Abstract
Telomeres are specialized chromatin structures found at the end of chromosomes and are crucial to the maintenance of eukaryotic genome stability. Human telomere DNA is comprised of the repeating sequence (T2AG3)n, which is predominantly double-stranded but terminates with a 3’ single-stranded tail. The guanine-rich tail can fold into secondary structures known as a G-quadruplexes (GQs) that may exist as a polymorphic mixture of anti-parallel, parallel, and several hybrid topological isomers. Using single-molecule Förster resonance energy transfer (smFRET), we have reconstructed distributions of telomere DNA GQ conformations generated by an in situ refolding protocol commonly employed in single-molecule studies of GQ structure, or using a slow cooling DNA annealing protocol typically used in the preparation of GQ samples for ensemble biophysical analyses. We find the choice of GQ folding protocol has a marked impact on the observed distributions of DNA conformations under otherwise identical buffer conditions. A detailed analysis of the kinetics of GQ folding over timescales ranging from minutes to hours revealed the distribution of GQ structures generated by in situ refolding gradually equilibrates to resemble the distribution generated by the slow cooling DNA annealing protocol. Interestingly, conditions of low ionic strength, which promote transient GQ unfolding, permit the fraction of folded DNA molecules to partition into a distribution that more closely approximates the thermodynamic folding equilibrium. Our results are consistent with a model in which kinetic partitioning occurs during in situ folding at room temperature in the presence of K+ ions, producing a long-lived non-equilibrium distribution of GQ structures in which the parallel conformation predominates on the timescale of minutes. These results suggest that telomere DNA GQ folding kinetics, and not just thermodynamic stability, likely contributes to the physiological ensemble GQ structures.
Highlights
The first solution structure of a human telomere Gquadruplex (GQ) revealed a fundamental structural architecture in which guanine bases are hydrogen bonded in a planar quartet geometry (Figure 1A) [1].Three consecutive intramolecular G-quartets may interact via stacking interactions and are topologically linked by short intervening DNA loop sequences (Figure 1B)
Distinct DNA folding protocols alter the observed distribution of GQ structures To analyze telomere DNA GQ folding, we designed a single molecule Förster Resonance Energy Transfer (FRET) (smFRET) construct comprised of the model telomere DNA sequence TAGGG(T2AG3)3
We have used smFRET to directly demonstrate the distribution of telomere DNA GQ structures generated during KCl in situ refolding on the timescale of minutes is demonstrably altered from the identical molecules prepared by KCl thermal annealing
Summary
The first solution structure of a human telomere Gquadruplex (GQ) revealed a fundamental structural architecture in which guanine bases are hydrogen bonded in a planar quartet geometry (Figure 1A) [1].Three consecutive intramolecular G-quartets may interact via stacking interactions and are topologically linked by short intervening DNA loop sequences (Figure 1B). Human telomere DNA GQ structure has been studied by numerous techniques including NMR [1,11,12,13,14], X-ray crystallography [15], circular dichroism [16,17,18,19], UV melting [13,20,21], force spectroscopy [22,23,24], and Förster Resonance Energy Transfer (FRET) [25,26,27,28].
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