The solution structures of higher-order human telomere G-quadruplex multimers

Abstract

Human telomeres contain the repeat DNA sequence 5′-d(TTAGGG), with duplex regions that are several kilobases long terminating in a 3′ single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate models proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5′-d(TTAGGG)n, with n = 8, 12 and 16, fold into multimeric structures containing the maximal number (2, 3 and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of ∼34 Å. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.

On the Road to Fight Cancer: The Potential of G-Quadruplex Ligands as Novel Therapeutic Agents

“…In addition, telomere-associated proteins, such as the heterodimer formed by the shelterin components POT1 and TPP1, have been found to bind and unwind telomeric G4 [8]. These observations together with the evidence that telomeric DNA can fold into different quadruplex structures (structural polymorphism) and that the different G4 topologies may mutually be in a dynamic equilibrium [9], support the notion that G4 structures may play an important biological role in the regulation of telomere function [6].…”

Vimentin binds to G-quadruplex repeats found at telomeres and gene promoters

“…Indeed, by increasing the number of TTAGGG repeats, the resulting oligonucleotide folds into one (TEL), two (2TEL), three (3TEL) or four (4TEL) adjacent G-quadruplexes. 22 Moreover, Vimentin association with telomeres has already been observed within living cells. 28 As expected, Vimentin did not bind to the single telomeric G4 (Figure 2C) while it interacted with the G4 repeats.…”

“…They were first characterized for the human telomeric sequence, where multiple adjacent G4s interact through transient π-π stacking of the external tetrads. 22 More recent studies highlighted the ability of gene promoter sequences to also give rise to G4 repeats. 23,24,25 Among them, the hTERT sequence, located within the core promoter of telomerase, folds into three interacting parallel three-quartet G4s, 23 the ILPR sequence, located within the promoter of insulin, folds into two cross-talking hybrid four-quartet G4s, 24 while KIT2KIT*, which is found within the core promoter of c-KIT, folds into two interacting parallel three-quartet and antiparallel two-quartet G4s.…”

Vimentin binds to G-quadruplex repeats found at telomeres and gene promoters

“…Therefore, to better characterize this interaction, we moved to the telomeric sequence, as it constitutes an easily tunable model for G4 repeats. Indeed, by increasing the number of TTAGGG repeats, the resulting oligonucleotide folds into one (TEL), two (2TEL), three (3TEL) or four (4TEL) adjacent G-quadruplexes ( 22 ). Moreover, Vimentin association with telomeres has already been observed within living cells ( 35 ).…”

“…G4 repeats comprise two or more adjacent G4 modules, which eventually give rise to end-to-end mutual interactions. They were first characterized for the human telomeric sequence, where multiple adjacent G4s interact through transient π–π stacking of the external tetrads ( 22 ). More recent studies highlighted the ability of gene promoter sequences to also give rise to G4 repeats ( 23–25 ).…”