Frustrated folding of guanine quadruplexes in telomeric DNA

Abstract

Human chromosomes terminate in long, single-stranded, DNA overhangs of the repetitive sequence (TTAGGG)n. Sets of four adjacent TTAGGG repeats can fold into guanine quadruplexes (GQ), four-stranded structures that are implicated in telomere maintenance and cell immortalization and are targets in cancer therapy. Isolated GQs have been studied in detail, however much less is known about folding in long repeat sequences. Such chains adopt an enormous number of configurations containing various arrangements of GQs and unfolded gaps, leading to a highly frustrated energy landscape. To better understand this phenomenon, we used mutagenesis, thermal melting, and global analysis to determine stability, kinetic, and cooperativity parameters for GQ folding within chains containing 8-12 TTAGGG repeats. We then used these parameters to simulate the folding of 32-repeat chains, more representative of intact telomeres. We found that a combination of folding frustration and negative cooperativity between adjacent GQs increases TTAGGG unfolding by up to 40-fold, providing an abundance of unfolded gaps that are potential binding sites for telomeric proteins. This effect was most pronounced at the chain termini, which could promote telomere extension by telomerase. We conclude that folding frustration is an important and largely overlooked factor controlling the structure of telomeric DNA.

Graphical Abstract

Mutagenesis, thermal melts and computational analyses show that folding frustration is a key determinant of guanine quadruplex folding in long telomeric repeat DNA.

Figure 1.

(A) Ribbon representation of a telomeric GQ structure (PDB 2JPZ (20)). Nucleobases and C3′ atoms for guanine residues are shown as red sticks and spheres, respectively. (B) Cartoon representation of the transition between folded (red) and unfolded (blue) states of a GQ. Each G-tract is represented by three consecutive circles. (C) 8-GQ and 7-GQ forms of a 32 telomeric repeat Tel₃₂ DNA sequence.

Figure 2.

(A) Fraction of potential GQs that are folded as a function of temperature, determined from spectroscopic absorbance measurements at 295 nm, for an oligonucleotide containing 12 telomeric repeats (Tel₁₂, red stars, maximum three GQs), as well as G-tract knockout mutants capable of forming one GQ (filled black symbols) or two adjacent GQs (blue open symbols). In the legend, x and | correspond to G-tracts containing GTG or GGG, as described in the text. Curves represent the best fit of a global thermodynamic folding model. Error bars are often smaller than the symbols used. (B) Unfolding probabilities at 310K for individual G-tracts in a WT Tel₁₂ DNA molecule calculated from the globally fitted thermodynamic parameters, where G-tracts 1, 2, 3, 4, 5, etc. correspond to GGG stretches beginning at nucleotides 4, 10, 16, 22, 28, etc. in the WT sequence.

Figure 3.

(A) Folding pathway of the Tel_8,ext DNA molecule. The four rows from top to bottom correspond to misfolded, fully unfolded, on-pathway intermediate, and fully folded states. The symbols | and – represent folded and unfolded G-tracts. x's represent G-tracts with GGG to GTG substitutions. (B) Thermal hysteresis folding/unfolding data for Tel_8,ext DNA and variants. Spectroscopic absorbance values obtained at 295 nm with temperature ramp rates of 4° min⁻¹ were converted to the fractions of folded GQs as a function of temperature. Data for G-tract knockout mutants capable of forming a single GQ are shown as triangles and diamonds, while those of the wild-type are shown as circles. Red and blue correspond to heating and cooling scans. Error bars are often smaller than the symbols used. (C) Isothermal refolding data for Tel_8,ext DNA. Tel_8ext refolding experiments. Spectroscopic absorbance at 295 nm for samples rapidly cooled from 373 K to 288, 293, 298 or 303 K, normalized to lie between 0 and 1, are plotted as a function of time and reflect conversion of misfolded 1-GQ states to the fully folded 2-GQ ground state. Curves in (B) and (C) correspond to the best fit to a global kinetic model.

Figure 4.

Unfolding probabilities for individual G-tracts calculated for (A) Tel₁₀₂₄ and (B) Tel₃₂ DNA molecules using Monte Carlo calculations or complete enumeration of the folding partition function, respectively, using experimental stability and cooperativity parameters. In (B), blue circles correspond to calculations with position-specific GQ stabilities and cooperativity included, while red squares have position-independent GQ stability and no cooperative effects. G-tract refers to the position along the nucleic acid molecule in terms of TTAGGG repeat.

Figure 5.

(A) Simulated folding of 10⁴Tel₃₂ DNA molecules using a discrete Markov chain model and experimental rate constants. The average fraction of G-tracts that are unfolded is plotted in solid colors as a function of time, with values for the most and least folded molecules in the ensemble shown with black dashed lines. (B) Unfolding probabilities for individual G-tracts calculated at 1 – 9 × 10^–1 (red), × 10⁰ (magenta), × 10¹ (blue), × 10² (green) and × 10³ (black) seconds. The unfolding probabilities at equilibrium are indicated with open circles. (C) Simulated folding of 500 (TGGG)₃₂T DNA molecules. The fractions of chains with ≥5, 6, 7 and 8 GQs are plotted as a function of time.