Guanine tetraplex topology of human telomere DNA is governed by the number of (TTAGGG) repeats

Abstract

Secondary structures of the G-rich strand of human telomere DNA fragments G3(TTAG3)n, n = 1-16, have been studied by means of circular dichroism spectroscopy and PAGE, in solutions of physiological potassium cation concentrations. It has been found that folding of these fragments into tetraplexes as well as tetraplex thermostabilities and enthalpy values depend on the number of TTAG3 repeats. The suggested topologies include, e.g. antiparallel and parallel bimolecular tetraplexes, an intramolecular antiparallel tetraplex, a tetraplex consisting of three parallel chains and one antiparallel chain, a poorly stable parallel intramolecular tetraplex, and both parallel and antiparallel tetramolecular tetraplexes. G3(TTAG3)3 folds into a single, stable and very compact intramolecular antiparallel tetraplex. With an increasing repeat number, the fragment tetraplexes surprisingly are ever less thermostable and their migration and enthalpy decrease indicate increasing irregularities or domain splitting in their arrangements. Reduced stability and different topology of lengthy telomeric tails could contribute to the stepwise telomere shortening process.

Figure 1

CD spectra of G₃TTAG₃ (left) and TAG₃TTAG₃T (right) measured at 25°C (dots) in 1 mM Na phosphate plus 0.3 mM EDTA, pH 7 immediately after thermal denaturation. Measurements in 10 mM potassium phosphate plus 0.15 M KCl were carried out (left): immediately (dashes) and 1, 3 and 10 days (from the thinnest to the thickest line) after K⁺ addition. The samples were kept and measured at 25°C; (right): after two days keeping at 0°C and measured at 0°C (dashes) and 25°C (dash–dots) and after three days keeping at 25°C and measured at 25°C (thick line). The thin full line spectra in both panels correspond to the samples kept for two days in 10 mM Na phosphate plus 0.15 M NaCl at 0°C and measured at 0°C. Inset: UV absorption spectra of G₃TTAG₃ in 10 mM K phosphate plus 0.15 M KCl at 91°C (dots) and 25°C (solid line). The sketch shows the antiparallel and parallel bimolecular tetraplexes of TAG₃TTAG₃T. The balls in the sketch refer to the oligonucleotide 5′ ends.

Figure 2

Electrophoresis run in the Robinson–Britton buffer plus 0.15 M KCl, pH 7.2, at 2°C. The samples were incubated one day in the electrophoretic buffer at 0°C (lanes 1) and at 37°C (lanes 2). The sample of G₃TTAG₃ was loaded immediately (lane 1) on the gel and after two days incubation (lane 2) at room temperature. The heteroduplexes G₃TTAG₃•C₃TAAC₃ and G₃(TTAG₃)₃•(C3TAA)₃C₃ were used as markers for mobility of 18 and 42 base long fragments, respectively.

Figure 3

CD spectra of G₃(TTAG₃)₃ and AG₃(TTAG₃)₃ in 1 mM Na phosphate plus 0.3 mM EDTA, pH 7, measured at 0°C (dashes) and in 10 mM K phosphate plus 0.15 M KCl (solid lines). The same spectra were obtained at 0°C and at room temperature. The sketch shows the tetraplex structure of G₃(TTAG₃)₃ or AG₃(TTAG₃)₃. The ball in the sketch refers to the oligonucleotide 5′ end.

Figure 4

CD spectra of G₃(TTAG₃)₄ in 1 mM Na phosphate plus 0.3 mM EDTA, pH 7 (dashes) and in 10 mM K phosphate plus 0.15 M KCl (solid line) measured at 0°C. Inset A: CD spectra of G₃(TTAG₃)₄ in 10 mM K phosphate plus 0.15 M KCl measured at 45, 61, 68, 73 and 79°C (from the solid to the dotted line). Inset B: CD spectrum of G₃T₄G₄ tetraplex measured in the same solution at 0°C. The sketch shows a suggested structure of G₃(TTAG₃)₄ tetraplex. The ball refers to the oligonucleotide 5′ end.

Figure 5

Upper panel: electrophoresis of the telomere fragments (primary structures given above the lanes) run in the Robinson-Britton buffer plus 0.15 M KCl, pH 7.2, at 2°C. The samples were incubated one day in the electrophoretic buffer before loading on the gel. The heteroduplexes with complementary C-rich strands serve as markers for 18, 42, 90 and 198 base long fragments. Bottom panel: R_f—mobility related to that of G₃(TTAG₃)₁₆•(C₃TAA)₁₆C₃ heteroduplex as a function of natural logarithm of fragment lengths: heteroduplexes serving as markers (diamonds); bimolecular tetraplexes (solid circles); intramolecular tetraplexes (open circles). The dotted line with open diamonds simulates mobility of single-stranded molecules.

Figure 6

CD spectra of G₃(TTAG₃)₇ in 1 mM Na phosphate plus 0.3 mM EDTA, pH 7 (dashed line) and 10 mM K phosphate plus 0.15 M KCl (solid line), both at 0°C. The sketches show two alternative arrangements of the G₃(TTAG₃)₇ tetraplex fitting the results obtained. The ball refers to the oligonucleotide 5′ end.

Figure 7

CD spectra of (A) G₃(TTAG₃)₅, (B) G₃(TTAG₃)₆ and (C) G₃(TTAG₃)₁₅ in 10 mM potassium phosphate plus 0.15 M KCl measured at 0°C. In addition, the (B) contains CD spectra of G₃(TTAG₃)₆ in the same solvent but measured at 37°C immediately after increasing temperature (short dashes), and after one day incubation at 37°C (long dashes).

Figure 8

Temperature dependences of the telomere DNA fragments in 10 mM potassium phosphate plus 0.15 M KCl, pH 7, monitored by changes in Δɛ₂₉₀. All points, with the exception of those marked with a cross, correspond to equilibrium values. The equilibrium was reached within 110 min with G₃TTAG₃ and 20 h with G₃(TTAG₃)₂ and G₃(TTAG₃)₆. Solid lines and open circles: G₃(TTAG₃)₃ (red), G₃(TTAG₃)₄ (blue), G₃(TTAG₃)₅ (green), and G₃(TTAG₃)₇ (black); dashed lines and solid circles: G₃(TTAG₃)₈ (yellow), G₃(TTAG₃)₉ (cyan); G₃(TTAG₃)₁₅ (violet solid circles) and G₃(TTAG₃)₁₆ (violet open circles); thin solid lines and triangles: G₃(TTAG₃)₂ (blue), G₃(TTAG₃)₆ (red), and G₃TTAG₃(black).