G-Quadruplex Motifs Arranged in Tandem Occurring in Telomeric Repeats and the Insulin-Linked Polymorphic Region

Abstract

To date, various G-quadruplex structures have been reported in the human genome. There are numerous studies focusing on quadruplex-forming sequences in general, but few studies have focused on two or more quadruplexes in the same molecule, which are most commonly found in telomeric DNA and other tandem repeats, e.g., insulin-linked polymorphic region (ILPR). Although the human telomere consists of a number of repeats, higher-order G-quadruplex structures are discussed less often because of the complexity of the structures. In this study, sequences consisting of 4-12 repeats of d(G(4)TGT), d(G(3)T(2)A), and/or d(G(4)T(2)A) have been studied by circular dichroism, ultraviolet spectroscopy, and temperature-gradient gel electrophoresis. These sequences serve as a model for the arrangement of quadruplexes in the telomere and ILPR in solution. Our major findings are as follows. (i) The number of G-rich repeats has a great influence on G-quadruplex stability. (ii) The evidence of quadruplex-quadruplex interaction is confirmed. (iii) For the first time, we directly observed the melting behavior of different conformers in a single experiment. Our results agree with other calorimetric and spectroscopic data and data obtained by single-molecule studies, atomic force microscopy, and mechanical unfolding by optical tweezers. We propose that the end of telomeres can be formed by only a few tandem quadruplexes (fewer than three). Our findings improve our understanding of the mechanism of G-quadruplex formation in long repeats in G-rich-regulating parts of genes and telomere ends.

Telomeric Overhang Length Determines Structural Dynamics and Accessibility to Telomerase and ALT-Associated Proteins

“…Although numerous studies have examined the folding kinetics of human G-quadruplex (GQ), studies of physiologically relevant telomeric substrates that have more than four repeats of TTAGGG are limited (Bauer et al, 2011; Lane et al, 2008; Vorlickova et al, 2005). Melting temperature and circular dichroism experiments have suggested that GQs are blocks that resemble “beads-on-a string” and that they can move independently of each other and are constrained only by connecting linkers (Petraccone et al, 2011; Yu et al, 2006).…”

Structure and Stability of Higher-Order Human Telomeric Quadruplexes

“…Several laboratories have reported that higher-order quadruplex assemblies are destabilized, with decreased melting temperatures, relative to their monomeric counterparts 34,37,44 . Our results (Table 1) are consistent with these reports, although we find that the melting of higher-order structures is not a simple two-state process as was assumed in these earlier studies.…”

“…The same study used FRET to show that two contiguous quadruplexes formed a 46 nt repeat sequence, in full accord with the detailed model proposed by Petraccone and coworkers 42,43 . Circular dichroism and thermal-gradient electrophoresis were recently used to study telomeric G-quadruplex motifs arranged in tandem 44 . Structures with two and three contiguous quadruplexes were observed, and quadruplex thermal stability was diminished upon formation of the higher-ordered structures.…”

Understanding the stability of DNA G-quadruplex units in long human telomeric strands

“…Nevertheless, the authors did not provide evidence of folding into contiguous G4s, hence it is not clear if the decrease in stability upon increasing the number of potential G4 units results from irregular folding of long telomeric strands or from other reasons. Bauer et al reported that (GGGTTA) 7,11 GGG structures display the same thermal stability and fold, each, into more than one conformers; they argued that the end of telomeres can form only a few tandem G4s (fewer than three) [34]. Petraccone et al reported that (TTAGGG) 8,12 and (TTAGGG) 8,12 TT sequences fold into two and three contiguous G4s and that the global structures display identical melting temperatures and complex melting behaviors; they suggested that the G4 units are not identical nor independent and that unfavorable interactions of an unknown nature could limit complete folding of the telomeric overhang into contiguous G4s [30].…”