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Recognition of 2′-Deoxy-l -ribonucleoside 5′-Triphosphates by Human Telomerase
Biochemical and Biophysical Research Communications 279, 475– 481 (2000) doi:10.1006/bbrc.2000.3982, available online at http://www.idealibrary.com on
Recognition of 2⬘-Deoxy-L-ribonucleoside 5⬘-Triphosphates by Human Telomerase 1 Toyofumi Yamaguchi,* ,† ,2 Rieko Yamada,* Aki Tomikawa,* Koichi Shudo,‡ Motoki Saito,§ Fuyuki Ishikawa,§ and Mineo Saneyoshi* ,† *Department of Biological Sciences and †Biotechnology Research Center, Teikyo University of Science and Technology, Uenohara, Yamanashi 409-0193, Japan; ‡National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan; and §Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
Received November 13, 2000
Telomerase is classified as one of the reverse transcriptases (RTs). To clarify whether L-enantiomers of natural 2ⴕ-deoxyribonucleoside 5ⴕ-triphosphates (dNTPs) are recognized by human telomerase, a quantitative telomerase assay based on the “stretch PCR” method was developed and used for kinetic analysis of the inhibitory effects of these compounds on the enzyme. Among the four L-enantiomers of dNTPs, L-dTTP and L-dGTP inhibited telomerase activity and the others showed slight or no inhibitory effect. Lineweaver– Burk plot analysis showed that the inhibition modes of L-dTTP and L-dGTP were partially competitive (mixed type) and competitive with the corresponding substrate dNTP, respectively. However, the K i values of L-dTTP and L-dGTP (21 and 15 M) were several times larger than the K m values (3– 6 M). These results suggest that the active site of telomerase is not able to discriminate strictly the chirality of dNTPs, although it is more discriminatory than HIV-1 RT. © 2000 Academic Press
Key Words: chiral discrimination; chirality; enantiomer; inhibitor; inhibition; L-dGTP; L-dTTP; nucleotide; telomerase.
Telomeres, the ends of eukaryotic chromosomes, contain the chromosomal DNA ends which consist of repeated guanine-rich sequences (1). Telomeres play an important role in chromosome organization and stability. The conventional DNA polymerases do not replicate the extreme ends of the linear DNA termi1 This paper constitutes Part 42 of the series “Synthetic Nucleosides and Nucleotides,” Part 41, Tomikawa, A., Kohgo, S., Ikezawa, H., Iwanami, N., Shudo, K., Kawaguchi, T., Saneyoshi, M., and Yamaguchi, T. (1997) Biochem. Biophys. Res. Commun. 239, 329 – 333. 2 To whom correspondence should be addressed. Fax: ⫹81-554-634431. E-mail: [email protected].
nus, and therefore DNA synthesis at chromosome ends results in progressive telomere shortening during successive rounds of cell division (2). Telomerase in human cells is able to extend the telomere DNA, (TTAGGG) n (3), since it is a ribonucleoprotein in which the internal RNA component serves as a template for directing the telomere DNA sequence onto the 3⬘-end of a telomere DNA primer (4, 5). Telomerase activity is very weak in most somatic cells, but telomerase is activated in about 90% of clinical cancers and cell lines (6 –11). It was suggested that this activation of telomerase contributes to tumor formation in vivo (12). Therefore, this enzyme is believed to be a good target for the development of antitumor agents (13–15). As described above, telomerase is classified as one of the reverse transcriptases (RTs). In the case of human immunodeficiency virus type 1 (HIV-1) RT, it has been reported that the enzyme binds and incorporates the L-enantiomer of the natural substrate dTTP when acting on a homopolymeric template-primer in vitro (16, 17). Thus, HIV-1 RT is considered as a low enantioselective enzyme. Indeed, an L-nucleoside analogue, (⫺)-2⬘-deoxy-3⬘thiacytidine (3-TC) (18, 19), has been approved for clinical use against HIV-1. However, it has already been reported that the L-enantiomers of carbocyclic dGTP (20) and 2⬘-fluoro-5-methylarabinofuranosyluracil 5⬘-triphosphate (21) are far less inhibitory against human telomerase, therefore suggesting the stereoselectivity of telomerase for nucleotide substrates. These studies prompted us to evaluate the inhibitory effects of L-enantiomers of natural substrates (Fig. 1) on human telomerase. In the present study, we established a quantitative assay procedure based on the ‘stretch PCR’ method (22) for kinetic analysis, and studied the enantioselectivity of HeLa cell telomerase for four dNTPs.
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FIG. 1.
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Nucleotide analogues examined in this study.
MATERIALS AND METHODS Enantiomers of the dNTPs, L-dTTP, L-dGTP, L-dATP and L-dCTP (16, 23), and 9--D-arabinofuranosylguanine 5⬘-triphosphate (araGTP) (24) were synthesized as reported previously. HeLa cell S100 extract was prepared as reported previously (22). RNase A (DNase free) and 2⬘,3⬘-dideoxyguanosine 5⬘-triphosphate (ddGTP) were purchased from Wako (Osaka, Japan). Taq polymerase (a thermophilic DNA polymerase), which was not associated with 3⬘-5⬘ exonuclease, was purchased from Takara (Otsu, Japan) or Sawady Technology (Tokyo, Japan). SYBR green I was purchased from FMC (Rockland, ME). Standard telomerase assay. A reaction mixture (20 l) for telomere DNA extension comprised 50 mM Tris–potassium acetate, pH 8.5, 1 mM MgCl 2 or 0.1 mM MnCl 2, 1 mM dithiothreitol (DTT), 1 M denatured TAG-U oligodeoxyribonucleotide (5⬘-GTA AAA CGA CGG CCA GTT TGG GGT TGG GGT TGG GGT TG-3⬘), 10 –200 M dGTP, 10 –200 M dTTP, 10 –200 M dATP, and HeLa cell S100 extract (1–1.7 g of protein). Incubation was performed for 10 min at 30°C followed by addition of 13 mM EDTA, pH 8.0 (40 l). The reaction
products were extracted with phenol/chloroform, and precipitated with 0.67 M ammonium acetate and 75% (v/v) ethanol (final concentrations) after addition of 8 g of yeast tRNA as carrier for removal of low-molecular-weight materials. The precipitated DNA was rinsed with cold 80% ethanol, dried and then dissolved in PCR buffer (25 l) for the amplification reaction. PCR buffer comprised 20 mM Tris– HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl 2, 0.1% Igepal CA630 (Sigma, St. Louis, MO), 0.2 M CTA-R oligodeoxyribonucleotide (5⬘-CAG GAA ACA GCT ATG ACC CCT AAC CCT AAC CCT AAC CCT-3⬘), and 250 M each of dATP, dGTP, dCTP and dTTP. The PCR and polyacrylamide gel electrophoresis were carried out according to the instructions in the telomerase assay kit (TeloChaser, Toyobo, Tokyo, Japan) based on the stretch PCR method, with slight modifications. The mixture was overlaid with mineral oil, heated at 95°C for 3 min, cooled to 85°C, and then mixed with 5 l of a solution of Taq polymerase (1 unit) which was preheated at 85°C for 1 min. The thermal cycle was 95°C, 30 s; 68°C, 30 s; 72°C, 45 s for 26 cycles. After treatment of the reaction mixture with 0.1 g RNase A, PCR products were separated by 10% polyacrylamide gel electrophoresis and detected by staining with SYBR green I. The amounts of PCR products constituting a DNA ladder were estimated with a fluorescent image analyzer FLA 3000 (Fuji Film, Tokyo, Japan) and were represented in linear arbitrary units (LAU). Inhibition study. Inhibition studies were performed by addition of varying concentrations of nucleotide analogues to the standard telomerase assay mixture. For determination of kinetic constants, Michaelis constant (K m) and inhibition constant (K i), and inhibition mode, the concentrations of both the inhibitor and the corresponding dNTP were varied in the presence of 200 M of the other dNTPs.
RESULTS Quantitative Telomerase Assay Based on the “Stretch PCR” Method for Kinetic Analysis As shown in Figs. 2A and 2C, the DNAs amplified from telomerase products by the stretch PCR method were detected as a ladder consisting of bands spaced
FIG. 2. Quantitative detection of telomerase activity. Time course of the extension reaction by telomerase (A and B). (A) Polyacrylamide gel electrophoresis of the DNA amplified from telomerase products by stretch PCR. HeLa cell S100 extracts including 1.6 and 0.8 g of protein were analyzed. The position of DNA size markers is shown on the left. (B) Relationship between incubation time and the amount of fluorescence from a DNA ladder amplified by stretch PCR. The amounts of PCR products were determined with a fluorescent image analyzer and are represented in linear arbitrary units (LAU). Relationship between telomerase activity in HeLa cell S100 extract and the amount of PCR products (C and D). (C) Polyacrylamide gel electrophoresis of the DNA amplified from telomerase products by stretch PCR. HeLa cell S100 extracts including 0, 0.2, 0.4, 0.8, and 1.6 g of protein were analyzed. (D) The LAU values estimated from the gel were plotted against the amounts of S100 extracts. 476
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FIG. 3. Lineweaver–Burk plot analyses of the inhibitory effects of ddGTP and araGTP on telomerase. Telomerase activity in the presence of the indicated concentrations of ddGTP (A) and dGTP (C). Lineweaver–Burk plot analyses of the inhibition by ddGTP (B) and araGTP (D). Relative reaction velocity (relative V) was calculated relative to the activity without inhibitor in the presence of 60 M dGTP, which was taken as 1.
six bases apart. Telomerase activity was abolished by pretreatment (30°C, 3 min) with 1 g of RNase A (data not shown). When the time course of telomerase products was examined, the amounts of the products (as LAU values) increased time-dependently and linearly during the time of incubation (Figs. 2A and 2B). From this result, a 10-min incubation time was chosen for the inhibition experiments. Additionally, LAU values of the products increased linearly when the amount of HeLa cell S100 extract was increased (Figs. 2C and 2D). By plotting the amounts of the products (as LAU values) against the number of PCR cycles, the kinetics of the PCR amplification were resolved. The kinetics showed an exponential increase in the PCR product until 27 cycles and partial saturation at 28 cycles (data not shown). Therefore, 26 cycles of amplification was chosen for routine assay. Inhibition by ddGTP and araGTP The results of representative gels of inhibition experiments with ddGTP and araGTP, which are known to be potent telomerase inhibitors (21, 25, 26), are shown in Fig. 3. As the concentration of ddGTP or araGTP increased in the presence of a fixed concentration of dGTP, the products became increasingly shorter and the total amount of the products decreased (Figs. 3A and 3C). We observed that addition of ddGTP or
araGTP after the telomerase extension reaction in the absence of ddGTP or araGTP produced no inhibitory effect (data not shown). By measuring the fluorescence intensity of the ladder in each lane in these gels (Figs. 3A and 3C), the relative velocity of reaction was determined and Lineweaver–Burk plot analyses were performed (Figs. 3B and 3D). The mode of inhibition of human telomerase by both compounds was shown to be competitive with dGTP. Inhibitory Effect of L-Enantiomers of Natural 2⬘-Deoxyribonucleoside 5⬘-Triphosphates (dNTPs) on HeLa Cell Telomerase Activity The inhibitory effects of the four L-enantiomers of natural dNTPs on human telomerase were examined. Figure 4A shows the gels of inhibition experiments with L-dNTPs in the presence of Mg 2⫹ as a divalent cation. Figure 4B shows the resulting inhibition curves determined by measuring the fluorescence intensity of the ladder in each lane in the gels of Fig. 4A. The remaining activity (%) of telomerase at various concentrations of L-dNTPs is indicated. As shown in Figs. 4A and 4B, human telomerase was inhibited moderately by L-dTTP and weakly by L-dGTP. In contrast, telomerase was not or only slightly inhibited by L-dATP or L-dCTP. We observed that addition of L-dTTP or L-dGTP after the telomerase extension reaction in the
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FIG. 4. Inhibition of telomerase activity by L-enantiomers of natural dNTPs in the presence of 1 mM Mg 2⫹as a divalent cation. Telomerase activity in the presence of the indicated concentrations of L-enantiomers (A). Telomerase activity was measured as described in the text in the presence of various concentrations of inhibitor, the D-dNTP (10 M) corresponding to the inhibitor, and the other two dNTPs (200 M each). When L-dCTP was examined, the concentrations of dGTP, dATP, and dTTP were 200, 200, and 10 M, respectively. The activity of the enzyme was estimated in the presence of the indicated concentrations of L-dNTPs. Activity without inhibitor was taken as 100%, and the activities remaining are shown (B). Lineweaver–Burk plot analysis of the inhibition by L-dTTP (C and D). Telomerase activity was measured in the presence of various concentrations of L-dTTP and dTTP, the other two dNTPs (200 M each). Relative reaction velocity (relative V) was calculated relative to the activity without inhibitor in the presence of 100 M dTTP, which was taken as 1.
absence of L-dTTP or L-dGTP produced no inhibitory effect (data not shown). Next, Lineweaver–Burk plot analysis of the inhibitory effect of L-dTTP on telomerase was carried out (Figs. 4C and 4D). Inhibition of L-dTTP was shown to be partially competitive (mixed type) with dTTP. In Fig. 5A, the inhibitory effects of L-dNTPs on telomerase in the presence of Mn 2⫹ as a divalent cation are shown. L-dTTP and L-dGTP exhibited significant inhibitory effect, and the other two L-nucleotides showed slight effect. From Lineweaver–Burk plot analysis, the modes of inhibition for telomerase by L-dTTP were shown to be partially competitive (mixed type) with dTTP (Fig. 5C) and competitive with dGTP (Fig. 5E), respectively. The K i values of L-dTTP and L-dGTP were estimated to be 21 M and 15 M, and the K m values of dTTP and dGTP were estimated to be 3.4 M and 5.9 M, respectively.
DISCUSSION Telomerase synthesizes telomere DNA and contributes to the maintenance of functional telomeres in transformed or highly proliferative cells. Telomerase is a ribonucleoprotein in which the internal RNA component serves as a template for directing the telomere DNA sequence (4, 5). Therefore, telomerase is classified as a reverse transcriptase (RT). In the case of viral RT, HIV-1 RT is able to recognize L-dTTP as a substrate when acting on poly(A)-oligo(dT) as the template-primer (16, 17). Accordingly, we planned to investigate the chiral discrimination of dNTPs by the cellular RT telomerase. Usually, telomerase activity is detected by the PCRbased TRAP (telomeric repeat amplification protocol) assay method. However, many investigators have experienced some difficulty in quantitative analysis of telomerase activity by means of the TRAP assay (6, 7,
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FIG. 5. Inhibition of telomerase activity by L-enantiomers of natural dNTPs in the presence of 0.1 mM Mn 2⫹as a divalent cation. Telomerase activity in the presence of the indicated concentrations of L-enantiomers (A). The activity of the enzyme was estimated in the presence of the indicated concentrations of L-dNTPs. Activity without inhibitor was taken as 100%, and the activities remaining are shown. Telomerase activity was measured in the presence of various concentrations of inhibitor, 10 M of the D-dNTP corresponding to the inhibitor, and the other two dNTPs (200 M each). When L-dCTP was examined, the concentrations of dGTP, dATP, and dTTP were 200, 200, and 10 M, respectively. Lineweaver–Burk plot analyses of the inhibition by L-dTTP (B and C) and L-dGTP (D and E). Relative reaction velocity (relative V) was calculated relative to the activities without inhibitor in the presence of 40 M dTTP (C) or 60 M dGTP (E), which were taken as 1.
10). Independently, Ishikawa and his co-workers developed a new PCR-based method, the ‘stretch PCR’ method, for measuring telomerase activity. They demonstrated that the stretch PCR method was quantitative over a wide range of 10 5-fold and that the length of PCR products did not decrease as the PCR cycles proceeded (22). In the present study, we have optimized the stretch PCR method to perform a kinetic analysis of telomerase. As described under Materials and Methods, the assay procedure was simplified as much as possible in order to obtain reproducible results. In the procedure, the use of a fluorescent image analyzer was essential to estimate the amounts of DNA stained with SYBR green I. With this assay method, Lineweaver–Burk plots were used to analyze the inhibition by ddGTP and araGTP, which are well known to be potent telomerase inhibitors (Fig. 3) (21, 25, 26). The mode of inhibition of telomerase by both compounds was competitive with dGTP. The K i values of ddGTP and araGTP were estimated to be 0.65 and 1.2 M, respectively. These K i
values are 10 –20 times smaller than the K m of dGTP (14 M), implying that the active site of human telomerase seems to prefer ddGTP or araGTP to dGTP. The K m values of dGTP and dTTP (14 and 8.3 M) were similar to those (around 8 M) of telomerase from human leukemic cells reported by Pai et al. (21). Thus, the present assay method has been proved to be quantitative enough to perform Lineweaver–Burk plot analysis. Human telomerase was inhibited partially competitively (mixed type inhibition) by L-dTTP in the presence of Mg 2⫹ or Mn 2⫹, suggesting that L-dTTP binds to the active site and/or to other regions of the telomerase molecule. On the other hand, telomerase was significantly inhibited by L-dGTP in the presence of Mn 2⫹. The mode of inhibition was competitive with dGTP. The competition between L-dGTP and dGTP indicates that both nucleotides bind to the same active site of the enzyme. The K i value of L-dGTP (15 M) is approximately three times larger than the K m of dGTP (5.9 M). In the case of HIV-1 RT, the K i value of L-dTTP
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(1.2 M) is 14 times smaller than the K m of dTTP (17 M) when poly(A)-oligo(dT) is used as the templateprimer in the presence of Mn 2⫹ (16). Thus, the active site of telomerase exhibits low chiral discriminatory ability, although not as low as that of HIV-1 RT. Generally, enzymes are thought to exhibit high substrate specificity and to be able to bind effectively to only one enantiomer of a chiral substrate. However, some exceptions have been shown among enzymes involved in nucleotide biosynthesis and DNA synthesis. Recently, Verri et al. have suggested that the enantioselectivity of enzymes involved in these reactions is less common than was believed until some years ago (27). In the case of telomerase, it has been reported that the L-enantiomers of carbocyclic-2⬘-deoxyguanosine 5⬘-triphosphate and 2⬘fluoro-5-methylarabinofuranosyluracil 5⬘-triphosphate exhibit less inhibitory effect (20, 21). These results suggest that telomerase is a highly stereoselective enzyme for nucleotide analogues. However, in the present study, two L-enantiomers of natural dNTPs were observed to be inhibitory. Our results may support the suggestion of Verri et al. (27). Whether L-enantiomers of natural dNTPs are able to be incorporated into telomere DNA by telomerase remains to be elucidated.
10.
11. 12.
13. 14. 15.
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ACKNOWLEDGMENTS This study was supported in part by Grants-in-Aid for Scientific Research (Category C, 10672097) and Scientific Research on Bioscience/Biotechnology Areas from the Ministry of Education, Science, Sports and Culture of Japan, and grants from the Promotion and Mutual Corporation for Private Schools of Japan and the TV Yamanashi Science Development Fund.
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Recognition of 2⬘-Deoxy-L-ribonucleoside 5⬘-Triphosphates by Human Telomerase 1 Toyofumi Yamaguchi,* ,† ,2 Rieko Yamada,* Aki Tomikawa,* Koichi Shudo,‡ Motoki Saito,§ Fuyuki Ishikawa,§ and Mineo Saneyoshi* ,† *Department of Biological Sciences and †Biotechnology Research Center, Teikyo University of Science and Technology, Uenohara, Yamanashi 409-0193, Japan; ‡National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan; and §Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
Received November 13, 2000
Telomerase is classified as one of the reverse transcriptases (RTs). To clarify whether L-enantiomers of natural 2ⴕ-deoxyribonucleoside 5ⴕ-triphosphates (dNTPs) are recognized by human telomerase, a quantitative telomerase assay based on the “stretch PCR” method was developed and used for kinetic analysis of the inhibitory effects of these compounds on the enzyme. Among the four L-enantiomers of dNTPs, L-dTTP and L-dGTP inhibited telomerase activity and the others showed slight or no inhibitory effect. Lineweaver– Burk plot analysis showed that the inhibition modes of L-dTTP and L-dGTP were partially competitive (mixed type) and competitive with the corresponding substrate dNTP, respectively. However, the K i values of L-dTTP and L-dGTP (21 and 15 M) were several times larger than the K m values (3– 6 M). These results suggest that the active site of telomerase is not able to discriminate strictly the chirality of dNTPs, although it is more discriminatory than HIV-1 RT. © 2000 Academic Press
Key Words: chiral discrimination; chirality; enantiomer; inhibitor; inhibition; L-dGTP; L-dTTP; nucleotide; telomerase.
Telomeres, the ends of eukaryotic chromosomes, contain the chromosomal DNA ends which consist of repeated guanine-rich sequences (1). Telomeres play an important role in chromosome organization and stability. The conventional DNA polymerases do not replicate the extreme ends of the linear DNA termi1 This paper constitutes Part 42 of the series “Synthetic Nucleosides and Nucleotides,” Part 41, Tomikawa, A., Kohgo, S., Ikezawa, H., Iwanami, N., Shudo, K., Kawaguchi, T., Saneyoshi, M., and Yamaguchi, T. (1997) Biochem. Biophys. Res. Commun. 239, 329 – 333. 2 To whom correspondence should be addressed. Fax: ⫹81-554-634431. E-mail: [email protected].
nus, and therefore DNA synthesis at chromosome ends results in progressive telomere shortening during successive rounds of cell division (2). Telomerase in human cells is able to extend the telomere DNA, (TTAGGG) n (3), since it is a ribonucleoprotein in which the internal RNA component serves as a template for directing the telomere DNA sequence onto the 3⬘-end of a telomere DNA primer (4, 5). Telomerase activity is very weak in most somatic cells, but telomerase is activated in about 90% of clinical cancers and cell lines (6 –11). It was suggested that this activation of telomerase contributes to tumor formation in vivo (12). Therefore, this enzyme is believed to be a good target for the development of antitumor agents (13–15). As described above, telomerase is classified as one of the reverse transcriptases (RTs). In the case of human immunodeficiency virus type 1 (HIV-1) RT, it has been reported that the enzyme binds and incorporates the L-enantiomer of the natural substrate dTTP when acting on a homopolymeric template-primer in vitro (16, 17). Thus, HIV-1 RT is considered as a low enantioselective enzyme. Indeed, an L-nucleoside analogue, (⫺)-2⬘-deoxy-3⬘thiacytidine (3-TC) (18, 19), has been approved for clinical use against HIV-1. However, it has already been reported that the L-enantiomers of carbocyclic dGTP (20) and 2⬘-fluoro-5-methylarabinofuranosyluracil 5⬘-triphosphate (21) are far less inhibitory against human telomerase, therefore suggesting the stereoselectivity of telomerase for nucleotide substrates. These studies prompted us to evaluate the inhibitory effects of L-enantiomers of natural substrates (Fig. 1) on human telomerase. In the present study, we established a quantitative assay procedure based on the ‘stretch PCR’ method (22) for kinetic analysis, and studied the enantioselectivity of HeLa cell telomerase for four dNTPs.
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Nucleotide analogues examined in this study.
MATERIALS AND METHODS Enantiomers of the dNTPs, L-dTTP, L-dGTP, L-dATP and L-dCTP (16, 23), and 9--D-arabinofuranosylguanine 5⬘-triphosphate (araGTP) (24) were synthesized as reported previously. HeLa cell S100 extract was prepared as reported previously (22). RNase A (DNase free) and 2⬘,3⬘-dideoxyguanosine 5⬘-triphosphate (ddGTP) were purchased from Wako (Osaka, Japan). Taq polymerase (a thermophilic DNA polymerase), which was not associated with 3⬘-5⬘ exonuclease, was purchased from Takara (Otsu, Japan) or Sawady Technology (Tokyo, Japan). SYBR green I was purchased from FMC (Rockland, ME). Standard telomerase assay. A reaction mixture (20 l) for telomere DNA extension comprised 50 mM Tris–potassium acetate, pH 8.5, 1 mM MgCl 2 or 0.1 mM MnCl 2, 1 mM dithiothreitol (DTT), 1 M denatured TAG-U oligodeoxyribonucleotide (5⬘-GTA AAA CGA CGG CCA GTT TGG GGT TGG GGT TGG GGT TG-3⬘), 10 –200 M dGTP, 10 –200 M dTTP, 10 –200 M dATP, and HeLa cell S100 extract (1–1.7 g of protein). Incubation was performed for 10 min at 30°C followed by addition of 13 mM EDTA, pH 8.0 (40 l). The reaction
products were extracted with phenol/chloroform, and precipitated with 0.67 M ammonium acetate and 75% (v/v) ethanol (final concentrations) after addition of 8 g of yeast tRNA as carrier for removal of low-molecular-weight materials. The precipitated DNA was rinsed with cold 80% ethanol, dried and then dissolved in PCR buffer (25 l) for the amplification reaction. PCR buffer comprised 20 mM Tris– HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl 2, 0.1% Igepal CA630 (Sigma, St. Louis, MO), 0.2 M CTA-R oligodeoxyribonucleotide (5⬘-CAG GAA ACA GCT ATG ACC CCT AAC CCT AAC CCT AAC CCT-3⬘), and 250 M each of dATP, dGTP, dCTP and dTTP. The PCR and polyacrylamide gel electrophoresis were carried out according to the instructions in the telomerase assay kit (TeloChaser, Toyobo, Tokyo, Japan) based on the stretch PCR method, with slight modifications. The mixture was overlaid with mineral oil, heated at 95°C for 3 min, cooled to 85°C, and then mixed with 5 l of a solution of Taq polymerase (1 unit) which was preheated at 85°C for 1 min. The thermal cycle was 95°C, 30 s; 68°C, 30 s; 72°C, 45 s for 26 cycles. After treatment of the reaction mixture with 0.1 g RNase A, PCR products were separated by 10% polyacrylamide gel electrophoresis and detected by staining with SYBR green I. The amounts of PCR products constituting a DNA ladder were estimated with a fluorescent image analyzer FLA 3000 (Fuji Film, Tokyo, Japan) and were represented in linear arbitrary units (LAU). Inhibition study. Inhibition studies were performed by addition of varying concentrations of nucleotide analogues to the standard telomerase assay mixture. For determination of kinetic constants, Michaelis constant (K m) and inhibition constant (K i), and inhibition mode, the concentrations of both the inhibitor and the corresponding dNTP were varied in the presence of 200 M of the other dNTPs.
RESULTS Quantitative Telomerase Assay Based on the “Stretch PCR” Method for Kinetic Analysis As shown in Figs. 2A and 2C, the DNAs amplified from telomerase products by the stretch PCR method were detected as a ladder consisting of bands spaced
FIG. 2. Quantitative detection of telomerase activity. Time course of the extension reaction by telomerase (A and B). (A) Polyacrylamide gel electrophoresis of the DNA amplified from telomerase products by stretch PCR. HeLa cell S100 extracts including 1.6 and 0.8 g of protein were analyzed. The position of DNA size markers is shown on the left. (B) Relationship between incubation time and the amount of fluorescence from a DNA ladder amplified by stretch PCR. The amounts of PCR products were determined with a fluorescent image analyzer and are represented in linear arbitrary units (LAU). Relationship between telomerase activity in HeLa cell S100 extract and the amount of PCR products (C and D). (C) Polyacrylamide gel electrophoresis of the DNA amplified from telomerase products by stretch PCR. HeLa cell S100 extracts including 0, 0.2, 0.4, 0.8, and 1.6 g of protein were analyzed. (D) The LAU values estimated from the gel were plotted against the amounts of S100 extracts. 476
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FIG. 3. Lineweaver–Burk plot analyses of the inhibitory effects of ddGTP and araGTP on telomerase. Telomerase activity in the presence of the indicated concentrations of ddGTP (A) and dGTP (C). Lineweaver–Burk plot analyses of the inhibition by ddGTP (B) and araGTP (D). Relative reaction velocity (relative V) was calculated relative to the activity without inhibitor in the presence of 60 M dGTP, which was taken as 1.
six bases apart. Telomerase activity was abolished by pretreatment (30°C, 3 min) with 1 g of RNase A (data not shown). When the time course of telomerase products was examined, the amounts of the products (as LAU values) increased time-dependently and linearly during the time of incubation (Figs. 2A and 2B). From this result, a 10-min incubation time was chosen for the inhibition experiments. Additionally, LAU values of the products increased linearly when the amount of HeLa cell S100 extract was increased (Figs. 2C and 2D). By plotting the amounts of the products (as LAU values) against the number of PCR cycles, the kinetics of the PCR amplification were resolved. The kinetics showed an exponential increase in the PCR product until 27 cycles and partial saturation at 28 cycles (data not shown). Therefore, 26 cycles of amplification was chosen for routine assay. Inhibition by ddGTP and araGTP The results of representative gels of inhibition experiments with ddGTP and araGTP, which are known to be potent telomerase inhibitors (21, 25, 26), are shown in Fig. 3. As the concentration of ddGTP or araGTP increased in the presence of a fixed concentration of dGTP, the products became increasingly shorter and the total amount of the products decreased (Figs. 3A and 3C). We observed that addition of ddGTP or
araGTP after the telomerase extension reaction in the absence of ddGTP or araGTP produced no inhibitory effect (data not shown). By measuring the fluorescence intensity of the ladder in each lane in these gels (Figs. 3A and 3C), the relative velocity of reaction was determined and Lineweaver–Burk plot analyses were performed (Figs. 3B and 3D). The mode of inhibition of human telomerase by both compounds was shown to be competitive with dGTP. Inhibitory Effect of L-Enantiomers of Natural 2⬘-Deoxyribonucleoside 5⬘-Triphosphates (dNTPs) on HeLa Cell Telomerase Activity The inhibitory effects of the four L-enantiomers of natural dNTPs on human telomerase were examined. Figure 4A shows the gels of inhibition experiments with L-dNTPs in the presence of Mg 2⫹ as a divalent cation. Figure 4B shows the resulting inhibition curves determined by measuring the fluorescence intensity of the ladder in each lane in the gels of Fig. 4A. The remaining activity (%) of telomerase at various concentrations of L-dNTPs is indicated. As shown in Figs. 4A and 4B, human telomerase was inhibited moderately by L-dTTP and weakly by L-dGTP. In contrast, telomerase was not or only slightly inhibited by L-dATP or L-dCTP. We observed that addition of L-dTTP or L-dGTP after the telomerase extension reaction in the
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FIG. 4. Inhibition of telomerase activity by L-enantiomers of natural dNTPs in the presence of 1 mM Mg 2⫹as a divalent cation. Telomerase activity in the presence of the indicated concentrations of L-enantiomers (A). Telomerase activity was measured as described in the text in the presence of various concentrations of inhibitor, the D-dNTP (10 M) corresponding to the inhibitor, and the other two dNTPs (200 M each). When L-dCTP was examined, the concentrations of dGTP, dATP, and dTTP were 200, 200, and 10 M, respectively. The activity of the enzyme was estimated in the presence of the indicated concentrations of L-dNTPs. Activity without inhibitor was taken as 100%, and the activities remaining are shown (B). Lineweaver–Burk plot analysis of the inhibition by L-dTTP (C and D). Telomerase activity was measured in the presence of various concentrations of L-dTTP and dTTP, the other two dNTPs (200 M each). Relative reaction velocity (relative V) was calculated relative to the activity without inhibitor in the presence of 100 M dTTP, which was taken as 1.
absence of L-dTTP or L-dGTP produced no inhibitory effect (data not shown). Next, Lineweaver–Burk plot analysis of the inhibitory effect of L-dTTP on telomerase was carried out (Figs. 4C and 4D). Inhibition of L-dTTP was shown to be partially competitive (mixed type) with dTTP. In Fig. 5A, the inhibitory effects of L-dNTPs on telomerase in the presence of Mn 2⫹ as a divalent cation are shown. L-dTTP and L-dGTP exhibited significant inhibitory effect, and the other two L-nucleotides showed slight effect. From Lineweaver–Burk plot analysis, the modes of inhibition for telomerase by L-dTTP were shown to be partially competitive (mixed type) with dTTP (Fig. 5C) and competitive with dGTP (Fig. 5E), respectively. The K i values of L-dTTP and L-dGTP were estimated to be 21 M and 15 M, and the K m values of dTTP and dGTP were estimated to be 3.4 M and 5.9 M, respectively.
DISCUSSION Telomerase synthesizes telomere DNA and contributes to the maintenance of functional telomeres in transformed or highly proliferative cells. Telomerase is a ribonucleoprotein in which the internal RNA component serves as a template for directing the telomere DNA sequence (4, 5). Therefore, telomerase is classified as a reverse transcriptase (RT). In the case of viral RT, HIV-1 RT is able to recognize L-dTTP as a substrate when acting on poly(A)-oligo(dT) as the template-primer (16, 17). Accordingly, we planned to investigate the chiral discrimination of dNTPs by the cellular RT telomerase. Usually, telomerase activity is detected by the PCRbased TRAP (telomeric repeat amplification protocol) assay method. However, many investigators have experienced some difficulty in quantitative analysis of telomerase activity by means of the TRAP assay (6, 7,
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FIG. 5. Inhibition of telomerase activity by L-enantiomers of natural dNTPs in the presence of 0.1 mM Mn 2⫹as a divalent cation. Telomerase activity in the presence of the indicated concentrations of L-enantiomers (A). The activity of the enzyme was estimated in the presence of the indicated concentrations of L-dNTPs. Activity without inhibitor was taken as 100%, and the activities remaining are shown. Telomerase activity was measured in the presence of various concentrations of inhibitor, 10 M of the D-dNTP corresponding to the inhibitor, and the other two dNTPs (200 M each). When L-dCTP was examined, the concentrations of dGTP, dATP, and dTTP were 200, 200, and 10 M, respectively. Lineweaver–Burk plot analyses of the inhibition by L-dTTP (B and C) and L-dGTP (D and E). Relative reaction velocity (relative V) was calculated relative to the activities without inhibitor in the presence of 40 M dTTP (C) or 60 M dGTP (E), which were taken as 1.
10). Independently, Ishikawa and his co-workers developed a new PCR-based method, the ‘stretch PCR’ method, for measuring telomerase activity. They demonstrated that the stretch PCR method was quantitative over a wide range of 10 5-fold and that the length of PCR products did not decrease as the PCR cycles proceeded (22). In the present study, we have optimized the stretch PCR method to perform a kinetic analysis of telomerase. As described under Materials and Methods, the assay procedure was simplified as much as possible in order to obtain reproducible results. In the procedure, the use of a fluorescent image analyzer was essential to estimate the amounts of DNA stained with SYBR green I. With this assay method, Lineweaver–Burk plots were used to analyze the inhibition by ddGTP and araGTP, which are well known to be potent telomerase inhibitors (Fig. 3) (21, 25, 26). The mode of inhibition of telomerase by both compounds was competitive with dGTP. The K i values of ddGTP and araGTP were estimated to be 0.65 and 1.2 M, respectively. These K i
values are 10 –20 times smaller than the K m of dGTP (14 M), implying that the active site of human telomerase seems to prefer ddGTP or araGTP to dGTP. The K m values of dGTP and dTTP (14 and 8.3 M) were similar to those (around 8 M) of telomerase from human leukemic cells reported by Pai et al. (21). Thus, the present assay method has been proved to be quantitative enough to perform Lineweaver–Burk plot analysis. Human telomerase was inhibited partially competitively (mixed type inhibition) by L-dTTP in the presence of Mg 2⫹ or Mn 2⫹, suggesting that L-dTTP binds to the active site and/or to other regions of the telomerase molecule. On the other hand, telomerase was significantly inhibited by L-dGTP in the presence of Mn 2⫹. The mode of inhibition was competitive with dGTP. The competition between L-dGTP and dGTP indicates that both nucleotides bind to the same active site of the enzyme. The K i value of L-dGTP (15 M) is approximately three times larger than the K m of dGTP (5.9 M). In the case of HIV-1 RT, the K i value of L-dTTP
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(1.2 M) is 14 times smaller than the K m of dTTP (17 M) when poly(A)-oligo(dT) is used as the templateprimer in the presence of Mn 2⫹ (16). Thus, the active site of telomerase exhibits low chiral discriminatory ability, although not as low as that of HIV-1 RT. Generally, enzymes are thought to exhibit high substrate specificity and to be able to bind effectively to only one enantiomer of a chiral substrate. However, some exceptions have been shown among enzymes involved in nucleotide biosynthesis and DNA synthesis. Recently, Verri et al. have suggested that the enantioselectivity of enzymes involved in these reactions is less common than was believed until some years ago (27). In the case of telomerase, it has been reported that the L-enantiomers of carbocyclic-2⬘-deoxyguanosine 5⬘-triphosphate and 2⬘fluoro-5-methylarabinofuranosyluracil 5⬘-triphosphate exhibit less inhibitory effect (20, 21). These results suggest that telomerase is a highly stereoselective enzyme for nucleotide analogues. However, in the present study, two L-enantiomers of natural dNTPs were observed to be inhibitory. Our results may support the suggestion of Verri et al. (27). Whether L-enantiomers of natural dNTPs are able to be incorporated into telomere DNA by telomerase remains to be elucidated.
10.
11. 12.
13. 14. 15.
16.
17.
ACKNOWLEDGMENTS This study was supported in part by Grants-in-Aid for Scientific Research (Category C, 10672097) and Scientific Research on Bioscience/Biotechnology Areas from the Ministry of Education, Science, Sports and Culture of Japan, and grants from the Promotion and Mutual Corporation for Private Schools of Japan and the TV Yamanashi Science Development Fund.
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