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3'-Azido-2',3'-dideoxynucleoside 5'-triphosphates inhibit telomerase activity in vitro, and the corresponding nucleosides cause telomere shortening in human HL60 cells.

Liu X, Takahashi H, Harada Y, Ogawara T, Ogimura Y, Mizushina Y, Saneyoshi M, Yamaguchi T - Nucleic Acids Res. (2007)

Bottom Line: To obtain more selective and potent inhibitors that can be employed as tools for studying telomerase, we investigated the telomerase-inhibitory effects of purine nucleosides bearing a 3'-down azido group: 3'-azido-2',3'-dideoxyguanosine (AZddG) 5'-triphosphate (AZddGTP), 3'-azido-2',3'-dideoxy-6-thioguanosine (AZddSG) 5'-triphosphate (AZddSGTP), 3'-azido-2',3'-dideoxyadenosine (AZddA) 5'-triphosphate (AZddATP) and 3'-azido-2',3'-dideoxy-2-aminoadenosine (AZddAA) 5'-triphosphate (AZddAATP).AZddGTP was significantly incorporated into the 3'-terminus of DNA by partially purified telomerase.However, AZddGTP did not exhibit significant inhibitory activity against DNA polymerases alpha and delta, suggesting that AZddGTP is a selective inhibitor of telomerase.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Center, Teikyo University of Science and Technology, Uenohara, Yamanashi, Japan.

ABSTRACT
Telomerase adds telomeric DNA repeats to the ends of linear chromosomal DNA. 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) is a known telomerase inhibitor. To obtain more selective and potent inhibitors that can be employed as tools for studying telomerase, we investigated the telomerase-inhibitory effects of purine nucleosides bearing a 3'-down azido group: 3'-azido-2',3'-dideoxyguanosine (AZddG) 5'-triphosphate (AZddGTP), 3'-azido-2',3'-dideoxy-6-thioguanosine (AZddSG) 5'-triphosphate (AZddSGTP), 3'-azido-2',3'-dideoxyadenosine (AZddA) 5'-triphosphate (AZddATP) and 3'-azido-2',3'-dideoxy-2-aminoadenosine (AZddAA) 5'-triphosphate (AZddAATP). Of these, AZddGTP showed the most potent inhibitory activity against HeLa cell telomerase. AZddGTP was significantly incorporated into the 3'-terminus of DNA by partially purified telomerase. However, AZddGTP did not exhibit significant inhibitory activity against DNA polymerases alpha and delta, suggesting that AZddGTP is a selective inhibitor of telomerase. We also investigated whether long-term treatment with these nucleosides could alter telomere length and growth rates of human HL60 cells in culture. Southern hybridization analysis of genomic DNA prepared from cells cultured in the presence of AZddG and AZddAA revealed reproducible telomere shortening.

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Utilization of dGTP and dATP analogs as substrates by cherry salmon telomerase. An 18-mer (TTAGGG)3 was extended by partially purified cherry salmon telomerase in the presence of 5 µM [α-32P]dTTP, 200 µM dATP, 0 or 5 µM dGTP and 0, 5 or 20 µM dGTP analogs for dGTP analog analysis, and 5 µM [α-32P]dTTP, 200 µM dGTP, 0 or 5 µM dATP and 0, 5 or 20 µM dATP analogs for dATP analog analysis.
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Figure 4: Utilization of dGTP and dATP analogs as substrates by cherry salmon telomerase. An 18-mer (TTAGGG)3 was extended by partially purified cherry salmon telomerase in the presence of 5 µM [α-32P]dTTP, 200 µM dATP, 0 or 5 µM dGTP and 0, 5 or 20 µM dGTP analogs for dGTP analog analysis, and 5 µM [α-32P]dTTP, 200 µM dGTP, 0 or 5 µM dATP and 0, 5 or 20 µM dATP analogs for dATP analog analysis.

Mentions: We analyzed the primer extension products synthesized by cherry salmon telomerase in the presence of ddGTP, araGTP, AZddSGTP or AZddGTP instead of dGTP, and in the presence of AZddAATP instead of dATP. The results are shown in Figure 4. These experiments were performed using an 18-mer of (5′-TTAGGG-3′)3 capable of incorporating two 32P-labeled dTMP molecules at the initial two residues, unlabled dAMP at the third and unlabled dGMP at the fourth position (22-mer) from the 3′-end of the primer. The primer extension experiment in the presence of [α-32P]dTTP, dATP and dGTP (complete reaction, lane 4 in Figure 4) gave long products of various lengths, as expected. Omission of dGTP (control reactions, lanes 1 and 6) gave products that migrated as 19-, 20- and 21-mer bands. Similarly, omission of dATP (control reaction, lane 15) gave products that migrated as 19- and 20-mer bands. When examined in the presence of [α-32P]dTTP, dATP with AZddGTP (lanes 11 and 12) or ddGTP (lanes 7 and 8) instead of dGTP gave a distinct 22-mer product, whereas AZddSGTP (lanes 2 and 3) did not give a clear 22-mer product. This means that AZddGTP, but not AZddSGTP, is effectively utilized by telomerase as a substrate and incorporated into the 3′-terminus of the primer strand, and that chain termination is elicited. When examined in the presence of [α-32P]dTTP, dGTP and AZddAATP (lanes 13 and 14) instead of dATP gave only 19- and 20-mer products, similar to the control reaction (lane 15). In this experiment, the telomerase employed was partially purified from a crude extract of immature testes of cherry salmon by diethylaminoethyl cellulose column chromatography and 70% ammonium sulfate fractionation, followed by dialysis against buffer. One explanation for the faint 22-mer bands in lanes 1, 2, 3 and 6 of Figure 4 is misincorporation of dGMP. However, we did not investigate this issue further.Figure 4.


3'-Azido-2',3'-dideoxynucleoside 5'-triphosphates inhibit telomerase activity in vitro, and the corresponding nucleosides cause telomere shortening in human HL60 cells.

Liu X, Takahashi H, Harada Y, Ogawara T, Ogimura Y, Mizushina Y, Saneyoshi M, Yamaguchi T - Nucleic Acids Res. (2007)

Utilization of dGTP and dATP analogs as substrates by cherry salmon telomerase. An 18-mer (TTAGGG)3 was extended by partially purified cherry salmon telomerase in the presence of 5 µM [α-32P]dTTP, 200 µM dATP, 0 or 5 µM dGTP and 0, 5 or 20 µM dGTP analogs for dGTP analog analysis, and 5 µM [α-32P]dTTP, 200 µM dGTP, 0 or 5 µM dATP and 0, 5 or 20 µM dATP analogs for dATP analog analysis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175342&req=5

Figure 4: Utilization of dGTP and dATP analogs as substrates by cherry salmon telomerase. An 18-mer (TTAGGG)3 was extended by partially purified cherry salmon telomerase in the presence of 5 µM [α-32P]dTTP, 200 µM dATP, 0 or 5 µM dGTP and 0, 5 or 20 µM dGTP analogs for dGTP analog analysis, and 5 µM [α-32P]dTTP, 200 µM dGTP, 0 or 5 µM dATP and 0, 5 or 20 µM dATP analogs for dATP analog analysis.
Mentions: We analyzed the primer extension products synthesized by cherry salmon telomerase in the presence of ddGTP, araGTP, AZddSGTP or AZddGTP instead of dGTP, and in the presence of AZddAATP instead of dATP. The results are shown in Figure 4. These experiments were performed using an 18-mer of (5′-TTAGGG-3′)3 capable of incorporating two 32P-labeled dTMP molecules at the initial two residues, unlabled dAMP at the third and unlabled dGMP at the fourth position (22-mer) from the 3′-end of the primer. The primer extension experiment in the presence of [α-32P]dTTP, dATP and dGTP (complete reaction, lane 4 in Figure 4) gave long products of various lengths, as expected. Omission of dGTP (control reactions, lanes 1 and 6) gave products that migrated as 19-, 20- and 21-mer bands. Similarly, omission of dATP (control reaction, lane 15) gave products that migrated as 19- and 20-mer bands. When examined in the presence of [α-32P]dTTP, dATP with AZddGTP (lanes 11 and 12) or ddGTP (lanes 7 and 8) instead of dGTP gave a distinct 22-mer product, whereas AZddSGTP (lanes 2 and 3) did not give a clear 22-mer product. This means that AZddGTP, but not AZddSGTP, is effectively utilized by telomerase as a substrate and incorporated into the 3′-terminus of the primer strand, and that chain termination is elicited. When examined in the presence of [α-32P]dTTP, dGTP and AZddAATP (lanes 13 and 14) instead of dATP gave only 19- and 20-mer products, similar to the control reaction (lane 15). In this experiment, the telomerase employed was partially purified from a crude extract of immature testes of cherry salmon by diethylaminoethyl cellulose column chromatography and 70% ammonium sulfate fractionation, followed by dialysis against buffer. One explanation for the faint 22-mer bands in lanes 1, 2, 3 and 6 of Figure 4 is misincorporation of dGMP. However, we did not investigate this issue further.Figure 4.

Bottom Line: To obtain more selective and potent inhibitors that can be employed as tools for studying telomerase, we investigated the telomerase-inhibitory effects of purine nucleosides bearing a 3'-down azido group: 3'-azido-2',3'-dideoxyguanosine (AZddG) 5'-triphosphate (AZddGTP), 3'-azido-2',3'-dideoxy-6-thioguanosine (AZddSG) 5'-triphosphate (AZddSGTP), 3'-azido-2',3'-dideoxyadenosine (AZddA) 5'-triphosphate (AZddATP) and 3'-azido-2',3'-dideoxy-2-aminoadenosine (AZddAA) 5'-triphosphate (AZddAATP).AZddGTP was significantly incorporated into the 3'-terminus of DNA by partially purified telomerase.However, AZddGTP did not exhibit significant inhibitory activity against DNA polymerases alpha and delta, suggesting that AZddGTP is a selective inhibitor of telomerase.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Center, Teikyo University of Science and Technology, Uenohara, Yamanashi, Japan.

ABSTRACT
Telomerase adds telomeric DNA repeats to the ends of linear chromosomal DNA. 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) is a known telomerase inhibitor. To obtain more selective and potent inhibitors that can be employed as tools for studying telomerase, we investigated the telomerase-inhibitory effects of purine nucleosides bearing a 3'-down azido group: 3'-azido-2',3'-dideoxyguanosine (AZddG) 5'-triphosphate (AZddGTP), 3'-azido-2',3'-dideoxy-6-thioguanosine (AZddSG) 5'-triphosphate (AZddSGTP), 3'-azido-2',3'-dideoxyadenosine (AZddA) 5'-triphosphate (AZddATP) and 3'-azido-2',3'-dideoxy-2-aminoadenosine (AZddAA) 5'-triphosphate (AZddAATP). Of these, AZddGTP showed the most potent inhibitory activity against HeLa cell telomerase. AZddGTP was significantly incorporated into the 3'-terminus of DNA by partially purified telomerase. However, AZddGTP did not exhibit significant inhibitory activity against DNA polymerases alpha and delta, suggesting that AZddGTP is a selective inhibitor of telomerase. We also investigated whether long-term treatment with these nucleosides could alter telomere length and growth rates of human HL60 cells in culture. Southern hybridization analysis of genomic DNA prepared from cells cultured in the presence of AZddG and AZddAA revealed reproducible telomere shortening.

Show MeSH
Related in: MedlinePlus