Purification of Tetrahymena Telomerase and Cloning of Genes Encoding the Two Protein Components of the Enzyme

Purification of Tetrahymena Telomerase and Cloning of Genes Encoding the Two Protein Components of the Enzyme

Cell, Vol. 81,677-686, June 2, 1995, Copyright © 1995 by Cell Press Purification of Tetrahymena Telomerase and Cloning of Genes Encoding the Two Protein Components of the Enzyme Kathleen Collins, Ryuji Kobayashi, RNA component contains a 9 nt sequence (3'-AACCCC- and Carol W. Greider AAC-5~ complementary to the 6 nt telomeric repeat, Cold Spring Harbor Laboratory d(TTGGGG) (Greider and Blackburn, 1989). This region Cold Spring Harbor, New York 11724 of the telomerase RNA templates the synthesis of telo- meres in vivo (Yu et al., 1990). Additional telomerase RNAs have been cloned from both ciliates and yeast (Shippen- Summary Lentz and Blackburn, 1990; Romero and Blackburn, 1991 ; Lingner et al., 1994; Melek et al., 1994; Singer and Gott- Telomerase is a ribonucleoprotein DNA polymerase schling, 1994). Aside from the presence of a template re- that catalyzes the de novo synthesis of telomeric sim- gion, these RNAs lack extensive primary sequence con- ple sequence repeats. We describe the purification of servation. Phylogenetic evidence suggests a conserved telomerase and the cloning of cDNAs encoding two secondary structure in the ciliate RNAs (Lingner et al., protein subunits from the ciliate Tetrahymena. Two 1994; Romero and Blackburn, 1991), but the function of proteins of 80 and 95 kDa copurified and coimmuno- the conserved structures is not yet known. precipitated with telomerase activity and the pre- Models for primer recognition by Tetrahymena telo- viously identified Tetrahymena telomerase RNA. The merase have proposed two primer-binding sites to account p95 subunit specifically cross-linked to a radiolabeied for the differences in elongation processivity and binding telomeric DNA primer, while the p80 subunit specifi- affinity of different primers (see, for example, Collins and cally bound to radiolabeled telomerase RNA. At the Greider, 1993; Lee and Blackburn, 1993). During pro- primary sequence level, the two telomerase proteins cessive elongation, the product 3' end is released from share only limited homologies with other polymerases the end of the template and repositioned at the beginning and polymerase accessory factors. of the template to allow addition of another repeat to the same substrate. When released from the template, prod- Introduction uct remains associated with telomerase by interaction at a second site (the anchor site). Binding at the second site The faithful replication of eukaryotic chromosomes re- is less sequence specific than binding of primer at the quires the activity of several DNA-dependent DNA poly- template site; however, telomeric or G-rich sequences are merases, a DNA-dependent RNA polymerase (primase), preferred (Collins and Greider, 1993; Lee and Blackburn, and the RNA-dependent DNA polymerase telomerase. 1993). Telomerase also catalyzes a nucleolytic cleavage Replication by DNA-dependent polymerases alone does activity. When primer or product is aligned at the extreme not completely duplicate the sequences at chromosome 5' end of the RNA template, residues at the 3' end of the termini, resulting in a loss of telomeric simple sequence substrate can be removed (Collins and Greider, 1993). repeats with each cell division. Telomerase compensates The processive elongation and nucleolytic cleavage cata- for this underreplication by de novo addition of simple se- lyzed by telomerase are similar to activities catalyzed by quence repeats (for review, see Greider, 1991). Conditions DNA-dependent RNA polymerases: in both cases, the 3' that affect the balance in number of telomeric simple se- end of the product, labile to nucleoiytic cleavage, is bound quence repeats also affect chromosome stability, gene at the template or active site, while a more 5' region of expression, and likely additional cellular processes (for product is bound at a second site on the enzyme (for RNA review, see Blackburn, 1994). Primary human cell cultures polymerase, see Kassavetis and Geiduschek, 1993). To that exhibit a loss of telomeric simple sequence repeats analyze telomerase in molecular detail, it was essential with proliferation do not possess detectable telomerase to identify and characterize the protein components of the activity (Counter et al., 1992). These cells become senes- enzyme. In this report, we describe the purification of Tet- cent after the number of population doublings required for rahymena telomerase and the cloning of cDNAs encoding shortening of telomeric simple sequence repeat tracts to the two protein subunits of this novel RNP polymerase. a predicted critical minimum size, independent of initial length (Counter et al., 1992; AIIsopp et al., 1992). Thus, Results telomeric simple sequence repeats have been proposed to act as a molecular clock for proliferation, counting down Purification of the Tetrahymena Telomerase RNP a set number of cell divisions. Many cancer cells, unlike Optimal purification of telomerase was obtained by chro- their somatic progenitors, possess telomerase activity matography of a cytoplasmic extract on hydroxylapatite, (Counter et al., 1994; Kim et al., 1994). If telomerase acti- spermine-agarose, Sepharose CL-6B, phenyl-Sepha- vation is a requirement for the continued growth of cancer rose, and DEAE-agarose or Q-Sepharose resins, followed cells, it would provide a potential target for therapeutic by glycerol gradient sedimentation (see Experimental Pro- treatment (Harley et al., 1994). cedures). Telomerase activity was assayed and the telo- Telomerase is unique among eukaryotic polymerases merase RNA component quantitated with each fractionation in that it functions as a stable ribonucleoprotein (RNP) step. Telomerase activity assayed across a representative complex. The 159 nt Tetrahymena thermophila telomerase glycerol gradient is shown in Figure 1A. Activity was con- Cell 678 A Figure 1. Purification of Tetrahymena Telo- merase (A) Telomerase activity across glycerol gradi- ent fractions. The indicated fractions were as- sayed for telomerase activity. The bottom of the gradient is fraction 1. (13) Copurification of p95 and p80 with telo- merase activity. The indicated fractions were B analyzed by SDS-PAGE and silver staining. ............ ~ kD The M lane contains markers of the indicated ..... 200 molecular masses. ..... ==~- 66 --- 45 5 6 7 8 9 I0 I 2 t3 4 16 17 M I 2 3 4 5 6 7 8 91011 1213141516 IT 18192D212-.2.?.3L:~252627 centrated in fractions 9-12 and peaked in fraction 10. The tions of telomerase were similarly assayed, p80 and p95 distribution of telomerase RNA coincided exactly with teio- were always present and could be clearly resolved from merase activity across the gradient (data not shown). Two other proteins, including another 80 kDa polypeptide (Fig- proteins cofractionated with telomerase activity and RNA ure 2D). (Figure 1B), migrating at 95 kDa (p95) and 80 kDa (p80). The stoichiometry of p80:p95:telomerase RNA was esti- In this gradient, more than one 80 kDa polypeptide was mated to be 1:1:1, on the basis of Coomassie staining of present in the fractions containing telomerase. These two proteins relative to markers of known concentration and 80 kDa species were shown to be distinct proteins by na- Northern blot analysis of the RNA relative to T7-tran- tive gel electrophoresis and immunoblot analysis (see be- scribed standards (data not shown). This stoichiometry low), as well as by centrifugation of peak gradient fractions would suggest a molecular mass of 80 kDa plus 95 kDa on a second glycerol gradient (data not shown). As shown plus 54 kDa, or approximately 230 kDa, which agrees with by a variety of purification schemes, only p80 and p95 the molecular mass of the complex estimated by analysis reproducibly copurified with telomerase activity (data not of gel filtration and glycerol gradient sedimentation (200- shown). 270 kDa; see Experimental Procedures). The gel filtration Active telomerase must contain protein(s) stably bound and velocity sedimentation properties of telomerase were to the telomerase RNA. To examine further whether p80 unchanged throughout the purification, suggesting that no and p95 were associated with the telomerase RNA, we components of the complex were lost. analyzed purified fractions by a two-dimensional gel assay. A glycerol gradient sample (fraction 10 described Cross-Linking of Telomeric Primer to p95 above) was divided and loaded in each of three lanes of To determine whether any individual proteins in telo- a native gel. After eiectrophoresis, one lane was analyzed merase preparations demonstrated specific binding to a for telomerase RNA by Northern blotting (Figure 2A); a telomeric primer DNA or to the telomerase RNA, we per- second lane was layered on top of and run into an SDS- formed cross-linking with 32P-labeled, iodouracil-substi- polyacrylamide gel to analyze proteins (Figure 2B); and a tuted DNA or RNA. Iodouracil substitution permits cross- third lane was transferred to nitrocellulose, divided into linking at relatively long wavelengths (312 versus 260 nm), segments, and assayed for telomerase activity (Figure reducing nonspecific excitation of unsubstituted groups 2C). In this native gel, telomerase RNA migrated to a posi- and protein denaturation due to photodamage (Willis et al., tion approximately one third to one half of the gel length 1993). Primers substituted with iodouracil were efficiently (Figure 2A). Both p80 and p95 migrated in the native gel elongated by telomerase, and T7-transcribed, iodouracil- to a position similar to that of the telomerase RNA and substituted telomerase RNA

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