Interrogation of G-quadruplex–protein interactions (original) (raw)

DNA and RNA quadruplex-binding proteins

International journal of molecular sciences, 2014

Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as w...

Quadruplex DNA: sequence, topology and structure

Nucleic Acids Research, 2006

G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes.

Recognition and binding of human telomeric G-quadruplex DNA by unfolding protein 1

2014

The specific recognition by proteins of G-quadruplex structures provides evidence of a functional role for in vivo G-quadruplex structures. As previously reported, the ribonucleoprotein, hnRNP Al, and it is proteolytic derivative, unwinding protein 1 (UP1), bind to and destabilize G-quadruplex structures formed by the human telomeric repeat d(TTAGGG) n. UP1 has been proposed to be involved in the recruitment of telomerase to telomeres for chain extension. In this study, a detailed thermodynamic characterization of the binding of UP1 to a human telomeric repeat sequence, the d[AGGG(TTAGGG) 3 ] Gquadruplex, is presented and reveals key insights into the UP1-induced unfolding of the G-quadruplex structure. The UP1−G-quadruplex interactions are shown to be enthalpically driven, exhibiting large negative enthalpy changes for the formation of both the Na + and K + Gquadruplex−UP1 complexes (ΔH values of −43 and −19 kcal/mol, respectively). These data reveal three distinct enthalpic contributions from the interactions of UP1 with the Na + form of Gquadruplex DNA. The initial interaction is characterized by a binding affinity of 8.5 × 10 8 M −1 (strand), 200 times stronger than the binding of UP1 to a single-stranded DNA with a comparable but non-quadruplex-forming sequence [4.1 × 10 6 M −1 (strand)]. Circular dichroism spectroscopy reveals the Na + form of the G-quadruplex to be completely unfolded by UP1 at a binding ratio of 2:1 (UP1:G-quadruplex DNA). The data presented here demonstrate that the favorable energetics of the initial binding event are closely coupled with and drive the unfolding of the G-quadruplex structure.

Existence and consequences of G-quadruplex structures in DNA

While the discovery of B-form DNA 60 years ago has defined our molecular view of the genetic code, other postulated DNA secondary structures, such as A-DNA, Z-DNA, H-DNA, cruciform and slipped structures have provoked consideration of DNA as a more dynamic structure. Four-stranded G-quadruplex DNA does not use Watson-Crick base pairing and has been subject of considerable speculation and investigation during the past decade, particularly with regard to its potential relevance to genome integrity and gene expression. Here, we discuss recent data that collectively support the formation of G-quadruplexes in genomic DNA and the consequences of formation of this structural motif in biological processes.

A New Pathway of DNA G-Quadruplex Formation

Angewandte Chemie International Edition, 2014

A new folding intermediate of Oxytricha nova telomeric Oxy-1.5 G-quadruplex was characterized in aqueous solution using NMR spectroscopy, native gel electrophoresis, thermal differential spectra (TDS), CD spectroscopy, and differential scanning calorimetry (DSC). NMR experiments have revealed that this intermediate (i-Oxy-1.5) exists in two symmetric bimolecular forms in which all guanine bases are involved in GG N1-carbonyl symmetric base pairs. Kinetic analysis of K +-induced structural transitions shows that folding of Oxy-1.5 G-quadruplex from i-Oxy-1.5 is much faster and proceeds through less intermediates than folding from single strands. Therefore, a new folding pathway of Oxy-1.5 Gquadruplex is proposed. This study provides evidence that Grich DNA sequences can self-assemble into specific preorganized DNA structures that are predisposed to fold into G-quadruplex when interacting with cations such as potassium ions.

Structural Diversity and Specific Recognition of Four Stranded G-Quadruplex DNA

Current Molecular Medicine, 2011

Structural multitude of nucleic acids serves basis for its multiple merits and applications. During structural transitions, significant to perform respective cellular functions, these DNA forms can vary from the single stranded to multi-stranded species. Hence, beyond the image of a monotonous DNA double-helix, there is now increasing interest in other polymorphic/ multi-stranded forms, the roles they may play in vivo and their potential use in therapeutics. Distinct guanine-rich nucleic acid sequences readily form a structurally diverse four-stranded architecture called G-quadruplexes. In addition to their presence at physical ends of chromosomes called telomeres, occurrence of these structural motifs in the upstream promoter regions of a number of genes, oncogenes and near transcription start sites, highlights that G-quadruplexes are involved in regulation of gene expression. Cancer cells typically possess shorter telomeres and have telomerase activity greatly exceeding that of normal cells. These differences create an opportunity to use anticancer therapies targeting telomerase and telomeres. The ability of small molecules to interact with and presumably stabilize Gquadruplex structures as a means of inhibiting telomerase has been a major drug design effort. Ligands, capable of interacting with four-stranded G-quadruplex have been generated. The discovery of proteins including transcription factors, recognizing G-quadruplexes, and conferring stabilization or unfolding them in biological systems, again makes G-quadruplexes, biologically pertinent structures. This review is an attempt to summarize the rapidly evolving literature exploring the amazing polymorphism of G-quadruplexes, and understanding their structure-specific-recognition and biological relevance, keeping in mind that G-tetraplexes are not only important drug targets, but may also act as gene regulatory elements. A pertinent detail of the challenges towards the rational design of structure-specific novel drugs has also been discussed.

Sequence specificity of inter- and intramolecular G-quadruplex formation by human telomeric DNA

Biopolymers, 2007

Human telomeric DNA consists of tandem repeats of the sequence 5′-d(TTAGGG)-3′. Guanine-rich DNA, such as that seen at telomeres, forms G-quadruplex secondary structures. Alternative forms of G-quadruplex structures can have differential effects on activities involved in telomere maintenance. With this in mind, we analyzed the effect of sequence and length of human telomeric DNA on G-quadruplex structures by native polyacrylamide gel electrophoresis and circular dichroism. Telomeric oligonucleotides shorter than four, 5′-d(TTAGGG)-3′ repeats formed intermolecular G-quadruplexes. However, longer telomeric repeats formed intramolecular structures. Altering the 5′-d(TTAGGG)-3′ to 5′-d(TTAGAG)-3′ in any one of the repeats of 5′-d(TTAGGG)4-3′ converted an intramolecular structure to intermolecular G-quadruplexes with varying degrees of parallel or anti-parallel-stranded character, depending on the length of incubation time and DNA sequence. These structures were most abundant in K+-containing buffers. Higher-order structures that exhibited ladders on polyacrylamide gels were observed only for oligonucleotides with the first telomeric repeat altered. Altering the sequence of 5′-d(TTAGGG)8-3′ did not result in the substantial formation of intermolecular structures even when the oligonucleotide lacked four consecutive telomeric repeats. However, many of these intramolecular structures shared common features with intermolecular structures formed by the shorter oligonucleotides. The wide variability in structure formed by human telomeric sequence suggests that telomeric DNA structure can be easily modulated by proteins, oxidative damage, or point mutations resulting in conversion from one form of G-quadruplex to another. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 74–84, 2007This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Preferential binding of a G-quadruplex ligand to human chromosome ends

Nucleic Acids Research, 2005

The G-overhangs of telomeres are thought to adopt particular conformations, such as T-loops or G-quadruplexes. It has been suggested that G-quadruplex structures could be stabilized by specific ligands in a new approach to cancer treatment consisting in inhibition of telomerase, an enzyme involved in telomere maintenance and cell immortality. Although the formation of G-quadruplexes was demonstrated in vitro many years ago, it has not been definitively demonstrated in living human cells. We therefore investigated the chromosomal binding of a tritiated G-quadruplex ligand, 3 H-360A (2,6-N,N 0 -methyl-quinolinio-3-yl)-pyridine dicarboxamide [methyl-3 H]. We verified the in vitro selectivity of 3 H-360A for G-quadruplex structures by equilibrium dialysis. We then showed by binding experiments with human genomic DNA that 3 H-360A has a very potent selectivity toward G-quadruplex structures of the telomeric 3 0 -overhang. Finally, we performed autoradiography of metaphase spreads from cells cultured with 3 H-360A. We found that 3 H-360A was preferentially bound to chromosome terminal regions of both human normal (peripheral blood lymphocytes) and tumor cells (T98G and CEM1301). In conclusion, our results provide evidence that a specific G-quadruplex ligand interacts with the terminal ends of human chromosomes. They support the hypothesis that G-quadruplex ligands induce and/or stabilize G-quadruplex structures at telomeres of human cells.