Coexistence of Two Distinct G-Quadruplex Conformations in the hTERT Promoter

Abstract

The catalytic subunit of human telomerase, hTERT, actively elongates the 3' end of the telomere in most cancer cells. The hTERT promoter, which contains many guanine-rich stretches on the same DNA strand, exhibits an exceptional potential for G-quadruplex formation. Here we show that one particular G-rich sequence in this region coexists in two G-quadruplex conformations in potassium solution: a (3 + 1) and a parallel-stranded G-quadruplexes. We present the NMR solution structures of both conformations, each comprising several robust structural elements, among which include the (3 + 1) and all-parallel G-tetrad cores, single-residue double-chain-reversal loops, and a capping A.T base pair. A combination of NMR and CD techniques, complemented with sequence modifications and variations of experimental condition, allowed us to better understand the coexistence of the two G-quadruplex conformations in equilibrium and how different structural elements conspire to favor a particular form.

DNA quadruplex folding formalism – A tutorial on quadruplex topologies

“…[23] 9. Prevalence of loop type combinations [24], (b) 1XAV [25], (c) 2LEE [26], (d) 2LBY [27], (e) 2KYP [28], (f) 2LPW [29], (g) 2KZE [30] and (h) 2LXQ [31] with examples determined in crowded conditions (2LD8, [32]) and in dimeric form stacked 5 0 -5 0 (2LE6, [33] and 2LXV, [31]). It is noteworthy that all these examples are three-stacked quadruplexes.…”

“…Second most prevalent overall is the Group II, antiparallel topology (ÀlÀlÀp), characterized by the anticlockwise progression of lateral, lateral and propeller loops with five examples: (a) 2JSL [34], (b) 2JPZ [35], (c) 2KZD [30], (d) 186D [36] and (e) 2F8U [37]. There are four examples of Group II (ÀpÀlÀl) topology (a) 2GKU [38], (b) 2HY9 [39] and (c) 2JSM [34].…”

How to study G-quadruplex structures

“…9). Alternatively, in order to force a single conformation in solution some guanosine residues can be substituted by 8-bromoguanosine or inosine (Lim et al, 2009, Lim et al, 2010. In fact, even if the methods above are used, about 10% of higher-order structures as well as an alternative conformation background is usually observed at 1 H NMR spectra.…”

Kinetics of DNA Refolding from Longitudinal Exchange NMR Spectroscopy

“…For example, human telomeric repeats are able to coexist in multiple G quadruplex forms, [4] which potentially exert different functions depending on the conformational state of the quadruplex. In addition, during homologous recombination, DNA undergoes a strand exchange process to form a Holliday junction.…”