ABSTRACT
A number of published studies indicate that telomerase may interact with oligonucleotide primers in a bipartite manner, with the 3'-end of the primer positioned at the catalytic site of the enzyme and a more 5' region of the primer binding to a second or `anchor' site of the enzyme. We systematically investigated the effects of mutations in the DNA primer on overall binding and polymerization by yeast telomerase. Our studies indicate that there is sequence-specific interaction between telomerase and a substantial region of the DNA primer. Mutations in the 3'-most positions of the primer reduced polymerization, yet had little effect on overall binding affinity. In contrast, mutations around the -20 position reduced binding affinity but had no effect on polymerization. Most strikingly, mutations centered around the -12 position of the DNA primer reduced overall binding affinity but dramatically enhanced primer extension, as well as primer cleavage. This finding suggests that reduced interaction with the -12 region of the DNA primer can facilitate a step in the catalytic region of yeast telomerase that leads to greater polymerization. A tripartite model of interaction between primer and telomerase is proposed to account for the distinct effects of mutations in different regions of the DNA primer.
Telomerase is a ribonucleoprotein (RNP) that is responsible for synthesis of the dG-rich strand of telomere terminal repeats (1-3). The enzyme was initially identified in Tetrahymena thermophila, a ciliated protozoan whose life cycle involves formation of a large number of telomeres. The original in vitro assay was based on the ability of telomerase to extend telomere-like oligodeoxynucleotides in the presence of dGTP and dTTP (4). The enzyme was subsequently found to be an unusual reverse transcriptase containing an integral RNA component, a small segment of which acts as the template for synthesis of the dGT-rich strand of telomeric repeats (5). Multiple repeats can be added to the input primer following initial binding, despite the minimal number of repeat units in the RNA template, suggesting that the enzyme must undergo a translocation step during processive DNA synthesis (6). Telomerase can also cleave the input primer under certain conditions (7-9), a property that is shared by a number of DNA and RNA polymerases.
Telomerase activity has been detected in a wide range of organisms, including protozoa (10), yeast (8, 11-13), mouse (14), Xenopus (15) and human (16). Genes encoding the RNA component of the enzyme complex have been cloned for many telomerases, such as that of yeast and human (17,18). Recently some of the polypeptide components of telomerase were cloned. In particular, a protein named p123 in Euplotes aediculatus and a homologous protein encoded by EST2 in yeast were shown to be the catalytic components of the respective telomerases (19,20); these two polypeptides exhibit significant homology to other reverse transcriptases and mutations that alter Est2p residues that are conserved among reverse transcriptases abolish telomerase activity in vitro and telomerase function in vivo. In addition, two polypeptides, p80 and p95, that co-purify with Tetrahymena telomerase have been cloned and been shown to interact with telomerase RNA and the DNA primer respectively (21). Mouse and human homologs of p80 have also been identified and been shown to associate with the respective telomerases (22,23). These recent developments should greatly facilitate structure-function analysis of telomerase.
A number of experiments indicate that telomerase may interact with oligonucleotide primers in a bipartite manner, with the 3'-end of the primer positioned at the catalytic site of the enzyme and a 5'-region binding to a second or `anchor' site of the enzyme. First, the processivity of Tetrahymena telomerase was greatly influenced by the length of the oligonucleotide: primers <10 nt were extended by only one repeat whereas longer primers were processively elongated (7,24). This suggests that binding to the upstream region (`anchor' site) of the primer prevents dissociation of the enzyme during translocation, which is needed for synthesis of long products. Second, a non-telomeric primer that was a poor substrate for telomerase elongation is a much better substrate when telomeric repeats were added to its 5'-end, again suggestive of an upstream interaction site in the enzyme (25-27). Third, the efficiency of primer utilization was altered when the 3'-sequence of the primer was held constant and the 5'-sequence was varied (24). Direct evidence for a telomerase `anchor' site came from a study where the -20 to -22 region of a DNA primer was shown to crosslink to a telomerase protein and the telomerase RNA from Euplotes aediculatus (28).
To further analyze primer-telomerase interactions we tested the effects of a large number of mutations (defined as deviations from canonical yeast telomere sequence) in the oligonucleotide primer on its ability to bind and to be extended by yeast telomerase. Our studies indicate that there is sequence-specific interaction between yeast telomerase and an extended region of the DNA primer. Mutations in different regions of the DNA primer have very distinct effects on binding and polymerization. A tripartite model of interaction between primer and yeast telomerase is proposed to account for these observations. A particularly interesting aspect of the model is that telomerase activity can be negatively regulated through an interaction with the -12 region of the DNA primer.
The haploid Saccharomyces cerevisiae strain DG338 was used throughout. The cells were grown in YPD medium (2% glucose, 1.0% Yeast Extract, 2% Bacto-peptone). The following protease inhibitors were included in all buffers: 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 2 µg/ml pepstatin A and 1 µg/ml leupeptin. Buffer TMG-15 contains 15% glycerol, 10 mM Tris-HCl, pH 8.0, 1.2 mM magnesium chloride, 0.1 mM EDTA, 0.1 mM EGTA, 1.5 mM DTT. Buffer TMG-10 is identical to TMG-15 except that glycerol was included at 10%. Buffer TMG-10(500) etc. denotes buffer TMG-10 plus the concentration of sodium acetate (mM) specified by the number in parentheses.
Yeast telomerase was prepared according to Cohn and Blackburn (8) with only slight modifications. Four liters of S.cerevisiae cultures were grown to late log phase, harvested and washed once with ice-cold water. Cell pellets were resuspended in an equal volume of TMG-15, mixed with a volume of glass beads equal to the total suspension volume and lysed by vortexing at maximum speed. Thirty seconds of vortexing was alternated with 30 s cooling in an ice-water bath for a total of 25 min. Cell lysates were collected and cleared by two successive centrifugations: first in a GSA rotor (Sorvall) at 8000 r.p.m. for 10 min and then in a T-865 rotor (Sorvall) at 32 000 r.p.m. for 1 h.
Telomerase activity was partially purified by passage through a DEAE-agarose (BioRad) column. A 5 ml DEAE column was equilibrated in TMG-10(0). The extract (20 ml) was loaded directly onto the column. The column was washed successively with 10 ml TMG-10(300) and 10 ml TMG-10(550) and the activity eluted with 10 ml TMG-10(900). Fractions were collected and used directly in telomerase assays.
A typical telomerase reaction was carried out in 30 µl containing the following: 10 mM Tris-HCl, pH 8.0, 2 mM magnesium acetate, ~300 mM sodium acetate (contributed by the protein fraction), 1 mM spermidine, 1 mM DTT, 5% glycerol (contributed by the protein fraction), 50 µM dTTP, 20 µCi [[alpha]-32P]dGTP (3000 Ci/mmol), 5 µM primer oligodeoxynucleotides and ~15 µl column fractions. Following incubation at 30°C for 1-1.5 h the reaction was stopped by adding 80 µl 10 mM Tris-HCl, pH 8.0, 20 mM EDTA, 0.1 mg/ml RNase A. Following incubation at room temperature for 10 min the products were further digested with 100 µl solution containing 10 mM Tris-HCl, pH 8.0, 0.5% SDS and 0.3 mg/ml proteinase K at 37°C for 20 min. The digested reaction products were precipitated with the addition of 750 µl absolute ethanol and 100 µl 7.5 M ammonium acetate containing 1 µg yeast tRNA and analyzed on a 15% TBE-urea polyacrylamide gel. Signals were quantified using a PhosphorImager system (Molecular Dynamics). For quantification of activity the signals from all labeled and RNase-sensitive products (including those that are shorter than the input primer) are summed.
DNA primers used for gel mobility shift experiments were radiolabeled with T4 polynucleotide kinase and purified through a Nick Column (Pharmacia). The gel mobility shift assay is based on a previously described protocol (29). Partially purified telomerase was incubated with 5 nM (0.4 ng) labeled primer in 16 µl buffer containing 10 mM Tris-HCl, pH 8.0, 2 mM MgCl2, 12% glycerol and 50 ng poly(dI·dC). Where indicated, cold competitor oligonucleotides were added before addition of telomerase. Binding was carried out at 4°C for 20 min and complex formation monitored by electrophoresis through a 4% polyacrylamide gel with running buffer containing 25 mM Tris-HCl, pH 8.3, 190 mM glycine, 5 mM MgCl2 and 1 mM EDTA. The gel was cast in running buffer supplemented with 10% (v/v) glycerol and 0.5 mM DTT.
One of us has previously reported a processive telomerase-like activity from S.cerevisiae that is TLC1-dependent (12,30). However, subsequent studies suggest that the fraction used may be contaminated with a second polymerase activity (data not shown). We therefore experimented with other published protocols for yeast telomerase derivation and found that a high salt eluate from a DEAE column, as reported by Cohn and Blackburn (8), has a robust RNase-sensitive polymerization activity that is TLC1-dependent (data not shown). Preparation of the fraction from a strain harboring point mutations in the TLC1 template region yielded an activity that has the expected property for nucleotide utilization. The TLC1-1 mutation substituted a GG dinucleotide for AC such that the enzyme can incorporate dCMP in addition to dGMP and dTMP (Fig. 1A; 17). To facilitate incorporation of dCMP by the mutant enzyme a primer that ends in a dGGG trinucleotide was used because this trinucleotide is immediately upstream of the expected dC residue. As shown in Figure 1, the enzyme preparation from the TLC1-1 strain supported extension of up to 3 nt in the presence of dCTP and dTTP (presumably the dCCT trinucleotide) and >7 nt in the presence of dCTP, dGTP and dTTP (the triangles in Fig. 1 denote the positions of `primer + 1'). A small amount of polymerization is also observed when labeled dGTP and unlabeled dTTP are used, presumably as a consequence of alternative alignment and/or primer cleavage. In contrast, the activity from a wild-type strain does not incorporate a significant amount of dCMP, even in the presence of both dGTP and dTTP. Consistent with earlier reports, we find that telomerase activity derived in this fashion acts non-processively in vitro and is not stimulated by ATP (data not shown). Since this fraction appears largely devoid of other contaminating activity, we have used it exclusively for subsequent assays.
To determine the nature and significance of the interaction between telomerase and different parts of the primer we tested the effects of defined sequence changes in the primer on its utilization by telomerase. As expected, point mutations in the 3'-most positions (-1 to -3) reduced the level of incorporation (Fig. 2A), consistent with the role of these nucleotides in aligning with the RNA template. In contrast, point mutations anywhere in the -4 to -14 region of a 15 nt primer consistently stimulated primer utilization (Fig. 2B and Tables 1 and 2). The effect was maximal with mutations around the -12 region (~20-fold; Fig. 2B and Table 2) and diminishes with mutations upstream or downstream. In addition to enhanced polymerization, the relative proportion of the products that were shorter than the input primer also increased (data not shown). Because >95% of the input primer remained undegraded (data not shown), the nuclease activity appeared to be tightly associated with yeast telomerase. It has previously been reported that primer/template mismatch can enhance primer cleavage by yeast telomerase (31). However, this series of primers, which end in TGGG, were designed to align with only 9 bases of the template. Thus the effects of mutations upstream of the -9 position cannot be accounted for by mismatch. Instead, altered interaction with a second site of the enzyme is likely to be responsible.
The nature of the mutation does not appear to greatly influence the degree of stimulation. For example, changing the dG residues at the -7 position to either dA, dC or dT resulted in comparable enhancement in primer utilization (5-fold for a G -> T change and 12-fold for a G -> A change). The same was true for mutations at the -12 position: changing the dG base to any one of the other three bases resulted in an ~20-fold stimulation of polymerization.
The time course of the reactions indicates that preferential utilization of mutant primers was due to an increase in the rate of DNA synthesis, rather than simply an increase in the overall extent of synthesis. As shown in Figure 2D, the rate of DNA synthesis for a wild-type primer (TEL15) was significantly greater than a mutant primer [TEL15(-12)G -> T] over a 20 min period. The reaction was essentially complete after 20 min and further incubation had little effect on the final signal (data not shown).
Because of the simultaneous increase in products that are both longer and shorter than the input primer, we tested the hypothesis that increased cleavage followed by elongation was responsible for the increase in the amount of longer products. A very efficient mutant primer TEL15(-12)G -> C and the wild-type primer TEL15 were tested for telomerase utilization in the presence of dTTP and ddGTP. If nuclease cleavage is obligatory for extension, then one would expect little or no product in the mutant reaction that is longer than the starting primer. However, as shown in Figure 2E, the TEL15(-12)G -> C reaction yielded a product at the `primer + 2' position that was ~20 times more intense than the equivalent band in the TEL15 reaction, consistent with direct extension of the input primer even in the case of the mutant primer. Thus mutations in the upstream region appear to stimulate two separate reactions by telomerase, direct polymerization and primer cleavage coupled with extension. Furthermore, the pattern of the short products in the dT + ddG reactions indicates that extension occurred at multiple positions, consistent with progressive exonucleolytic cleavage or endonucleolytic cleavage at multiple positions.
The stimulatory effects of primer mutations were not dependent upon particular reaction parameters. For example, when labeled dGTP was used in combination with unlabeled dTTP the same degree of stimulation was observed (data not shown). Varying the sodium acetate (from 30 to 300 mM) and magnesium (from 1 to 5 mM) concentration also had little effect on the magnitude of stimulation (data not shown).
All of the primers used in the previous section are designed to align with TLC1 RNA such that the 3'-end residue is positioned at the +9 position of the template (see Fig. 1A for an illustration). To rule out the possibility that the stimulatory effect is specific for this type of primer we tested oligonucleotides with a different 3'-sequence that should support formation of a 12 base hybrid (Table 1, series 3). As shown in Figure 3A and Table 2, mutations in the upstream region of these 19 nt oligonucleotides again stimulated polymerization by telomerase, with the maximal effect observed for a `-12 & -13' double mutant [TEL19(-12)G -> C(-13)T -> A].
Because there is no reason to suspect that yeast telomerase will bind more strongly to all the mutant primers, we hypothesized that there is a negative correlation between the strength of the upstream interaction (centered around the -12 position of the primer) and activity of the primers in standard polymerization assays. If this is indeed the case, shorter primers that lack this upstream interaction region may better support polymerization. Primers with the same 3'-end sequence but ranging in size from 6 to 15 nt were synthesized and tested in polymerization assays (Fig. 4 and Tables 1 and 2). Consistent with our hypothesis, an 11 nt primer (TEL11) was 12 times and a 9 nt primer (TEL9) 4 times more active than TEL15. The 6 nt primer (TEL6) was only twice as active, possibly because it did not efficiently or stably form a hybrid with telomerase RNA.
To obtain direct evidence for reduced binding between telomerase and short or mutant primers we measured the overall affinity of yeast telomerase for the various primers in gel mobility shift experiments. When the telomerase-containing fraction was incubated with a labeled single-stranded telomere oligonucleotide (TEL15) and then electrophoresed through a non-denaturing polyacrylamide gel a number of distinct complexes could be visualized. Only the complex with the lowest mobility, which migrated as an ~1 kb double-stranded DNA, was abolished when the fraction was pretreated with RNase A (Fig. 5A, marked with a dot). Furthermore, addition of an RNase inhibitor prior to RNase A preserved the low mobility complex (RNase + Inh, Fig. 5A), whereas addition of the inhibitor following RNase A treatment failed to restore complex formation [RNase (Inh), Fig. 5A]. A number of observations indicate that this RNase-sensitive binding activity is specific for the G-rich strand of yeast telomeres. First, the C-rich strand of yeast telomere on its own did not support complex formation (data not shown). Second, converting the single-stranded probe to a double-stranded oligonucleotide abolished complex formation (Fig. 5B, lane CA). Finally, adding a number of non-telomeric oligonucleotides or telomeric oligonucleotides from other species had little effect on the pattern of mobility shift (Fig. 5B, lanes H4CSPRA, TOPSEQ2, TELH4 and PTEL3). These observations, together with the fact that the RNase-sensitive complex co-migrates with TLC1 RNA (data not shown), strongly suggest that the low mobility complex is due to telomerase.
Since telomerase must capture the DNA primer before polymerization can take place, it may seem surprising that the lowered affinity of the mutant primers does not result in reduced DNA synthesis. However, in standard polymerization assays the primer is present at micromolar concentrations, orders of magnitude higher than the estimated Kd. Thus an effect of lower binding affinity is likely to be masked. However, such an effect should be revealed when the primers are tested at concentrations approaching the dissociation constants (~3 nM; data not shown). This is indeed the case: when the polymerization assays were done with 1.6-5 nM primer the effect of a `-12' mutation is only 3-fold, rather than the 20-fold seen at micromolar concentrations (Fig. 6).
Our results suggest that the interaction between yeast telomerase and oligonucleotide primer is tripartite, as illustrated in Figure 7. The 3'-end of the primer forms a DNA-RNA hybrid with the TLC1 telomerase RNA. The 5'-region of the primer can be functionally subdivided into two parts: the -4 to -14 region of the primer interacts with one site of the enzyme (site A in Fig. 7), while the -16 to -21 region of the primer interacts with another site (site B in Fig. 7). The location of the target of site B in our model suggests that it may correspond to the previously characterized `anchor' site in ciliate telomerase (28). Both site A and site B interactions appear to contribute to the overall binding energy between telomerase and primer. However, only site A interaction influences polymerization at the catalytic site; loss of site A interaction facilitates polymerization. Possibly, conformational alteration of the enzyme at site A can facilitate an important conformational or chemical step at the catalytic site. Loss of site A interaction also appears to facilitate primer cleavage coupled with extension. How these two types of stimulation are related is not known.
We thank Vicki Lundblad and David Garfinkel for the provision of yeast strains, and Stewart Shuman for constructive comments on an earlier version of the manuscript. This study was supported in part by a Basil O'Connor Starter Scholar Award.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Yeast strains, media and buffers
Preparation of yeast telomerase
Primer extension assays for S.cerevisiae telomerase
Oligonucleotide labeling and gel mobility shift assay
Results
A TLC1-directed yeast telomerase activity
Mutations in an upstream region of the DNA primer stimulate primer extension
The stimulatory effect of upstream mutations is not specific for a particular template position or for a particular primer length
Short primers lacking the upstream interaction site support greater polymerization by yeast telomerase
Mutations throughout the upstream region of the DNA primer diminish its overall affinity for yeast telomerase
The magnitude of stimulation of DNA synthesis by upstream mutations in the primer is reduced at low primer concentrations
Discussion
References
REFERENCES
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