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The Catalytic Subunit of Yeast Telomerase (Telomeres͞reverse Transcriptase)

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The Catalytic Subunit of Yeast Telomerase (Telomeres͞reverse Transcriptase) Proc. Natl. Acad. Sci. USA Vol. 94, pp. 9202–9207, August 1997 Genetics The catalytic subunit of yeast telomerase (telomeresyreverse transcriptase) CHRISTOPHER M. COUNTER*†,MATTHEW MEYERSON*†‡,ELINOR NG EATON*, AND ROBERT A. WEINBERG*§ *Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142; and ‡Department of Pathology, Massachusetts General Hospital, Boston, MA 02114 Contributed by Robert A. Weinberg, June 16, 1997 ABSTRACT Telomerase is an RNA-directed DNA poly- mena telomerase and have been found to associate with either merase, composed of RNA and protein subunits, that repli- enzyme activity or the RNA component (15). A mammalian cates the telomere ends of linear eukaryotic chromosomes. homolog of p80, TP1yTLP1, has now been cloned and shown Using a genetic strategy described here, we identify the to be physically associated with telomerase activity; however, product of the EST2 gene, Est2p, as a subunit of telomerase in the expression of TP1yTLP1 in different tissues does not the yeast Saccharomyces cerevisiae. Est2p is required for en- correlate with activation of the enzyme (16, 17). Additionally, zyme catalysis, as mutations in EST2 were found to result in it is unknown whether any of these proteins are essential for the absence of telomerase activity. Immunochemical experi- enzyme function. ments show that Est2p is an integral subunit of the telomerase Surprisingly, there are no obvious sequences homologous to enzyme. Critical catalytic residues present in RNA-directed p80 or p95 encoded within the one eukaryotic genome that has DNA polymerases are conserved in Est2p; mutation of one been entirely sequenced, that of the yeast Saccharomyces such residue abolishes telomerase activity, suggesting a direct cerevisiae (18). Because the telomerase enzyme of yeast re- catalytic role for Est2p. quires both RNA and protein components (19, 20), either yeast possess telomerase protein subunit(s) that are unrelated to, or Telomeres are DNA–protein complexes that cap the ends of have diverged from, p95 and p80, or the core components of eukaryotic chromosomes and maintain chromosome stability. telomerase remain to be identified in Tetrahymena. Tandem arrays of simple guanine-rich repeat sequences con- Although the RNA subunit of yeast telomerase is known to stitute the telomeric DNA (1, 2). In most eukaryotes, these be encoded by the gene TLC1 (20) and is essential for telomeric repeats are synthesized by telomerase, an enzyme telomerase catalysis (19), no yeast protein has heretofore been composed of one or more protein subunits and an RNA found to be essential for telomerase enzyme activity. TLC1 subunit that provides the template for telomeric DNA synthe- mutants (20) exhibit two phenotypes that are shared by sis (3–5). Thus, telomerase is an RNA-directed DNA poly- mutants of the genes EST1 (21) and CDC13 (22–24): a gradual merase, or reverse transcriptase, that includes the RNA tem- decrease in telomere length and a consequent decline in cell plate as a subunit. viability. However, EST1 is not essential for telomerase activity Interest in telomeres and telomerase has heightened in (19), and mutational analysis of CDC13 suggest that the recent years with the discovery that telomerase is present at product of this gene may function as a telomere capping low or undetectable levels in most human somatic tissues while protein (22). Therefore, despite the similar mutant phenotype being readily detectable in germ-line cells and in the vast of EST1 and CDC13, these genes do not appear to encode majority of tumor cell samples (6, 7). Somatic cell lineages that essential components of the telomerase enzyme. A third ORF lack telomerase lose telomere segments progressively as they that exhibits a similar mutant phenotype, EST2 (ever shorter pass through successive replication cycles (8, 9), ultimately telomeres gene 2), was recently identified (25). We and others limiting their lifespan. Conversely, cell populations that res- (26) now show that this gene encodes the catalytic subunit of urrect telomerase expression ensure maintenance of telomere telomerase. length, thereby removing a barrier to their further unlimited replication and resulting in the process termed immortaliza- MATERIALS AND METHODS tion (10, 11). These observations have led to a model which proposes that the process of telomere shortening limits the Yeast Strains. Strain Y0025, in which RAD52 gene expres- replicative potential of most human somatic cell lineages and sion could be regulated, was constructed by replacing the that cancer cells overcome this limitation by activating telom- RAD52 gene of strain L3853 (a gift of G. Fink of the erase expression during the course of tumor progression (10, Whitehead Institute; MATa, ura3–52, lys2-D201, leu2–3,112, 11). trp1-D1, his3-D200) with HIS3 by homologous recombination Validation of this model has been held back by the difficul- and introducing the plasmid pYCPR1, which encodes the ties associated with identifying the mechanism by which te- RAD52 gene under its own promoter and the selectable lomerase activity is regulated. Studies of telomerase regulation marker URA3 (27). The tlc1Dyrad52D strain expressing have focused on the identification of the enzyme subunits. The Rad52p from a plasmid mentioned in Fig. 1A was derived by TLC1 LEU2 telomerase RNA subunit gene has been cloned from many replacing the gene with in a Y0025 background. tlc1 C species (4), including humans (12), but the expression of the Similarly, the D strain mentioned in Fig. 1 was generated by replacing the TLC1 locus with a URA3 marker in the L3853 mammalian telomerase RNAs does not correlate well with strain. The EST2-HA strain described in the legend of Fig. 2 telomerase activity (12–14). Two proteins, p95 and p80, have was constructed by introducing an HA-URA3-HA cassette also been identified from highly purified fractions of Tetrahy- in-frame into the 39 end of the EST2 gene by homologous The publication costs of this article were defrayed in part by page charge Abbreviations: Est2p, protein product of the EST2 gene; FOA, payment. This article must therefore be hereby marked ‘‘advertisement’’ in 5-fluoroorotic acid; HA, influenza hemagglutinin. accordance with 18 U.S.C. §1734 solely to indicate this fact. †C.M.C. and M.M. contributed equally to this work. © 1997 by The National Academy of Sciences 0027-8424y97y949202-6$2.00y0 §To whom reprint requests should be addressed. e-mail: weinberg@ PNAS is available online at http:yywww.pnas.org. wi.mit.edu. 9202 Downloaded by guest on September 27, 2021 Genetics: Counter et al. Proc. Natl. Acad. Sci. USA 94 (1997) 9203 recombination, and then counter-selecting on 5-fluoroorotic site. The resulting product was then digested with DpnI, which acid (FOA) to excise the URA3 gene, generating three copies cuts only methylated DNA, before ligation and bacterial of the influenza hemagglutinin (HA) tag in frame with EST2 transformation. The BamHI EST2-HA fragment containing (28). The construction of this epitope-tagged version deleted the point mutation was subcloned into the plasmid p426-GAL the 10 carboxyl-terminal amino acids of the protein product of (31) and the insert was sequenced. Clones containing only the the EST2 gene (Est2p); these codons contain a TA repeat desired mismatch were used to transform either est2::Tn3 sequence that is present in multiple copies in the yeast genome. rad52D or est2::Tn3 yeast strains. A derivation of this strain, EST2-HA tlc1D (Fig. 2B) was generated by replacing the TLC1 gene with a URA3 marker. RESULTS AND DISCUSSION Yeast Mutagenesis. Approximately 230 mg of the appropri- ately prepared yeast mini-Tn3::lacZ::LEU2 genomic library A Genetic Screen Identifies Genes Required for Telomerase (29) was introduced by homologous recombination into strain Activity. Because neither genetic nor sequence homology Y0025. Approximately 1 3 106 uracil-independent leucine- approaches had defined a protein subunit of yeast telomerase, independent clones representing successful transposon inser- we initiated a screen for yeast genes that are essential for tions were pooled, and replated to a total number of '1 3 106 telomerase catalytic function. We reasoned that mutations in clones to ‘‘age’’ the yeast. Then 1 3 105 of such yeast were such genes would lead to three phenotypes: (i) absence of reseeded for the rad52D synthetic lethal screen, as described in telomerase enzymatic activity, (ii) a consequent decrease in the text. telomere length, and (iii) dependence on the RAD52 gene Sequence Analysis. Genomic DNA flanking the transposon product for prolonged viability. This third prediction is based insertion sites was rescued as described (29), sequenced, and on the observation that the loss of telomeric DNA observed in compared with the Saccharomyces cerevisiae Genome Data- yeast lacking a functional telomerase RNA component can be base (http:yygenome-www.stanford.eduySaccharomycesy) compensated by the DNA recombination machinery, of which with the BLAST sequence comparison algorithm. Est2p se- RAD52 is a central component. Accordingly, the simultaneous quences were compared using BLAST to multiple genome inactivation of RAD52 and the telomerase RNA component databases, including the Arabidopsis thaliana Database results in cell lethality once telomeres become critically short (http:yygenome-www.stanford.eduyArabidopsisy). in both S. cerevisiae (25) and Kluyveromyces lactis (32). Southern Hybridization. Genomic DNA was isolated and To determine if mutants in telomerase function could be digested with the restriction enzyme XhoI, which liberates the revealed by a genetic screen for yeast that require RAD52 majority of telomeres as small ('1.3-kbp) heterogeneous function, we generated a yeast strain that lacked a functional fragments. Samples (2 mg) of each DNA were resolved on 0.8% TLC1 telomerase RNA subunit gene but also carried an agarose gels for 570 Vzh and hybridized as described (10), using inactive chromosomal rad52D allele, which was complemented a 32P kinase-labeled yeast telomeric oligonucleotide CACCA- by a plasmid-borne wild-type RAD52 gene linked to a URA3 CACCCACACACCACA.
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