Extension of G-quadruplex DNA by ciliate telomerase
2006; Springer Nature; Volume: 25; Issue: 5 Linguagem: Inglês
10.1038/sj.emboj.7601006
ISSN1460-2075
AutoresLiana Oganesian, Ian K. Moon, Tracy M. Bryan, Michael B. Jarstfer,
Tópico(s)RNA Interference and Gene Delivery
ResumoArticle2 March 2006free access Extension of G-quadruplex DNA by ciliate telomerase Liana Oganesian Liana Oganesian Children's Medical Research Institute, Sydney, Australia Search for more papers by this author Ian K Moon Ian K Moon School of Pharmacy, Division of Medicinal Chemistry and Natural Products, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Tracy M Bryan Corresponding Author Tracy M Bryan Children's Medical Research Institute, Sydney, Australia Search for more papers by this author Michael B Jarstfer Corresponding Author Michael B Jarstfer School of Pharmacy, Division of Medicinal Chemistry and Natural Products, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Liana Oganesian Liana Oganesian Children's Medical Research Institute, Sydney, Australia Search for more papers by this author Ian K Moon Ian K Moon School of Pharmacy, Division of Medicinal Chemistry and Natural Products, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Tracy M Bryan Corresponding Author Tracy M Bryan Children's Medical Research Institute, Sydney, Australia Search for more papers by this author Michael B Jarstfer Corresponding Author Michael B Jarstfer School of Pharmacy, Division of Medicinal Chemistry and Natural Products, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Author Information Liana Oganesian1, Ian K Moon2, Tracy M Bryan 1 and Michael B Jarstfer 2 1Children's Medical Research Institute, Sydney, Australia 2School of Pharmacy, Division of Medicinal Chemistry and Natural Products, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA *Corresponding authors: Children's Medical Research Institute, 214 Hawkesbury Road, Westmead, Sydney, NSW 2145, Australia. Tel.: +61 2 9687 2800; Fax: +61 2 9687 2120; E-mail: [email protected] of Pharmacy, Division of Medicinal Chemistry and Natural Products, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, USA. Tel.: +1 919 966 6422; Fax: +1 919 966 0204; E-mail: [email protected] The EMBO Journal (2006)25:1148-1159https://doi.org/10.1038/sj.emboj.7601006 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Telomeric DNA can fold into four-stranded structures known as G-quadruplexes. Here we investigate the ability of G-quadruplex DNA to serve as a substrate for recombinant Tetrahymena and native Euplotes telomerase. Inter- and intramolecular G-quadruplexes were gel-purified and their stability examined using native gel electrophoresis, circular dichroism (CD) and thermal denaturation. While intermolecular G-quadruplexes were highly stable, they were excellent substrates for both ciliate telomerases in primer extension assays. In contrast, intramolecular G-quadruplexes formed in K+ exhibited biphasic unfolding and were not extended by ciliate telomerases. Na+-stabilised intramolecular G-quadruplexes were extended by telomerase owing to their rapid rate of dissociation. The Tetrahymena telomerase protein component bound to inter- but not intramolecular K+-stabilised G-quadruplexes. This study provides evidence that parallel intermolecular G-quadruplexes can serve as substrates for telomerase in vitro, their extension being mediated through direct interactions between this higher-order structure and telomerase. Introduction Telomeres are protein–DNA structures at the ends of eukaryotic chromosomes that protect chromosome ends from fusion and are vital in safeguarding genomic stability. The 3′ strand of telomeres is composed of tandem repeats of short G-rich sequences that protrude as a single-stranded DNA overhang. These repeats are T2AG3 in humans and T2G4 and T4G4, respectively, in the ciliates Tetrahymena and Euplotes (Blackburn and Gall, 1978; Klobutcher et al, 1981). Single strands of telomeric DNA can adopt higher order structures, known as G-quartets, under physiological conditions in vitro (Henderson et al, 1987; Sundquist and Klug, 1989). These structures are defined by the coordination of four guanine residues in a cyclic array, stabilised by Hoogsteen hydrogen-bonding and a centrally located cation (Figure 1A) (Williamson et al, 1989). Multiple layers of G-quartets stack to form G-quadruplexes, in which one or more strands are assembled together in either an intra- or intermolecular configuration (Figure 1). G-quadruplexes exhibit extensive structural polymorphism (reviewed in Simonsson, 2001). The DNA strand orientation may be either parallel (Figure 1C and D), antiparallel (Figure 1B) or in some cases both configurations (Figure 1E). Figure 1.(A) Structure of a G-quartet. Hydrogen bonds between guanines and interaction with a monovalent cation, located centrally, are shown. (B) Solution structure of antiparallel Oxy 3.5 G-quadruplex in sodium (Wang and Patel, 1995) and potassium (Smith et al, 1995). (C, D) Predicted structure of parallel intermolecular Oxy 1.5 G-quadruplex in potassium (C) and 12GT in sodium (D) (this work). (E) Solution structure of antiparallel intramolecular 24GG G-quadruplex in sodium (Wang and Patel, 1994). Download figure Download PowerPoint Evidence supporting the physiological relevance of G-quadruplex structures in vivo is rapidly mounting. Notably, several proteins have been isolated that drive intermolecular G-quadruplex assembly. For example, the β subunit of Oxytricha telomere binding protein has been demonstrated to accelerate formation of dimer and tetramer quartets (Fang and Cech, 1993). Similarly, RAP1, a major dsDNA telomeric binding protein from Saccharomyces cerevisiae, not only binds to but also promotes the formation of a parallel G-quadruplex in the presence of potassium (Giraldo and Rhodes, 1994; Giraldo et al, 1994). Furthermore, numerous factors that resolve G-quadruplex structure (Baran et al, 1997; Harrington et al, 1997; Sun et al, 1998, 1999; Enokizono et al, 2005) and some that specifically cleave G-quadruplex proximal DNA (Liu et al, 1993; Sun et al, 2001) have been identified. The most direct evidence for the existence of these secondary structures in vivo comes from the generation of antibodies against antiparallel G-quadruplex DNA that react with the ciliated protozoan Stylonychia lemnae macronuclei (Schaffitzel et al, 2001). The telomere binding proteins TEBPα and TEBPβ are both required for the formation of these G-quadruplexes at Stylonychia telomeres in vivo (Paeschke et al, 2005). Telomerase is a ribonucleoprotein enzyme, which adds telomeric repeats to the chromosome end by reverse transcription of an integral RNA template (Greider and Blackburn, 1985). The observation that >85% of all cancers exhibit telomerase activity has attracted significant attention to this enzyme (Shay and Bacchetti, 1997). Initially, Zahler et al (1991) demonstrated that telomerase from the ciliate Oxytricha nova does not require primer folding into a G-quadruplex for elongation, and proposed that stabilisation of a primer's secondary structure may in fact inhibit telomere elongation in vivo. However, this seminal research used crude extracts containing telomerase in which G-quadruplex-interacting proteins may have been present, as well as crude mixtures of G-quadruplex DNA. Using purified telomerase isolated from Euplotes aediculatus and purified recombinant Tetrahymena thermophila telomerase, we have re-evaluated the ability of telomerase to extend G-quadruplex DNA. We utilised telomeric oligonucleotides capable of forming both intramolecular and intermolecular conformations, stabilised by either K+ or Na+ ions. Owing to the heterogeneous nature of G-quadruplex conformations, we were careful to isolate particular conformations by gel purification and verify their identity. In the case of intramolecular antiparallel G-quadruplexes, our results validated the initial observation that increasing stabilisation of telomeric secondary structure inhibits telomerase-catalysed primer extension (Zahler et al, 1991). However, intermolecular parallel G-quadruplexes can be extended by both Euplotes and Tetrahymena telomerase, to a greater extent than would be predicted by their slow dissociation. Results Folding and characterisation of G-quadruplex DNA To examine the ability of telomerase to extend G-quadruplex DNA, we prepared and isolated G-rich oligonucleotides folded in either K+ or Na+. Tetrahymena telomeric oligonucleotides (Table I) migrated according to their size on a native gel with no salt, indicating a lack of secondary structure (Figure 2A). In general, folding G-rich DNA gives rise to a heterogeneous population of structures. Indeed, in our hands, most telomeric oligonucleotides folded into heterogeneous mixtures when analysed by nondenaturing PAGE (Figures 2 and 3). In the presence of both Na+ and K+ salts, a large proportion of the 21 and 24-nucleotides (nt) Tetrahymena oligonucleotides migrated faster than expected, indicating that they have a compact structure, which is likely to be an intramolecular G-quadruplex, as has been observed previously (Figure 2B and C; Williamson et al, 1989). Figure 2.Native gel electrophoresis of the indicated oligonucleotides (1 μM) of Tetrahymena telomeric sequence in the absence of salt (A), in 50 mM NaGlu (B) or KGlu (C). T15 is an unstructured molecular weight (MW) marker. Download figure Download PowerPoint Figure 3.Characterisation of gel-purified G-quadruplexes. (A) Native gel electrophoresis of G-quadruplexes formed by Tetrahymena oligonucleotide 24TT in the presence of 50 mM KGlu. Lane 1: T15 unstructured MW marker. Lanes 2 and 3: 24TT oligonucleotide folded in the presence of 50 mM KGlu at 4 and 100 μM concentrations, respectively, prior to gel purification. The intra- and intermolecular bands were isolated from 4 and 100 μM 24TT G-quadruplex mixtures, respectively, and their post-purification profiles are shown in lanes 4 and 5. (B) Native gel electrophoresis of 12GT intermolecular G-quadruplex in 100 mM NaCl pre- (lane 2) and post-gel purification (lane 4). Lanes 1 and 3: T15 MW marker. (C) CD spectra of gel-purified intermolecular 24TT, 21GG, and intramolecular 24TT G-quadruplexes in 50 mM KGlu and intermolecular 12GT G-quadruplex in 100 mM NaCl. The DNA concentrations were 17 μM for intermolecular 24TT and 12GT, 10 μM for intermolecular 21GG and 2.6 μM for intramolecular 24TT. (D) Native gel electrophoresis of gel-purified G-quadruplexes formed by Euplotes oligonucleotides Oxy 3.5 (50 μM, in the presence of 50 mM K+ or NaGlu) and Oxy 1.5 (200 μM, in the presence of 50 mM KGlu). MW markers (M) are 5′-end labelled Poly-T (T10, T20) or non-telomeric DNA (19-mer and 30-mer) stained with Sybr Green. (E) CD spectra of gel-purified 5 μM Oxy 3.5 intramolecular G-quadruplexes (50 mM K+ or NaGlu) and 5 μM Oxy 1.5 intermolecular G-quadruplex (50 mM KGlu). Download figure Download PowerPoint Table 1. Oligonucleotides used in this study Nomenclature Sequence Length Tetrahymena oligonucleotides 6TT 5′-GGGGTT-3′ 6 6GG 5′-TTGGGG-3′ 6 12GT 5′-TGGGGTTGGGGT-3′ 12 24TT 5′-GGGGTTGGGGTTGGGGTTGGGGTT-3′ 24 24GG 5′-TTGGGGTTGGGGTTGGGGTTGGGG-3′ 24 21GG 5′-GGGGTTGGGGTTGGGGTTGGG-3′ 21 48CC 5′-(AACCCC)8-3′ 48 48AA 5′-(CCCCAA)8-3′ 48 30AA 5′-(CCCCAA)5-3′ (RNA) 30 Biot-24TT 5′-Biotin-GGGGTTGGGGTTGGGGTTGGGGTT-3′ 24 Biot-21GG 5′-Biotin-GGGGTTGGGGTTGGGGTTGGG-3′ 21 Euplotes oligonucleotides Oxy 1.5 5′-GGGGTTTTGGGG-3′ 12 Oxy 3.5 5′-GGGGTTTTGGGGTTTTGGGGTTTTGGGG-3′ 28 Ea23 5′-TTTTGGGGTTTTGGGGTTTTGGG-3′ 23 EaTR 5′-CAAAACCCCAAAACC-3′ (RNA) 15 Non-telomeric oligonucleotides Biot-PBR 5′-Biotin-AGCCACTATCGACTACGCGATCAT-3′ 24 T10 5′-TTTTTTTTTT-3′ 10 T15 5′-TTTTTTTTTTTTTTT-3′ 15 T20 5′-TTTTTTTTTTTTTTTTTTTT-3′ 20 In the presence of K+, the 24TT and 21GG oligonucleotides also demonstrated slower mobility species, indicative of intermolecular G-quadruplexes consisting of two or more DNA strands (Figure 2C). The proportion of this band increased with increasing DNA concentrations, as would be expected for an intermolecular complex (data not shown). 24GG did not show a prominent intermolecular G-quadruplex band in K+ (Figure 2C), even though it has been reported that this oligonucleotide can form an intermolecular G-quadruplex in the presence of K+ (Hardin et al, 1991); the reason for this difference under our reaction conditions is unknown. The Tetrahymena telomeric oligonucleotide 12GT also demonstrated a mixture of bands in the presence of Na+, including a band of slower mobility than the T15 marker (Figure 3B). Crosslinking analysis of this band after gel purification indicates that it is a four-stranded intermolecular structure (Figure 1D, Supplementary Figure 1). Since the pattern of G-quadruplexes formed by these oligonucleotides is heterogeneous, it was important to isolate individual conformations for further study. After gel purification, both inter- and intramolecular G-quadruplex conformations of 24TT in K+ were well preserved (Figure 3A, lanes 4 and 5; ∼95% purity), as was the intermolecular form of 12GT in Na+ (Figure 3B, lane 4, ∼99% purity). Structures formed from 21GG were less well-preserved after purification, yielding 60–85% purity (data not shown). The contaminating bands consisted of other G-quadruplexes, not linear DNA. All subsequent analyses were carried out using these gel-purified structures. We confirmed that the observed bands represent G-quadruplexes using circular dichroism (CD). Parallel G-quadruplexes exhibit a positive CD peak at ∼265 nm and a negative CD peak at ∼240 nm, while antiparallel G-quadruplexes exhibit a positive CD peak at ∼295 nm and a negative CD peak at ∼260 nm (Williamson, 1994; Keniry, 2000). The gel-purified intermolecular conformations displayed strong positive and negative peaks at 260/240 nm, respectively, and negligible signals at 295 nm, supporting their assignment as parallel G-quadruplexes (Figure 3C). On the other hand, the isolated intramolecular 24TT G-quadruplex had a strong positive peak at 295 nm and no signal at 260 nm, indicative of an antiparallel conformation (Figure 3C). An intramolecular antiparallel structure has previously been observed by NMR and platination studies for 24GG in Na+ (Wang and Patel, 1994; Redon et al, 2001); the structures of the other Tetrahymena telomeric oligonucleotides used in this study have not been solved. As substrates for Euplotes telomerase, we chose to study Oxy 1.5 and Oxy 3.5 (Table I), which form well-characterised G-quadruplexes. Oxy 3.5 folded in both K+ and Na+ demonstrated a major compact species that migrated below the T20 marker, supporting intramolecular G-quadruplex formation (Figure 3D). Oxy 1.5 folded in K+ showed a shift toward a slower moving species (∼48 nt), supporting a multimeric, possibly four-stranded, intermolecular quadruplex (Figure 3D). G-quadruplex formation of these oligonucleotides was also verified by CD (Figure 3E). Oxy 3.5 folded in K+ or Na+ exhibited positive and negative CD peaks at 291/262 nm and 296/264 nm, respectively. These results verify the formation of antiparallel, intramolecular G-quadruplexes as previously characterised (Smith et al, 1995; Wang and Patel, 1995; Petraccone et al, 2004). Oxy 1.5 folded in K+ showed positive and negative CD peaks at 262 and 238 nm, suggesting the formation of a parallel G-quadruplex. The major compact or multimeric species from each oligomer was isolated by gel purification and used for further characterisations. Stability of Tetrahymena G-quadruplexes The stability of gel-purified G-quadruplexes was measured by examining the melting temperature (Tm) of the folded structures and by employing a complementary strand 'trap' assay (Raghuraman and Cech, 1990). In the 'trap' assay, an excess of a C-rich complementary strand is added to the G-quadruplex and samples are analysed over time by nondenaturing electrophoresis. Provided that the concentration of the C-strand is high enough to trap any unfolded G-strand molecules, the rate of Watson-Crick (WC) duplex formation is indicative of the rate of G-quadruplex unfolding. The rates we observed (below) were independent of the concentration of C-strand oligonucleotide, providing evidence that this strand is not actively invading the quadruplex (data not shown). Upon addition of a 10-fold excess of C-rich (48CC) strand, ∼98 and 100% of 24TT and 21GG Na+-stabilised intramolecular G-quadruplexes, respectively, hybridised to the complementary strand within 1.5 min (Supplementary Figure 2A and D). This suggests that G-quadruplexes formed from both sequences in Na+ are highly unstable and unfold rapidly. Both G-quadruplexes exhibited a monophasic mode of unfolding with a half-life (t1/2) of <1.5 min. The intramolecular 21GG G-quadruplex stabilised in K+ displayed biphasic unfolding kinetics. Approximately 40% of this G-quadruplex hybridised to the complementary strand within 1 min of the reaction; over the following 60 min a further 30% of this G-quadruplex slowly unfolded (Figure 4A and D). The unfolding profile of this G-quadruplex fitted to a double exponential equation, yielding an unfolding rate of 0.95±0.13 h−1 and a t1/2 of 0.7±0.1 h for the slower unfolding population. The population that was immediately accessible to the C-rich strand had a t1/2 of <1 min. Figure 4.Complementary strand trap method for determining the rate of unfolding of gel-purified Tetrahymena G-quadruplexes. The indicated G-quadruplexes were incubated with 10- to 20-fold excess of complementary single-stranded DNA (48AA) or RNA (30AA) and at the indicated time intervals aliquots were loaded onto a native 12% polyacrylamide gel containing 50 mM KGlu (A, B) or 100 mM NaCl (C). T15: MW marker; WC: preannealed Watson–Crick duplex. Note that later time points have less time to migrate into the gel. Plots D–F show the quantitation of the corresponding gels; error bars represent standard deviation from an average of at least two experiments. (A) Unfolding of 32P-labelled intramolecular K+-stabilised 21GG G-quadruplex at 0.54 μM (85% purity). (B) Unfolding of 32P-labelled intermolecular K+-stabilised 21GG G-quadruplex at 3 μM (63% purity). (C) Unfolding of 32P-labelled intermolecular Na+-stabilised 12GT G-quadruplex at 11 μM (99% purity). Download figure Download PowerPoint The intramolecular 24TT K+-stabilised G-quadruplex also demonstrated biphasic unfolding with an initial burst (∼32%) of hybridisation 1.5 min after addition of the C-rich strand followed by slower unfolding (Supplementary Figure 2B and E). Fitting this data to a double exponential equation yielded an unfolding rate of 0.15±0.02 h−1 for the slower unfolding population, which corresponds to a t1/2 of 4.8±0.8 h. The rapidly hybridisable population had a t1/2 of <1 min. Thus, for both oligonucleotides, there are two distinct species: one that is readily hybridisable by the complementary strand and one that is not. Intermolecular G-quadruplexes formed from 21GG, 24TT or 12GT sequences followed a monophasic dissociation trend. Within the 62 min of the hybridisation reaction <6% of the total intermolecular 21GG G-quadruplex contributed to WC duplex formation (Figure 4B and E). The majority of the duplex formation can be attributed to the contaminating intramolecular counterpart of this G-quadruplex. Similarly, <7% of the gel-purified intermolecular 24TT G-quadruplex unfolded within 63 min of incubation with its complementary strand (Supplementary Figure 2C and F). Notably, in unfolding assays using the intermolecular 12GT quadruplex, which is much more pure than the other two, no WC duplex formation is detectable even after 60 min (Figure 4C and F). For all three intermolecular G-quadruplexes, the unfolding rate was too slow to fit to a single exponential equation. Observations from 'trap' assays described above were confirmed by measuring melting temperatures of the intermolecular 21GG and 24TT G-quadruplexes. Both G-quadruplexes displayed high Tm values that increased with increasing G-quadruplex concentration, indicative of intermolecular complexes (Table II). Overall, it is apparent that the parallel intermolecular G-quadruplexes are highly stable. Table 2. Tm values for Tetrahymena and Euplotes gel-purified G-quadruplexesa G-quadruplex Cation Strand orientation Strand stoichiometry Tm (°C)b Tm (°C)c Oxy 3.5 Na+ Antiparallel Intramolecular 60 — Oxy 3.5 K+ Antiparallel Intramolecular 85 — Oxy 1.5 K+ Parallel Intermolecular 88 — 21GG K+ Parallel Intermolecular 82 88 24TT K+ Parallel Intermolecular 77 90 aUV melting curves were monitored at 295 nm in the appropriate folding buffer. Heating and cooling curves were superimposable with only slight hysteresis. b10 μM strand concentration. c20 μM strand concentration. Stability of Euplotes G-quadruplexes Similar measurements of Tm and complementary strand trap assays were carried out to determine the stability of the Euplotes intramolecular G-quadruplexes. In these trap assays, however, an RNA oligonucleotide consisting of the 15-nt Euplotes telomerase RNA template sequence (EaTR) was used as the trap, in order to mimic a telomerase assay. The K+-stabilised Oxy 3.5 intramolecular G-quadruplex was incubated in the presence of increasing EaTR template in K+ reaction buffer (Figure 5A). Lone G-quadruplex exhibited a single band while pre-annealed Oxy 3.5-EaTR showed multiple band shifts, indicating that several higher-order species had formed. The presence of multiple species is likely due to several different binding modes of the RNA template to the DNA primer in 1:1 and 2:1 stoichiometries. These higher-order interactions were not characterised individually, but were treated as contributors to the total shifted species. Figure 5.Euplotes complementary strand 'trap' assays used to determine G-quadruplex stability in the presence of telomerase RNA template (EaTR). 5′ end-labelled 10 μM Oxy 3.5 intramolecular G-quadruplexes in 50 mM KGlu (A) or NaGlu (B) were incubated with 1 or 100 equivalents of unlabelled EaTR template for 1 or 30 min at 25°C and electrophoresed on 20% nondenaturing polyacrylamide gels (in 50 mM K+ (A) or NaGlu (B)). Lane 1: 5′ end-labelled unstructured poly-T markers (M). Lane 2: lone G-quadruplex. Lane 3: preannealed Oxy 3.5-EaTR polymorphic species. Download figure Download PowerPoint To determine if the amount of shifted species changed over time, time course incubations of 1 and 100 molar equivalents of EaTR template at 1 and 30 min time points were conducted (Figure 5A). At 1 min with 100 equivalents of EaTR template, 78% of the K+-stabilised Oxy 3.5 G-quadruplex was conserved, while at 30 min with 100 equivalents of template, 76% of the quadruplex was conserved. This limited gain in shifted species over 29 min indicates that a small population (∼22%) of G-quadruplex was available to rapidly bind to the template, followed by a slower unwinding of the remaining hybridisable population. Thus, like the K+-stabilised Tetrahymena intramolecular G-quadruplex, this structure exhibits biphasic unfolding kinetics. The Na+-stabilised Oxy 3.5 G-quadruplex time course revealed that with 100 equivalents of EaTR template at 1 and 30 min of hybridisation, 45 and 35% of the quadruplex was conserved, respectively (Figure 5B). Thus, like the Tetrahymena intramolecular G-quadruplexes, the Na+-stabilised Oxy 3.5 G-quadruplex is less stable than the K+ form. 'Trap' assays with the Oxy 1.5 intermolecular G-quadruplex were unsuccessful due the radioactive end label interfering with intermolecular G-quadruplex assembly (Uddin et al, 2004); however, the stability of this Euplotes G-quadruplex proved to be very high as determined from Tm measurements (Table II). Extension of folded G-quadruplexes by Tetrahymena telomerase Telomerase activity assays were performed to determine if the G-quadruplexes characterised in the previous sections act as substrates for their respective telomerase enzymes. Na+-stabilised intramolecular G-quadruplexes formed from either 21GG or 24TT were readily extendable by recombinant Tetrahymena telomerase (Figure 6A, Supplementary Figure 3A). This was also the case for 24GG (data not shown). Extension of these G-quadruplexes is likely due to the fact that they are in rapid equilibrium with their linear forms. These G-quadruplexes exhibited higher Km values and equivalent or lower relative Vmax values than their linear counterparts (Table III). This indicates a lower relative specificity (kcat/Km) of the enzyme for these G-quadruplex substrates. This supports the idea that if a Na+-stabilised intramolecular G-quadruplex is in constant equilibrium with its unfolded state, then less linear DNA is available for binding and extension by telomerase. Figure 6.Telomerase activity assays using in vitro reconstituted recombinant Tetrahymena telomerase and G-quadruplexes formed from Tetrahymena oligonucleotides. For all panels, LC=loading control (32P-labelled 100-mer), * represents the unextended 5′ 32P-labelled gel-purified G-quadruplex, GP and UP refer to gel-purified and unpurified G-quadruplexes, respectively. All assays were conducted at 25°C for 60 min (A–C), 10 min (D) or 15 min (E) using ∼2 nM enzyme. (A) Telomerase extension of 32P-labelled intramolecular 21GG ('GP Intra'; 1.8 μM, 85% purity) and unlabelled 21GG (1.8 μM) in 50 mM NaGlu. (B) Telomerase extension of 32P-labelled intramolecular 21GG (1.1 μM, 78% purity) and unlabelled 21GG (1.1 μM) in 150 mM KGlu. (C) Telomerase extension of 32P-labelled intermolecular 21GG (0.7 μM, 66% purity) and unlabelled 21GG (0.7 μM) in 150 mM KGlu. (D) Telomerase extension of 32P-labelled intermolecular 21GG (0.09–1.5 μM, 63% purity) in 50 mM KGlu. The control is unlabelled linear 21GG over the same concentration range, in the absence of KGlu. (E) Telomerase extension of intermolecular 12GT (0.06–8 μM, 99% purity) in 100 mM NaCl. Linear 12GT (−NaCl) and denatured 12GT (+NaCl) G-quadruplex over the same concentration range were used as controls. Download figure Download PowerPoint Table 3. Km values and relative Vmax ratios (G-quadruplex/linear DNA) for gel-purified intramolecular Tetrahymena G-quadruplexes stabilised in 50 mM NaGlu and their linear counterparts Primer Conformation Km±s.d. (nM) Relative Vmax (folded/linear) 24TT Intramolecular 1240±750 0.34±0.09 24TT Linear 560±60 24GG Intramolecular 880±420 1.19±0.4 24GG Linear 160±50 K+-stabilised gel-purified intramolecular G-quadruplexes were not extended by telomerase (Figure 6B, Supplementary Figure 3B). Some very faint extension products were discernable, which could be attributed to the presence of contaminating intermolecular G-quadruplex. This result was unexpected given the immediate hybridisation of 32–40% of this G-quadruplex to its complementary strand followed by a much slower dissociation of the remainder of the population (see Figure 4A and D, Supplementary Figure 2B and E). It is improbable that the absence of accessory protein(s) in the in vitro reconstituted preparation of Tetrahymena telomerase resulted in an inability of telomerase to extend K+-stabilised intramolecular G-quadruplexes, since partially purified native Tetrahymena telomerase also failed to utilise the K+-stabilised intramolecular 24GG G-quadruplex as a primer (data not shown). Intermolecular G-quadruplexes, unlike their intramolecular counterparts, were excellent substrates for Tetrahymena telomerase (Figure 6C, D and E, Supplementary Figure 3B and C). Their extension is not likely due to spontaneous dissociation of the G-quadruplex into its linear state, since it is evident from the unfolding analyses that only a very small proportion (at most ∼6%) of the G-quadruplex unfolds within the first 10 min of the reaction (Figure 4E and F, Supplementary Figure 2F). If the extension of 21GG intermolecular G-quadruplex that is seen in Figure 6D is due to this unfolding then it should be equivalent to the level of extension of linear DNA of the same sequence at 6% of a given concentration of G-quadruplex. However, the extension of linear DNA at 0.09 μM, for example, is 3.8-fold lower than the extension of G-quadruplex at 1.5 μM. The same holds true for the 24TT intermolecular G-quadruplex (Supplementary Figure 3C). This argument is even stronger for the 12GT intermolecular G-quadruplex, since it is extended by telomerase (Figure 6E) in the absence of any detectable unfolding (Figure 4C). To further characterise intermolecular G-quadruplexes as candidate substrates for telomerase, we measured the affinity (Km) and rate constants (kcat) of gel-purified intermolecular 24TT stabilised in K+ and its linear control. Telomerase has a reduced affinity for the intermolecular G-quadruplex, with a Km of 3160±1100 nM, as compared to that of the linear control at 450±230 nM. The kcat values for the G-quadruplex and its linear counterpart measure 0.4±0.1 and 0.18±0.01 min−1, respectively. Despite a higher kcat for the G-quadruplex, its kcat/Km remains lower than that of the linear primer (2.1±0.9 × 103 versus 6.7±2.5 × 103 s−1 M−1). Thus, while the G-quadruplex is a good substrate, its linear counterpart is favoured by telomerase. Extension of folded G-quadruplexes by Euplotes telomerase G-quadruplexes folded from Oxy 3.5 and Oxy 1.5 were tested for their ability to act as primers for native Euplotes telomerase. All telomerase assays i
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