High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase Erez Romi*, Nava Baran*, Marina Gantman*, Michael Shmoish†, Bosun Min‡, Kathleen Collins‡, and Haim Manor*§ Departments of *Biology and †Computer Science, Technion–Israel Institute of Technology, Haifa 32000, Israel; and ‡Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204
Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the Km for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the templateadjacent single-stranded DNA binding site within a cycle of repeat synthesis. specific cleavage of proteins 兩 telomerase–primer interaction 兩 UV crosslinking
T
elomerase is a unique reverse transcriptase (RT) that extends the single-stranded 3⬘ overhangs of telomeres by copying a template within the integral RNA component of the enzyme (1). Some telomerase enzymes can also use this internal template to direct the synthesis of telomeres at nontelomeric sites of chromosome fragmentation (2). In addition to the telomerase RNA subunit (TER), the enzyme contains a catalytic protein subunit, designated telomerase RT (TERT), and accessory proteins (3, 4). Telomerase was first discovered in extracts of the ciliate Tetrahymena thermophila (5), and telomerase from this organism remains an excellent model system for studies of enzyme structure and function. Its RNA subunit (tTER) of 159 nt contains the repeat-complementary sequence 3⬘-AACCCCAAC-5⬘ and other motifs required for ribonucleoprotein (RNP) assembly and activity (1, 3). T. thermophila TERT (tTERT) consists of 1,117 amino acids, including a region between residues 518 and 881 that is conserved among RTs and designated as the RT domain (6). The N-terminal half of TERT contains motifs conserved among TERTs but not viral RTs. It constitutes two independently folded domains: the TERT essential N-terminal domain (TEN) and the TERT high-affinity TER binding domain (TRBD). In tTERT, residues 1–195 can be considered to constitute the TEN domain, whereas residues 196–528 comprise the TRBD (7–9). Telomerase specificity of interaction with single-stranded DNA has been studied by monitoring the elongation of primers of varying lengths, sequences and concentrations. Differences in the primer concentration-dependence and repeat addition processivity of product synthesis indirectly suggest that extensive contacts to the enzyme are made by primer regions 5⬘ of the www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703157104
template hybrid (2). More direct physical assays have also been used to investigate enzyme–primer interactions. Our previous interference footprinting studies indicated that functionally nonredundant interactions of primer with enzyme occur primarily in the six or seven 3⬘-terminal primer nucleotides (10). In addition, atomic-resolution structure determined for residues 13–176 of the tTERT TEN domain revealed a surface groove with features suggestive of a channel for binding single-stranded DNA (11). Mutagenesis of some channel residues (Q168A, F178A) strongly reduced recombinant telomerase activity, and activity was eliminated by an adjacent substitution (W187A) in the C-terminal ‘‘tail’’ of TEN that adopted alternative structures. These same substitutions each reduced the specificity of TERT crosslinking to a radiolabeled, iodouracil-derivatized DNA primer (11). Here we investigate the sites of tTERT interaction with DNA by mapping covalent crosslinks induced by UV light. For functional significance, we designed the assays to map tTERT interaction site(s) for a nucleotide of the primer at the boundary of the primer-template hybrid in catalytically active telomerase complexes (see below). These interaction site(s) can be studied most readily with recombinant core enzyme containing tTER and tTERT, because the important template-adjacent DNA contacts are not overshadowed by the additional DNA interactions that may occur in telomerase holoenzyme complexes (12). We characterized a site-specific DNA crosslink to tTERT tryptophan 187 (W187). Another crosslink site was also detected within the tTERT segment spanning residues 192–411 of the TRBD. Primer extension activity assays revealed that W187 by itself was not essential for recombinant enzyme activity, but substitutions in the neighboring residue Q168 altered primer Km for elongation. Our data are the first to directly map a specific TERT–DNA interaction at a single amino acid resolution within catalytically active telomerase RNP. Overall, the data provide new information about the positioning of DNA and TERT domains in functional telomerase complexes. Results Mapping of DNA Crosslinking Sites in Catalytically Active Telomerase.
We performed site-specific DNA crosslinking to TERT, using the 6-nt primer 5⬘-G(IdU)TGGG-3⬘ (IdU5), in which a thymidine was substituted with 5-Iododeoxyuridine (IdU) at the (⫺5) Author contributions: E.R., K.C., and H.M. designed research; E.R., N.B., M.G., and B.M. performed research; E.R., N.B., M.S., K.C., and H.M. analyzed data; and K.C. and H.M. wrote the paper. The authors declare no conflict of interest. Abbreviations: RT, reverse transcriptase; TEN, TERT essential N-terminal domain; TER, telomerase RNA subunit; tTER, Tetrahymena telomerase RNA subunit; TERT, telomerase catalytic protein subunit; tTERT, Tetrahymena telomerase catalytic protein subunit; TRBD, TERT high-affinity TER binding domain. §To
whom correspondence should be addressed. E-mail:
[email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/ 0703157104/DC1. © 2007 by The National Academy of Sciences of the USA
PNAS 兩 May 22, 2007 兩 vol. 104 兩 no. 21 兩 8791– 8796
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Communicated by E. Peter Geiduschek, University of California at San Diego, La Jolla, CA, April 5, 2007 (received for review October 20, 2006)
position relative to the 3⬘OH end. This position was previously shown to be involved in primer interaction with telomerase (10, 13). A complex of reconstituted core enzyme (tTERT and tTER) with primer was irradiated with a long-wavelength UV light (⬎295 nm), thereby favoring crosslinks of the IdU with aromatic amino acids in close proximity. Next, 32P-dGTP was added to the mixture and the crosslinked DNA was extended with a single 32P-dGMP by the enzyme molecule to which it was bound (Fig. 1A). Thus, only the crosslinked primer molecules that were properly aligned within a functional RNP were radiolabeled. The reactions were analyzed for tTERT crosslinking (Fig. 1B Upper) and for enzyme activity (Fig. 1B Lower). In complete reactions with substituted primer and UV, a single radiolabeled protein having the expected mobility of tTERT was observed, and the expected 7-nt extension product was detected as well (lane 1). Similar experiments performed with unsubstituted primer, or without UV treatment, gave barely detectable signal of crosslinked tTERT, or no signal, despite efficient elongation (lanes 2 and 4). A reaction carried out in the absence of primer produced no detectable radiolabeled protein or DNA (lane 3), as did reactions carried out with radiolabeled dCTP instead of dGTP (lane 5) or with tTER or tTERT missing from the enzyme reconstitution (lanes 6–7). Taken together, these results demonstrate that the observed crosslinking product reflects site-specific interaction of the primer and tTERT in a catalytically active complex. The yield of crosslinked products obtained in our assays (⬇1 fmol) was too low to allow definition of the crosslinking sites by using mass spectrometry. Therefore, we used a mapping method involving partial protein degradation that was previously used in studies of RNA polymerase elongation complexes (14, 15). After primer crosslinking and radiolabeling by nucleotide addition, tTERT molecules were immunopurified and digested with cyanogen bromide (CNBr). This reagent cleaves polypeptides on the carboxyl side of methionine residues, of which there are 18 in tTERT (Fig. 1C). Partial digestion was performed such that most tTERT molecules were either cleaved once or not cleaved at all (‘‘single-hit’’ cleavage). Each of the tTERT molecules cleaved at a single site was expected to yield two fragments, of which only the fragment containing the crosslink would be radiolabeled. Assuming that single-hit digestion occurred with equal probability at all methionines of tTERT, the ensemble of digested molecules should encompass 18 pairs of radiolabeled and unlabeled fragments. Crosslinking at each segment of tTERT delineated by sequential methionines should generate a unique pattern of 32P-labeled fragments, which was simulated by computer as shown in Fig. 1D. Partial digestion was performed over a time course and the ratio of uncleaved and cleaved tTERT molecules was determined to most closely approximate conditions of single-hit digestion (Fig. 1E, lanes 2–3). The resolution of the SDS/PAGE gels used here was optimal in the range of fragments of 20–55 kDa. Smaller fragments were masked by the large amounts of radioactivity from uncrosslinked product DNA and unincorporated dGTP (data not shown). Comparison of experimentally observed fragments in Fig. 1E to the simulated fragment patterns in Fig. 1D indicated compatibility only with a crosslinking site in the segments of amino acids 2–13 or 14–194. The corresponding region in the yeast TERT Est2p has been also proposed to interact with DNA primers (16). The presence of a weakly labeled fragment in lane 1 (undigested tTERT control) that comigrated with the 26.0-kDa fragment in lanes 2–4 prevented the unambiguous attribution of the 26.0-kDa fragment to a digestion product of tTERT. If digestion did not produce a 26.0-kDa fragment, then the crosslinking site could map either in the segment 2–194, or in the segment 195–235. Subsequently, we carried out another experiment in which 8792 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703157104
Fig. 1. Site-specific crosslinking of telomerase and DNA primer. (A) Scheme of the crosslinking experiment. x designates the IdU substitution. X designates the crosslinked IdU. g is the newly added [32P]-dGMP. Nucleotide numbers at the boundaries of the RNA template region are indicated. (B) (Upper) SDS/ PAGE analysis of crosslinked tTERT. Size markers are depicted on the right with masses in kilodaltons. (Lower) Analysis of the noncrosslinked DNA products. Samples from the crosslinking reactions were withdrawn, and noncrosslinked extended DNA primers (⬎99% of the products) were analyzed by ureapolyacrylamide gel electrophoresis. (C) CNBr cleavage map of tTERT. CNBr cleavage occurs at methionine residues, whose positions are indicated. (D) Simulation of the SDS/PAGE patterns expected in a CNBr single-hit digestion of crosslinked tTERT. The 18 lanes present the gel patterns expected for the 18 possible locations of the crosslinking site. Size markers are depicted along the y axis, whereas the cleavage sites are depicted along the x axis. Each lane is located between two successive cleavage sites and reflects the gel pattern expected if the crosslink would occur between these two sites. The simulation refers to fragments larger than 20 kDa, which were resolvable by SDS/PAGE. The arrowheads point at fragments that were useful for mapping of the crosslinking site. The simulation tool was implemented as the R-function (25) and is available upon request from M.S. (E) SDS/PAGE of fragments generated by partial digestion of crosslinked tTERT with CNBr. The arrowheads indicate the fragments that provided the map position of the crosslinking site. The fragments are marked by their masses in kilodaltons. (F) SDS/PAGE of fragments generated by extensive CNBr digestion of WT or M194L crosslinked tTERT. The arrows indicate fully and partially digested fragments. The fragments are marked by their map positions in tTERT.
Romi et al.
Romi et al.
Fig. 2. Fine mapping of the crosslinks. (A) A scheme illustrating the fine mapping. (Upper) A complete map of the CNBr cleavage sites in the WT enzyme is shown with enlargement of the region including the crosslinking site. This scheme also shows the two methionines that have been substituted with leucine in the double mutant M194L/M235L and the tryptophan residue W187 that has been identified as the major crosslinking site. (Lower) Schematized fragments produced by CNBr cleavage of additional tTERT variants, which included a third substitution in addition to M194L/M235L. The numbers represent the fragments masses produced by complete cleavage with CNBr and bold double arrows designate the major radiolabeled fragments that were observed. The numbers in parentheses are the combined masses of fragments and DNA, which are the actual species analyzed in the gel. (B) SDS/PAGE of fragments from extensive digestion assays performed with the tTERT mutants shown in A, crosslinked to the primer IdU5. The arrows indicate the major radiolabeled fragments produced by complete digestion with CNBr. The empty arrowheads indicate the minor radiolabeled fragments generated by partial cleavage. The filled arrowhead points at the fragment that contains the second crosslinking site. (C) A scheme illustrating the template alignment of the three substituted primers used for the assays shown in B, D, and E. The stars indicate the active site. x designates the IdU substitution. (D) SDS/PAGE of fragments from extensive digestion assays performed with the tTERT mutants shown in A, crosslinked to the primer IdU3. (E) SDS/PAGE of fragments from extensive digestion assays performed with the tTERT mutants shown in A, crosslinked to the primer IdU8. The filled arrowhead points at the fragment containing the second crosslinking site.
We also studied crosslinks generated by using two additional IdU-substituted primers 5⬘-GGG(IdU)TG-3⬘ (IdU3) and 5⬘(IdU)TGGGGTT-3⬘ (IdU8). To become radiolabeled subsequent to crosslinking, these two primers must align at the beginning or end of the template, respectively (Fig. 2C). As in the IdU5 primer used above, the IdU substitutions are positioned to parallel the template-region residue A51. We found, using extensive digestion assays (Fig. 2 D and E), that these primers also crosslink to W187. Additionally, the primer IdU8 generated a minor crosslinked species similar to that of IdU5 (Fig. 2E, filled arrowhead), but we could not conclude whether IdU3 generated this minor species as well. We also carried out crosslinking assays, using primers with IdU substitutions at nucleotide residues aligned with the RNA template residues A50 and A45, respectively. The first substitution gave a considerably lower yield of crosslinking, and the second substitution gave an indetectable crosslinking signal (data not shown). PNAS 兩 May 22, 2007 兩 vol. 104 兩 no. 21 兩 8793
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tTERT was extensively digested with CNBr (Fig. 1F). Under these conditions, the majority of the tTERT molecules were digested at all of the cleavage sites. This digestion gave rise to a major radiolabeled fragment of ⬇23.5 kDa, which is the expected mass of the 14–194 peptide crosslinked to the elongated primer. Also produced was a minor 28.5-kDa fragment, which is the expected mass of the 14–235 peptide crosslinked to the elongated primer and likely results from incomplete digestion at M194. To further validate these results, we generated tTERT with M194 substituted with leucine (M194L), thereby eliminating this cleavage site. The substitution was expected to cause an increase of ⬇5 kDa in the size of the peptide that crosslinked to the primer, which was the observed result (Fig. 1F; compare lanes 2 and 4). A minor fragment was apparently produced by incomplete digestion of the M194L tTERT at M235, generating a radiolabeled fragment with the expected mass of the 14–411 peptide crosslinked to the primer. To determine the location of the crosslinking site at single amino acid resolution, we produced additional substitution variants of tTERT and used them in extensive digestion assays. First, we generated a tTERT in which the methionine residues 194 and 235 were substituted with leucine. This created a methionine-free segment spanning amino acids 14–411 (Fig. 2A Upper). Using the double mutant tTERT, we generated a series of triple mutants in which nonconserved amino acids located at various positions along the segment 145–191 were substituted with methionine. These substitutions were designed to enable cleavage of the 14–411 segment into two asymmetric fragments resolvable by SDS/PAGE, only one of which should be radiolabeled. The expected pairs of fragments that would result from full digestion are shown in Fig. 2 A Lower. SDS/PAGE of crosslinking assays analyzed by extensive digestion revealed that each tTERT variant, except W187M, produced one major radiolabeled fragment and one or two additional minor radiolabeled fragments. The major radiolabeled fragment produced by the tTERT double mutant M194L/M235L (Fig. 2B, lane 1) had a mass consistent with the expected 49.4-kDa segment spanning amino acids 14–411 crosslinked to the DNA (Fig. 2 A). The major radiolabeled fragments produced by digestion of the tTERT triple mutants with the substitutions E145M, L167M, and K186M (Fig. 2B, lanes 2–4) migrated as expected if they share a common C terminus at M411 and N-termini at the positions specified by the third mutation (Fig. 2 A, bold doublearrowhead lines). The major radiolabeled fragments produced by digestion of the tTERT triple mutants with the substitutions Y188M, K189M, and N191M (Fig. 2B, lanes 6–8) migrated as expected if they share a common N-terminal end at L14 and C-termini at the positions specified by the third mutation (Fig. 2 A, bold double-arrowhead lines). These data suggest that the major crosslinking site maps between K186 and Y188 at a tryptophan residue, W187. The crosslinking site W187 was confirmed by assaying the tTERT triple mutant with the substitutions M194L/M235L and W187M, which did not generate a major radiolabeled peptide (Fig. 2B, lane 5). In addition to the major products described above (Fig. 2B, marked by arrows), these assays generated distinct minor species (Fig. 2B, marked by empty and filled arrowheads). The lengths of the two species marked by empty arrowheads suggest that they are partial digestion products. However, the single species marked by a filled arrowhead could not be generated by partial digestion. Therefore, it must represent a second crosslinking site that maps in the segment spanning the amino acids 192–411. Of the tTERT triple mutant series sharing M194L/M235L (Fig. 2 A), this second crosslink was detected with mutants containing W187M, Y188M, K189M, and N191M (Fig. 2B, lanes 5–8); in the assays of mutants containing E145M, L167M, and K186M, the second crosslink could not be detected because it resides on the fragment that also includes the first crosslink.
Fig. 3. Activity assays of tTERT mutants. (A) Catalytic activity of telomerase reconstituted in vitro with C-terminally FLAG-tagged WT or W187A tTERT, or with untagged WT, Q168A or Q168E tTERT. (Upper) SDS/PAGE analysis of the tTERT protein synthesis reactions used for activity assays is shown. Each tTERT was expressed comparably. (Lower) Activity assay reactions performed in the presence of 50 M GTTGGG primer, dGTP, and dTTP were analyzed by denaturing gel electrophoresis. (B) Double reciprocal plot of the rate of product synthesis (v) versus primer concentration ([S]) for enzymes with C-terminally FLAG-tagged WT and W187-substituted tTERTs. C-terminal FLAG-tagged tTERT proteins were used for direct comparison with crosslinking assays, which revealed covalent linkage of W187 to DNA. (C) Double reciprocal plot of the rate of product synthesis (v) versus primer concentration ([S]) for enzymes with untagged WT and Q168-substituted tTERTs.
Telomerase Activity Assays of tTERT Molecules with Altered Amino Acids at or near the Crosslinking Site. We next carried out telom-
erase activity assays of tTERT variants in W187 and adjacent amino acids. We first synthesized tTERT with the substitution W187A. This substituted tTERT was reconstituted into recombinant core enzyme and assayed for catalytic activity by direct primer extension, using the unsubstituted version of the 8794 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703157104
crosslinked DNA 5⬘-GTTGGG-3⬘. In reactions containing dTTP and radiolabeled dGTP, we found that the W187A enzyme synthesized a profile of products that was indistinguishable from the comparably tagged WT enzyme (Fig. 3A, lanes 1 and 2). This remained true for reactions performed with dGTP alone, with a longer primer (GTTGGG)3, with primers bearing different 3⬘ end permutations relative to the template and with a broad range of primer concentrations [shown in supporting information (SI) Fig. 5; additional data not shown]. Furthermore, no significant reduction in the radioactivity incorporated into the longer extension products relative to the shorter products in the mutant versus the WT enzyme was observed (SI Fig. 5C). Thus, the W187A substitution does not appear to have a substantial effect on the enzyme processivity. The various assays comparing the activities of the WT enzyme and the W187A variant were performed by using several different preparations of each of the enzymes. The reason for the contrast between our data and the severe catalytic defect reported for W187A in ref. 11 is not known. To additionally characterize a potential primer binding defect of the W187A enzyme, we determined the primer Km for elongation. We used reactions containing radiolabeled dGTP alone, so that product turnover was forced to occur after primer extension by a single nucleotide. We found that primer Km was little if at all affected by the W187A substitution (Fig. 3B). These results could be due to the existence of additional interactions between the primer and the enzyme that maintain the catalytic activity of the W187 variant. The finding that tTERT W187 is not essential for catalytic activity is consistent with our observation that covalent crosslinking of W187 to primer still allowed primer elongation. Substitutions of tTERT were also made at residues flanking W187. These substitutions were similarly assayed for catalytic activity. SI Fig. 5 reveals that a substitution of the adjacent tyrosine residue W188A did not substantially affect the activity of the enzyme. We also tested tTERT alanine substitutions of each of the four lysine residues, K183, K185, K186, and K189. These individual substitutions, substitutions of pairs of these lysine residues, or triple mutants of W187A, Y188A, and each one of the lysine residues did not cause a substantial reduction in the activity of the enzyme (data not shown). We did observe a significant decrease in the activity of the substitution F158A and of the triple substitution K185I/K186Q/K189A. Interestingly, Jacobs et al. (11) observed a decrease in the activity of the triple mutant K183A/K185A/K186A. Studies have reported that substitution of Q168 strongly compromised catalytic activity (11, 17). We found that product synthesis by tTERT Q168A and Q168E enzymes could be more readily detected if activity assays contained high primer concentration (Fig. 3A, lanes 3–5). Assays of the primer concentration-dependence of product synthesis for the WT and the Q168A and Q168E enzymes revealed a ⬎10-fold increase in primer Km upon Q168A substitution and a ⬇4-fold increase in primer Km upon Q168E substitution (Fig. 3C). Importantly, among the various tTERT substitutions in this region described in this work, only the Q168 substitutions were found to have a specific impact on primer Km. These results suggest that the Q168 side-chain has an influence on the binding affinity of single-stranded DNA. Discussion We present here the first direct physical mapping at a single amino acid resolution of DNA-TERT contacts in functional telomerase complexes. Our crosslinking data clearly demonstrated that in such complexes a close proximity, i.e., a chemical interaction, occurs between tTERT W187 and a DNA primer nucleotide that aligns with the 3⬘ terminus of the RNA template region. Furthermore, the same interaction was found to occur with three different primers that had their 3⬘ end aligned at Romi et al.
different positions along the template (Fig. 2 B–E). This apparent uncoupling of the register of DNA–TERT interaction from the register of DNA–template interaction is consistent with results from our previous DNA footprinting and kinetic assays (10, 18, 19). In the absence of a high resolution crystal structure of a complete telomerase enzyme, our data provide novel information on the folding of the multidomain TERT in the enzyme. Specifically, the data allow, for the first time, estimation of the distances between TERT residues at the boundary of the TEN domain and TERT residues in the RT domain active site (Fig. 4). The precise spatial location of the DNA interaction with W187 relative to the RNA template is defined through the alignment of the IdU substitution with the tTER residue A51. Thus, based on the anticipated length and geometry of an RNA-DNA hybrid, we can estimate the distance between W187 and the aspartic acids in the active site of the telomerase. This distance varies from ⬇17 to ⬇27 Å, depending on whether the primer 3⬘ end aligns with the template 3⬘ end or 5⬘ end, respectively (Fig. 4B). The three primers used for the crosslinking assays (Fig. 2C) simulate the sequential stages of product elongation by telomerase. The results of these assays suggest that during telomere synthesis, the TEN and possibly the TRBD domains are displaced relative to the active site. Such displacement could be accommodated by structural flexibility of the region at the C terminus of the TEN domain (11). The maximal accommodated displacement would set an upper limit on the length of the template-product hybrid, possibly accounting for the dissociation of base-pairs formed at the template 3⬘ end before the template 5⬘ end can be copied (20–22). Curiously, mutation of a tTER motif flanking the template 3⬘ end led to the stalling of product synthesis at the first template position proposed to require unpairing of the template-product hybrid (23). This region of tTER 3⬘ of the template has been proposed to contact the TEN domain directly (8), potentially on a surface distinct from the groove for binding to single-stranded DNA (11). Therefore, we speculate that TEN domain displacements could Romi et al.
coordinate the repositioning of both the template 3⬘-flanking region of tTER and the template-adjacent DNA binding site of tTERT relative to the active site during primer elongation. Compared with previous assays of TERT–DNA interaction, the strength of our procedure is that it ensures that the identified contacts are functionally relevant. No crosslinked TERT–DNA complexes would be labeled in our assays unless the primer is properly aligned at the active site of an active enzyme. This specificity is lacking in experiments using prelabeled DNA molecules, which are bound to various telomerase complexes and then crosslinked. The data obtained in such experiments could represent binding of the DNA primers to inactive enzyme molecules, or nonproductive binding to active molecules in a manner irrelevant to the catalytic process. For similar reasons, data on binding of DNA oligonucleotides to purified TERT do not necessarily apply to functional enzyme complexes. In addition to the contact with W187, the DNA must interact with TERT by making other contacts, because the W187A substitution does not substantially affect the activity of the enzyme. One additional contact detected in our assays occurs with a site located in a segment spanned by the amino acids 192–411. Additionally, we have shown that substitutions made in the tTERT amino acid Q168 strongly affect the Km for primer elongation by the reconstituted core enzyme. These substitutions may influence primer Km directly through altering protein-DNA contact, although we cannot preclude some impact of Q168 substitutions on local protein conformation. The recently solved TEN domain structure places the Q168 side chain in a surface groove predicted to bind single-stranded DNA (11). Interestingly, W187 is located at the end of this groove (11). Finally, we note that in the context of telomerase holoenzyme, tTERT and/or other telomerase-associated proteins are likely to provide additional sites for DNA interaction that contribute to primer binding specificity and to processive elongation. Future studies will be required to investigate the full spectrum of telomerase interaction sites for single-stranded DNA. Materials and Methods Telomerase Reconstitution and Crosslinking. Telomerase core enzyme was reconstituted in rabbit reticulocyte lysate, as described PNAS 兩 May 22, 2007 兩 vol. 104 兩 no. 21 兩 8795
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Fig. 4. Model of catalytically active telomerase complexes. (A) Linear scheme of tTERT with conserved motifs. Motifs 1, 2, A, B⬘, C, D, and E are common to RTs and telomerases. Motifs T and T2 are common to all telomerases. Motifs CP and CP2 are best conserved among TERT proteins from ciliates. D618, D815, and D816 are conserved aspartic acid residues at the active site of the enzyme, and W187 is the site of covalent tTERT-DNA crosslinking. TEN, telomerase essential N-terminal domain; TRBD, telomerase RNA binding domain; RT, reverse transcriptase domain. (B) A cartoon illustrating schematic models for folded structures of the telomerase–DNA complexes that form at the beginning and at the end of a repeat synthesis cycle. In both, a primer nucleotide in the same template register contacts tTERT W187. The blue asterisk indicates the position of the active site in the RT domain. The distance of ⬇17 Å was calculated based on the geometry of the RNA-DNA duplex of 3 base pairs. The distance of ⬇27 Å was calculated for an RNA-DNA duplex of 8 bp. However, the actual distance could be slightly larger because of unpairing of one or more nucleotides at the 3⬘ end of the template.
in ref. 24, using tTER purified after transcription by T7 RNA polymerase and tTERT encoding plasmids. The tTERT polypeptides used for crosslinking studies harbored a C-terminal FLAG tag. Complexes for crosslinking were generated by incubating reconstituted telomerase with an IdU-substituted DNA primer (Operon Biotechnologies, Huntsville, AL) for 10 min at 4°C. A typical 30-l crosslinking reaction mixture contained 50 mM Tris䡠HCl (pH 8.0), 100 mM sodium acetate, 2 mM MgCl2, 1 mM spermidine, 0.2 units RNasin, 10–40 M primer, and 15 l of enzyme in rabbit reticulocyte lysate. Crosslinking was subsequently performed in a 96-well microtiter plate by irradiating the mixtures for 30 min with long-wavelength UV light (⬎295 nm). The UV source was an USH-200dp mercury lamp (Ushio, Simi Valley, CA) that emitted light at 20–25 mW/cm2. A filter was used to block the radiation at wavelengths ⬍295 nm. The temperature of the irradiated mixtures was kept at 4–12°C by placing the plate on top of a HPA100–12 cooler (Melcor). Next, the crosslinked primer molecules were extended with the telomerase by incubation with 0.16 M [␣32P]-dGTP (3,000 Ci/mmol; GE Healthcare, Piscataway, NJ) for 2 h at 25°C. Crosslinking Site Mapping. For cleavage of crosslinked complexes,
tTERT was immunopurified on FLAG antibody M2 resin (Sigma, St. Louis, MO), as described in ref. 18. Briefly, a reaction mixture of 120–150 l containing crosslinked sample was swirled with 20 l of 1:1 bead slurry for 16 h at 4°C. The beads were then thoroughly washed and tTERT was eluted by incubating the beads for 5 min at 95°C in 20 l of 1% SDS. Partial degradation with CNBr was carried out by modifications of published procedures (14, 15). Eluted crosslinked tTERT (35 l) was used for each reaction. Tris䡠HCl (pH 8.0) and BSA were added to final concentrations of 32 mM and 22 g/ml respectively. Cleavage was then achieved by the addition of 1.5 l of 1 M HCl and 1.5 l of 1 M CNBr (dissolved in acetonitrile), and the mixtures were incubated for 10, 20, or 30 min at 25°C. To stop the reactions, Tris䡠HCl (pH 8.8), 2-mercaptoethanol, glycerol, and bromophenol blue were added to final concentrations of 0.1 M, 4%, 10%, and 0.02%. The mixtures were analyzed by electrophoresis in 12% polyacrylamide (29:1)/SDS gels (14, 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
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15). Detection was done by phosphorimaging dried gels. For extensive degradation, a 35-l solution of eluted crosslinked tTERT, 1.5 l of 1 M HCl, and 1.5 l of 9 M CNBr were combined and each mixture was incubated for 20 h at 25°C. The reactions were stopped and analyzed as above by SDS/PAGE. Mutations in plasmids encoding tTERT were generated by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The sequence of the whole tTERT gene in these plasmids was determined at least twice to ensure that mutations only occurred in the desired locations. Activity Assays. For activity assay comparisons, [35S]methionine
was added to the protein expression reactions. Primer extension assays were performed largely as described in ref. 24. For single dGTP addition reactions, 0.6 M of [␣32P]-dGTP (800 Ci/mmol) (GE Healthcare) was used. For complete repeat synthesis reactions, this radiolabeled dGTP was supplemented with 2 M extra unlabeled dGTP and 0.2 mM dTTP. Reactions were performed at room temperature, stopped, and assayed by urea polyacrylamide gel electrophoresis (18). Dried gels were imaged and product intensities were quantified by using the Typhoon (GE Healthcare) and ImageQuant software. Sets of tTERT RNPs were assayed in parallel, and their reaction products were run on the same gel and quantified at the same time. Rates in arbitrary units of phosphorimager signal intensity were calculated by linear fit from a time course of 5-, 10-, and 15-min reactions, well within the linear range. Best linear fit to data in the doublereciprocal plots was used to calculate the Km. We thank Prof. Joseph Salzman, Dr. Boris Meyler, Gregory Avrushchenko, and Ron Praguer from the Microelectronics Research Center– Department of Electrical Engineering, Technion–Israel Institute of Technology for the design and construction of the apparatus for UV irradiation and for the use of facilities in their laboratory. This work was supported by Israel Science Foundation Grant No. 378/03, the Niedersachsen Foundation, the Technion Research and Development Foundation (H.M.), and National Institutes of Health Grant GM54198 (to K.C.). 15. Mustaev A, Zaychikov E, Grachev M, Kozlov M, Severinov K, Epshtein V, Korzheva N, Bereshchenko O, Markovtsov V, Lukhtanov, E. et al. (2003) Methods Enzymol 371:191–206. 16. Lue NF (2005) J Biol Chem 280:26586–26591. 17. Miller MC, Liu JK, Collins K (2000) EMBO J 19:4412–4422. 18. Baran N, Haviv Y, Paul B, Manor H (2002) Nucleic Acids Res 30:5570 –5578. 19. Manor H, Haviv I, Baran N (2002) in Telomeres and Telomerases: Cancer and Biology, eds Krupp G, Parwaresch R, (Landes Bioscience, Austin, TX). 20. Wang H, Gilley D, Blackburn EH (1998) EMBO J 17:1152–1160. 21. Kelleher C, Teixeira MT, Forstemann K, Lingner J (2002) Trends Biochem Sci 27:572–579. 22. Hammond PW, Cech TR (1998) Biochemistry 37:5162–5172. 23. Cunningham DD, Collins K (2005) Mol Cell Biol 25:4442–4454. 24. Collins K, Gandhi L (1998) Proc Natl Acad Sci USA 95:8485–8490. 25. R Development Core Team (2005). R: A language and environment for statistical computing. (R Foundation for Statistical Computing, Vienna, Austria) ISBN 3-900051-07-0, www.R-project.org.
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