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Pif1 regulates telomere length by preferentially removing telomerase from long telomere ends.

Li JR, Yu TY, Chien IC, Lu CY, Lin JJ, Li HW - Nucleic Acids Res. (2014)

Bottom Line: Using its RNA component as a template, telomerase uses its reverse transcriptase activity to extend the 3'-end single-stranded, repetitive telomeric DNA sequence.Pif1, a 5'-to-3' helicase, has been suggested to regulate telomerase activity.We used single-molecule experiments to directly show that Pif1 helicase regulates telomerase activity by removing telomerase from telomere ends, allowing the cycling of the telomerase for additional extension processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, National Taiwan University, Taiwan.

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Observation of the multiple-rounds telomerase-mediated telomere extension. (A–D) BM histograms of the control custom-prepared DNA substrates mimicking the telomerase-extended products. (A) BM histogram of the mimicked ‘single-round’ DNA substrates. (B) Mimicked ‘double-rounds’ BM histogram. Since the oligo probe can anneal to two repetitive sequences in the ‘double-rounds’ DNA substrate, there are two BM peaks with the higher BM peak representing the ‘double-rounds’ population. (C) Mimicked ‘three-rounds’ BM histogram. (D) Mimicked ‘four-rounds’ BM histogram. (E) TPM method to monitor the in vitro multiple rounds telomerase extension activity. DNA substrates with same telomere sequence in the 3′-end were incubated at 30°C with bead-labeled telomerase and specified nucleotides. The telomerase-mediated telomere extension reactions were stopped by protease K and RNase H. The purified DNA substrates were then immobilized in glass surface and annealed with oligo probes, followed by TPM analysis. (F) BM histogram of DNA without telomerase incubation. (G–J) BM histogram of DNA substrates incubated with telomerase in the presence of dTTP and dGTP for 0.5 h, 1 h, 3 h and overnight. The percentage of the lowest BM peak (non-extended and extended once) decreases with the reaction time.
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Figure 1: Observation of the multiple-rounds telomerase-mediated telomere extension. (A–D) BM histograms of the control custom-prepared DNA substrates mimicking the telomerase-extended products. (A) BM histogram of the mimicked ‘single-round’ DNA substrates. (B) Mimicked ‘double-rounds’ BM histogram. Since the oligo probe can anneal to two repetitive sequences in the ‘double-rounds’ DNA substrate, there are two BM peaks with the higher BM peak representing the ‘double-rounds’ population. (C) Mimicked ‘three-rounds’ BM histogram. (D) Mimicked ‘four-rounds’ BM histogram. (E) TPM method to monitor the in vitro multiple rounds telomerase extension activity. DNA substrates with same telomere sequence in the 3′-end were incubated at 30°C with bead-labeled telomerase and specified nucleotides. The telomerase-mediated telomere extension reactions were stopped by protease K and RNase H. The purified DNA substrates were then immobilized in glass surface and annealed with oligo probes, followed by TPM analysis. (F) BM histogram of DNA without telomerase incubation. (G–J) BM histogram of DNA substrates incubated with telomerase in the presence of dTTP and dGTP for 0.5 h, 1 h, 3 h and overnight. The percentage of the lowest BM peak (non-extended and extended once) decreases with the reaction time.

Mentions: We first tested whether the observed BM amplitudes of the TPM extension assay could reflect the telomerase-mediated telomere extension. Here we prepared various 5′-digoxigenin labeled DNA substrates of different lengths that mimicked the ssDNA extension products after one, two, three and four rounds of extensions by telomerase (see Supplementary Table SI (D) for DNA substrates). The DNA substrates were immobilized onto the anti-digoxigenin-coated slide surface, and were then annealed to oligo probes coated by 220 nm polystyrene beads. These individually tethered DNA-beads undergo BM, which can be visualized under an optical microscope, and BM distributions were then analyzed. The mimicked ‘single-round’ extension DNA substrate showed a single BM peak at 11.1 ± 3.0 nm (mean ± SD, Figure 1A). In the case of a multiple-rounds extension, the probe oligos can anneal to several different positions, due to the repetitive nature of the telomere sequences. As expected, DNA substrates that mimicked ‘double-rounds’ of extensions showed two peaks, with an additional, higher BM peak at ∼19.1 nm (Figure 1B). Similarly, DNA substrates with ‘three-rounds’ extensions showed peaks at ∼10.6, 18.0 and 23.9 nm, and those with ‘four-rounds’ extensions showed peaks at ∼11.1, 17.2, 24.0 and 33.3 nm (Figure 1C and D). Significantly, we found a linear relationship between the number of extended rounds and the observed BM peak values (Supplementary Figure S1A). Thus, our TPM-based assay is capable of detecting multiple-rounds of telomerase-mediated telomere extension products.


Pif1 regulates telomere length by preferentially removing telomerase from long telomere ends.

Li JR, Yu TY, Chien IC, Lu CY, Lin JJ, Li HW - Nucleic Acids Res. (2014)

Observation of the multiple-rounds telomerase-mediated telomere extension. (A–D) BM histograms of the control custom-prepared DNA substrates mimicking the telomerase-extended products. (A) BM histogram of the mimicked ‘single-round’ DNA substrates. (B) Mimicked ‘double-rounds’ BM histogram. Since the oligo probe can anneal to two repetitive sequences in the ‘double-rounds’ DNA substrate, there are two BM peaks with the higher BM peak representing the ‘double-rounds’ population. (C) Mimicked ‘three-rounds’ BM histogram. (D) Mimicked ‘four-rounds’ BM histogram. (E) TPM method to monitor the in vitro multiple rounds telomerase extension activity. DNA substrates with same telomere sequence in the 3′-end were incubated at 30°C with bead-labeled telomerase and specified nucleotides. The telomerase-mediated telomere extension reactions were stopped by protease K and RNase H. The purified DNA substrates were then immobilized in glass surface and annealed with oligo probes, followed by TPM analysis. (F) BM histogram of DNA without telomerase incubation. (G–J) BM histogram of DNA substrates incubated with telomerase in the presence of dTTP and dGTP for 0.5 h, 1 h, 3 h and overnight. The percentage of the lowest BM peak (non-extended and extended once) decreases with the reaction time.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Observation of the multiple-rounds telomerase-mediated telomere extension. (A–D) BM histograms of the control custom-prepared DNA substrates mimicking the telomerase-extended products. (A) BM histogram of the mimicked ‘single-round’ DNA substrates. (B) Mimicked ‘double-rounds’ BM histogram. Since the oligo probe can anneal to two repetitive sequences in the ‘double-rounds’ DNA substrate, there are two BM peaks with the higher BM peak representing the ‘double-rounds’ population. (C) Mimicked ‘three-rounds’ BM histogram. (D) Mimicked ‘four-rounds’ BM histogram. (E) TPM method to monitor the in vitro multiple rounds telomerase extension activity. DNA substrates with same telomere sequence in the 3′-end were incubated at 30°C with bead-labeled telomerase and specified nucleotides. The telomerase-mediated telomere extension reactions were stopped by protease K and RNase H. The purified DNA substrates were then immobilized in glass surface and annealed with oligo probes, followed by TPM analysis. (F) BM histogram of DNA without telomerase incubation. (G–J) BM histogram of DNA substrates incubated with telomerase in the presence of dTTP and dGTP for 0.5 h, 1 h, 3 h and overnight. The percentage of the lowest BM peak (non-extended and extended once) decreases with the reaction time.
Mentions: We first tested whether the observed BM amplitudes of the TPM extension assay could reflect the telomerase-mediated telomere extension. Here we prepared various 5′-digoxigenin labeled DNA substrates of different lengths that mimicked the ssDNA extension products after one, two, three and four rounds of extensions by telomerase (see Supplementary Table SI (D) for DNA substrates). The DNA substrates were immobilized onto the anti-digoxigenin-coated slide surface, and were then annealed to oligo probes coated by 220 nm polystyrene beads. These individually tethered DNA-beads undergo BM, which can be visualized under an optical microscope, and BM distributions were then analyzed. The mimicked ‘single-round’ extension DNA substrate showed a single BM peak at 11.1 ± 3.0 nm (mean ± SD, Figure 1A). In the case of a multiple-rounds extension, the probe oligos can anneal to several different positions, due to the repetitive nature of the telomere sequences. As expected, DNA substrates that mimicked ‘double-rounds’ of extensions showed two peaks, with an additional, higher BM peak at ∼19.1 nm (Figure 1B). Similarly, DNA substrates with ‘three-rounds’ extensions showed peaks at ∼10.6, 18.0 and 23.9 nm, and those with ‘four-rounds’ extensions showed peaks at ∼11.1, 17.2, 24.0 and 33.3 nm (Figure 1C and D). Significantly, we found a linear relationship between the number of extended rounds and the observed BM peak values (Supplementary Figure S1A). Thus, our TPM-based assay is capable of detecting multiple-rounds of telomerase-mediated telomere extension products.

Bottom Line: Using its RNA component as a template, telomerase uses its reverse transcriptase activity to extend the 3'-end single-stranded, repetitive telomeric DNA sequence.Pif1, a 5'-to-3' helicase, has been suggested to regulate telomerase activity.We used single-molecule experiments to directly show that Pif1 helicase regulates telomerase activity by removing telomerase from telomere ends, allowing the cycling of the telomerase for additional extension processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, National Taiwan University, Taiwan.

Show MeSH