G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding

Abstract

Human telomeres terminate with a single-stranded 3' G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K(+), POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA's access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na(+), in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.

Keywords: DNA damage response; single molecule imaging; telomere protection.

Fig. 1.

Steady-state equilibrium of POT1-mediated GQ unfolding in the presence of either 150 mM K⁺ or 150 mM Na⁺. (A) Schematic of smFRET assay under TIRF illumination to monitor the folding/unfolding of a model telomeric GQ. (B) smFRET data (yellow bars) showing unfolding of GQ at varying concentrations of POT1 in 150 mM Na⁺. A multi-Gaussian fitting (blue curve) determines three distinct FRET peaks (F1, P, and 2P). F1 (E_F = 0.70, light green) represents antiparallel GQ. P (E_F = 0.32 pink) and 2P (E_F = 0.16, blue) correspond to one and two POT1-bound unfolded ssTEL4, respectively. (C) smFRET data showing unfolding of GQ at varying concentrations of POT1 in 150 mM K⁺. Unlike Na⁺, two folded populations, F1 (E_F = 0.70, light green) and F2 (E_F = 0.80, dark green), and one unfolded population (2P, E_F = 0.16, blue) were observed. F2 is consistent with the parallel GQ conformation. (D) In 150 mM K⁺, POT1 is unable to unfold the F2 population, which remains nearly constant (red line) from 0 to 2,000 nM POT1. (E) The percentage of P and 2P at 150 mM Na⁺ as a function of POT1 concentration. Global fitting of the data to fractional occupancy of two distinguishable sites as a function of substrate concentration yields K_eq = 620 ± 210 nM and cooperativity of −1.5 k_BT. Increasing the parameters of the fit for the cooperative model significantly improves the fit (F test, F = 11.2, α = 0.05), and hence is justifiable. (F) At 150 mM K⁺, the percentage of the 2P population (blue circles) increases as a function of POT1 concentration, owing to the unfolding of the F1 conformation. The 2P population is fitted to a Hill equation (red line).

Fig. 2.

The effect of TPP1 on POT1-mediated GQ unfolding in the presence of either 150 mM K⁺ or 150 Na⁺. (A) Unfolding of telomeric GQ as a function of TPP1 concentration in 150 mM K⁺ and 1 µM POT1. (B) In 150 mM K⁺, the 2P population increases 1.3% per μM of TPP1 (blue line) and the F1 population decreases at a similar rate (1.2% per μM of TPP1, red line). (C) Unfolding of telomeric GQ as a function of TPP1 concentration in the presence of 150 mM Na⁺ and 200 nM POT1. (D) In 150 mM Na⁺, the percentage of unfolded populations (P + 2P) increases by a factor of 2 when TPP1:POT1 ratio is increased from 1:1 to 2:1 at 200 nM POT1.

Fig. 3.

Competition between RPA versus POT1 or POT1/TPP1 to bind ssTEL4 in 150 mM K⁺. (A) FRET histograms display unfolding of GQ and binding of RPA to the unfolded ssTEL4 (R peak at E_F = 0.10, lavender); ∼90% of GQ molecules are unfolded at 10 nM RPA. (B) Langmuir binding isotherm analysis (red curve) of RPA-mediated unfolding for the RPA-only case. (C) Unfolding of GQ and binding of RPA to the unfolded ssDNA in the presence of 1 µM POT1; ∼90% of GQ molecules are unfolded at 100 nM RPA concentration. (D) Langmuir binding isotherm analysis (red curve) of RPA-mediated unfolding in the presence of 1 µM POT1. (E) Unfolding of GQ by RPA in the presence of 1 µM POT1/TPP1; ∼47% of GQ molecules are unfolded at 1 µM RPA. Peak position of R (0.10) is distinct from 2P (0.16). Peak positions of F1 and F2 are similar to the POT1-only case. (F) Langmuir binding isotherm analysis (red curve) of RPA-mediated unfolding in the presence of 1 µM POT1/TPP1.

Fig. 4.

Competition between RPA versus POT1 to bind ssTEL in 150 mM Na⁺. (A) FRET histograms display unfolding of GQ and binding of RPA to the unfolded ssTEL4. All GQ molecules are unfolded at 10 nM RPA. (B) Langmuir binding isotherm analysis (red curve) of RPA-mediated unfolding for the RPA-only case. (C) Unfolding of GQ and binding of RPA to the unfolded ssDNA in the presence of 200 nM POT1. All GQ molecules are unfolded at 10 nM RPA. (D) Langmuir binding isotherm analysis (red curve) of RPA-mediated unfolding in the presence of 200 nM POT1. The difference between α and K_eq values obtained from the fit to those obtained in the RPA-only case is within the experimental error, suggesting that the GQ is not protected by POT1 in 150 mM Na⁺.

Fig. 5.

POT1 and RPA compete for the 3′ overhang of ssTEL4 in 150 mM K⁺. (A) The RPA-mediated GQ unfolding was significantly reduced using the ssTEL construct terminating with a short TT-3′ overhang (ssTEL4^TT), rather than the TTAG-3′ overhang of ssTEL4. Even at 1,000 nM RPA, only ∼50% of the GQ molecules remain folded. (B) The RPA-mediated GQ unfolding at 1 µM POT1. (C and D) The Langmuir isotherm fits to GQ unfolding in the absence and presence of POT1, respectively. POT1 addition did not significantly affect K_eq for GQ unfolding, compared with the 12-fold increase observed in ssTEL4 terminating with a TTAG overhang.

Fig. 6.

A model for protection of ssTEL4 against RPA-mediated unfolding. (A) In the absence of POT1/TPP1, RPA unfolds both antiparallel (F1) and parallel (F2) conformations at a low concentration either in Na⁺ or in K⁺. (B) In 150 mM Na⁺, ssTEL4 forms only antiparallel GQ. Binding of one (P) or two (2P) POT1 unfolds the antiparallel conformation. Addition of RPA displaces POT1/TPP1 from ssTEL4. In 150 mM K⁺, POT1/TPP1 binds to both GQ folding patterns and unfolds antiparallel GQ, whereas parallel GQ remains folded. Binding of two POT1s to ssTEL4 is required to stabilize the unfolded state. POT1/TPP1 binding effectively suppresses RPA binding to ssTEL4 even at high RPA concentrations. In particular, the majority of parallel GQ remains stably folded against RPA-mediated unfolding.