Telomeric i-motifs and C-strands inhibit parallel G-quadruplex extension by telomerase

Abstract

Telomeric C-rich repeated DNA sequences fold into tetrahelical i-motif structures in vitro at acidic pH. While studies have suggested that i-motifs may form in cells, little is known about their potential role in human telomere biology. In this study, we explore the effect of telomeric C-strands and i-motifs on the ability of human telomerase to extend G-rich substrates. To promote i-motif formation at neutral pH, we use telomeric sequences where the cytidines have been substituted with 2'-fluoroarabinocytidine. Using FRET-based studies, we show that the stabilized i-motifs resist hybridization to concomitant parallel G-quadruplexes, implying that both structures could exist simultaneously at telomeric termini. Moreover, through telomerase activity assays, we show that both unstructured telomeric C-strands and telomeric i-motifs can inhibit the activity and processivity of telomerase extension of parallel G-quadruplexes and linear telomeric DNA. The data suggest at least three modes of inhibition by C-strands and i-motifs: direct hybridization to the substrate DNA, hybridization to nascent product DNA resulting in early telomerase dissociation, and interference with the unique mechanism of telomerase unwinding and extension of a G-quadruplex. Overall, this study highlights a potential inhibitory role for the telomeric C-strand in telomere maintenance.

Graphical Abstract

Figure 1.

Model systems designed for this study. (A) Side-by-Side and Offset models portraying the folding at pH 7 of araF-C rich telomeric sequences into i-motifs (F-IM) and araF-G rich telomeric sequences into parallel G-quadruplexes (G4). At pH 7, the unmodified telomeric C-rich sequence (C-ss) remains single-stranded. (B) Structures of araF-G and araF-C nucleoside modifications used to stabilize parallel G-quadruplexes and i-motifs, respectively.

Figure 2.

¹H NMR spectra acquired at different times and at 5°C for an equimolar mixture of ‘offset’ model (A) parallel G-quadruplex + F-IM and (B) unmodified G-quadruplex + C-ss. Individual strands were pre-folded before mixing in 20 mM KP_i, 70 mM KCl, pH 7.0.

Figure 3.

Biophysical studies of G- and C-strands alone. (A) CD spectra of F-IM and C-ss in telomerase assay buffer conditions (pH 7), at 5 and 20°C. (B) CD spectra of 35G3 G-quadruplex in telomerase assay buffer pH 7 from 5 to 65°C. (C) UV-based thermal denaturation data showing changes in normalized absorbance of the 35G3 G-quadruplex with increasing temperature, when measured in KP_i/KCl buffer pH 7 (pink) or telomerase assay buffer pH 7 (green).

Figure 4.

(A) Representative ‘side-by-side’ models to measure the extent of telomeric i-motif folding (cy5-conjugated strand) by FRET, in the presence of a concomitant telomeric parallel G-quadruplex (cy3-conjugated strand), 35G3-Cy3. (B) FRET ratios calculated from fluorescence emissions across different temperatures following the mixing of pre-folded, equimolar amounts of parallel G-quadruplex, 35G3-Cy3, with either C-ss-Cy5 or F-IM-Cy5 sequences at pH values of 6.0, 7.0, and 8.5. Samples were pre-folded and mixed in KP_i/KCl buffer, with a duplex concentration of 5 μM, followed by dilution in telomerase assay buffer to make a final duplex concentration of 1 μM.

Figure 5.

Telomerase extension assays at pH 7 and 20°C, in the presence of radiolabeled α-³²P-dGTP, analyzed using denaturing polyacrylamide gel electrophoresis. (A) The extension products of 1 μM 35G3 or 56G3 alone, or in the presence of 1 μM of F-IM, C-ss, HP or pT. The dashed line represents the omission of irrelevant lanes. (B) The extension products of 1 μM 35G3 alone, or in the presence of 1 μM of F-IM, C-ss, F-IM-u or C-ss-u. Loading/recovery control: 5′-³²P-labeled synthetic 30 nt DNA. (C) and (D) represent quantitations of relative overall telomerase extension activity in (A) and (B), respectively, in comparison to the extension of 35G3 or 56G3 alone; n = 2–7 independent experiments.

Figure 6.

Telomerase extension assays at pH 6, 7, and 8.5 and at 20°C, in the presence of radiolabeled α-³²P-dGTP. Lanes 1–3: The extension products with varying pH of 1 μM of linear telomeric control (33-LinG) alone, which cannot fold into a G-quadruplex (Table 1). Lanes 4–12: The extension products with varying pH of 1 μM of 35G3 alone, or in the presence of 1 μM of F-IM or C-ss. The inset represents a darker exposure of lanes 4–9. Loading/recovery control: 5′-³²P-labeled synthetic 30 nt DNA.

Figure 7.

Telomerase extension assays at pH 7 and 20°C, in the presence of radiolabeled α-³²P-dGTP. (A) Bands correspond to extension products of 1 μM ‘side-by-side’ 29-LinG, ‘side-by-side’ parallel G-quadruplex or untethered 33-LinG alone, or in the presence of 1 μM of tethered or untethered F-IM or C-ss. Loading/recovery control: 5′-³²P-labeled synthetic 30 nt DNA. (B) represents quantitation of relative overall telomerase extension activity in (A), in comparison to the extension of 29-LinG, 35G3, or 33-LinG alone. (C) represents changes in the intensity of bands corresponding to telomerase extension products, normalized to the intensity of the band corresponding to the addition of the second hexanucleotide repeat.

Figure 8.

(A) Non-telomeric i-motifs, IM-a and IM-b, designed to test the sequence and structure dependence of the inhibitory effects of telomeric i-motifs. (B) Telomerase extension assays at pH 7 and 20°C, in the presence of radiolabeled α-³²P-dGTP. The extension products of 1 μM parallel G-quadruplex (‘side-by-side’) alone, or in the presence of 1 μM of complementary strands (lanes 2–11) were analyzed using denaturing polyacrylamide gel electrophoresis. Loading/recovery control: 5′-³²P-labeled synthetic 30 nt DNA.

Figure 9.

Proposed mechanism for C-strand mediated inhibition of telomerase extension of human telomeric G-rich substrates. (A) Summary of proposed modes of inhibition of extension of parallel G-quadruplex or linear G-strand telomerase substrates. (B) Proposed models for i-motif and unstructured C-strand mediated inhibition of extension of a G-quadruplex substrate, proceeding via the binding to the loop of a partially unfolded G-quadruplex, leading to abrupt telomerase dissociation.