpha}$ , primase and Pol ${delta}$ , but not Pol ${varepsilon}$ (23). Although the exact roleof Pol ${varepsilon}$ in replication is not yet clear (9, 12, 73), mutationof pol ${varepsilon}$ (cdc20-M10) in both fission yeast and budding yeast,similar to fission yeast rad2, induced shortening of telomeres(Fig. 2B, lane 5) (59). This suggests that in fission yeast,pol ${varepsilon}$ ⁺ and rad2⁺ regulate telomere length through a differentmechanism from that of the pol ${alpha}$ ⁺, pol ${delta}$ ⁺, and ligase genes. SincePol ${alpha}$ , primase, and Pol ${delta}$ are all thought to be involved in lagging-strandsynthesis, a different kind of telomere length regulation seenin the pol ${varepsilon}$ mutant may reflect a unique and distinct role ofPol ${varepsilon}$ in replication.

How might the replication complex influence the telomere homeostasis? Studies of telomeres in the ciliate Euplotes (28, 63, 65) andbudding yeast (1, 2, 15, 23, 27, 47, 64) have led to the proposalthat G-strand extension and C-strand synthesis require a coordinatedregulation. Since C-strand synthesis requires the lagging-strandreplication proteins, it is reasonable to assume that synthesisof the two telomeric strands also requires a tight coordinationof the lagging-strand replication complex and the telomerase-containingtelomeric complex (27). Studies of budding yeast have shownthat Pol1p (Pol ${alpha}$ ) interacts with Cdc13p by two-hybrid analysis;mutations in an N-terminal region of Pol1p abolish the interactionand result in longer telomeres (64). Cdc13p also interacts withEst1p (64), which also associates with the telomerase RNA (26,40, 56). This interaction chain implies that Cdc13p might recruitboth telomerase and Pol ${alpha}$ to the telomeres in budding yeast; henceCdc13p may be a key linchpin in telomere homeostasis.

In this study, we have shown that the Trt1 and Pol ${alpha}$ proteinscoexist in a complex in vivo (Fig. 5), and the physical and/orfunctional status of Pol ${alpha}$ significantly affects the telomeraseprotein stability and telomere length maintenance (Fig. 7 and8). It is not yet known whether the two proteins directly interactor whether other proteins mediate the interaction. One S. pombeprotein, Taz1, has been identified as a telomere binding protein(21, 29). Taz1 is an ortholog of human telomere repeat bindingfactors (TRFs). Taz1 is neither a structural nor a functionalhomolog of Cdc13p (20, 39). Another telomere end binding protein,Pot1, was recently identified in fission yeast and humans (10,11). It is not yet clear whether Pot1 is an ortholog of Cdc13and whether Pot1 is a linchpin of the interaction between thelagging strand replication complex component Pol ${alpha}$ and Trt1. Thus,the coordination of G- and C-strand syntheses in S. pombe maybe mediated by a yet-to-be-identified protein. It is also possiblethat the coordination of the synthesis of G and C strands infission yeast is by a direct interaction between the Pol ${alpha}$ -containinglagging-strand replication complex and the telomerase-containingtelomeric complex.

The pol ${alpha}$ ts13 mutant at 25°C has a growth rate similar tothat of the wild type; however, pol ${alpha}$ ts13 cells exhibit a slightcdc phenotype with a normal nuclear morphology (13), indicatingthat 25°C is the semipermissive temperature of this mutant.Moreover, at 25°C, the pol ${alpha}$ ts13 mutant exhibits an elevatedmutation rate (41); hence, at this temperature, Pol ${alpha}$ is semidysfunctional.Here we showed that at 25°C, the pol ${alpha}$ ts13 mutant allele inducedan aberrant lengthening of telomeres (Fig. 1), a mild reductionof TPE (Fig. 3), a severely compromised interaction with Trt1(Fig. 7B), and a significant decrease in the cellular Trt1 proteinlevel (Fig. 7B and C). It is possible that a semidysfunctionalPol ${alpha}$ adversely affects the coordination between the lagging strandreplication complex and the telomerase complex. The impairedphysical coordination between these two protein complexes wouldmake Trt1 prone to degradation, resulting in a decrease in thecellular Trt1 level, and a mild reduction in silencing at thetelomere loci due to a change of the heterochromatin structureat the telomere. In support of this hypothesis, ectopic expressionof wild-type pol ${alpha}$ at the restrictive temperature can progressivelyrestore the cellular level of Trt1 protein and reduce telomerelength to near-wild-type length (Fig. 8). However, the recoveryrates of Trt1 protein level and the telomere length inducedby ectopic expression of pol ${alpha}$ ⁺ in the pol ${alpha}$ ts13 are not perfectlycorrelated. This could be due to a delayed telomere effect.Furthermore, the telomere length also seemed to have reacheda new steady-state level, almost as short as the wild-type telomerelength (Fig. 8C). This telomere-shortening phenomenon was alreadyseen at the first time point after establishment of the pol ${alpha}$ ⁺transformants (Fig. 8C, lane 4) and is most likely due to thepreselection of pol ${alpha}$ ⁺ transformants at 36°C. The fact thattelomeres do not quite revert to wild-type length could alsobe due to an altered extension of heterochromatin in the regionnear the telomere during the previous phase when the telomereswere abnormally long. If this were the case, it would requiremany generations to restore the chromatin near telomeres tothe original state. Nonetheless, these results suggest thatthe Pol ${alpha}$ protein plays a significant role in maintaining telomericcomplex stability and telomere homeostasis.

There are two paradoxical findings in our studies. First, Pol ${alpha}$ and Trt1 interact in early S phase (Fig. 6). At this phase ofthe cell cycle, no telomere replication presumably takes place.Budding yeast telomere addition at the de novo end occurs inM phase, although telomerase activity can be measured in vitroin both G₁- and M-phase cells (23). The finding that Pol ${alpha}$ andTrt1 coimmunoprecipitate at low levels throughout S phase suggeststhat a fraction of Pol ${alpha}$ may constitutively associate with telomeraseduring the entire S phase. This also implies that another essentialfactor or factors required for the telomerase-mediated telomereaddition may be associating with telomerase in a cell cycle-dependentmanner (23).

The second paradox is how the pol ${alpha}$ ts13 myc-trt1⁺ mutant, witha significantly reduced level of Trt1 protein, can exhibit lengthenedtelomeres. A likely explanation is that cells might have excessamounts of potentially active telomerase, which is usually tightlyregulated by the coordination between the telomerase complex,the replication complex, and/or perhaps other proteins. In thepol ${alpha}$ ts13 mutant, a dysfunctional Pol ${alpha}$ might perturb the tightcoordination of these proteins in regulating the telomerase-mediatedtelomere addition. Furthermore, a perturbed telomeric proteincomplex in the pol ${alpha}$ ts13 mutant may allow the chromosome endsto be more accessible to extension by low levels of residualtelomerase in an aberrant manner (27). Thus, it is possiblethat the abnormal telomere lengthening observed in the pol ${alpha}$ ts13strain is a consequence of perturbation of the organizationof protein complexes at the telomere end.

Mutations of replication proteins not only contribute to telomere dysfunction but also promote genomic instability. Telomere dysfunction due to the absence of functional telomerasehas been shown to increase mutation rate, impair DNA repair,enhance ionizing radiation sensitivity, and induce genomic instabilityin tumorigenesis (7, 17, 35, 70, 76). We have previously reportedthat specific mutations of S. pombe pol ${alpha}$ , pol ${delta}$ , primase (spp1and spp2), and ligase (cdc17-K42) confer a mutator phenotypecharacterized by deletion of sequences flanked by short directrepeats and small sequence alterations (41). Deletion of thefission yeast rad2⁺ gene, similar to deletion of the homologousgene in budding yeast, RAD27 (71), induces duplication of sequencesflanked by short direct repeats (41). Comparison of the panelof replication mutant alleles that confer a mutator phenotypeto those that exhibit telomere length alterations shows thatall of the replication mutants (especially those implicatedin lagging-strand synthesis) that confer a mutator phenotypealso exhibit telomere length deregulation (41) (Fig. 1 and 2).However, some replication mutant alleles, such as those in thetwo pol ${delta}$ mutants (pol ${delta}$ ts2 and cdc6-23), and a pol ${varepsilon}$ mutant (cdc20-M10),exhibit deregulation of telomere length, but do not displaya mutator phenotype (41). These results suggest that the replicationgenes contribute to telomeric complex stability as well as genomicmutation avoidance in an allele-specific manner. The underlyingmechanisms in these mutants causing a mutator phenotype mayalso differ from those causing telomere deregulation. Studiesof budding yeast est1 ${Delta}$ cells have suggested that telomerase contributesto inhibition of chromosome instability (35). Studies of mammaliancells and rodent models without telomerase RNA have shown thattelomerase dysfunction in certain genetic contexts can facilitatecancer development by compromising chromosome integrity (6-8,17, 37, 76). In this study, we have shown that mutations ofreplication proteins can affect the homeostasis of telomeres.Using Pol ${alpha}$ as a paradigm, we demonstrate that mutation of Pol ${alpha}$ has a significant effect on telomere length maintenance andtelomerase protein levels. It is not yet known to what extentthe observed mutator phenotypes induced by replication mutantsare indirectly contributed by a telomere homeostasis abnormality.Nonetheless, the results of this study and our previous studies(41) suggest that mutations of replication genes, especiallythose whose products are involved in the lagging-strand synthesis,could have a major impact on the overall genomic stability.

ACKNOWLEDGMENTS

We thank members of the Wang laboratory for helpful discussionand Thomas R. Cech for providing us the CF830 and CF248 strainsand Robin Allshire for providing the FY1872 strain. We especiallythank Toru Nakamura, Peter Baumann, and Steven Artandi for valuableadvice during the course of the study.

This study was supported by grants from National Cancer Instituteof the National Institutes of Health to T.S.W and grants fromthe Swedish Cancer Fund (2163-B00-11XAC) and the Swedish ResearchCouncil (K2002-31X-14197-01A) to P.S. M.D. was a predoctoralfellow supported by the Sweden-America Foundation, the SwedishInstitute, and Lennander's Foundation.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5324. Phone: (650) 725-4907. Fax: (650) 725-6902. E-mail: twang{at}cmgm.stanford.edu.

REFERENCES

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