Quadruplex in



Keywords: quadruplex in
Description: Pierre Murat 1,2 Shankar Balasubramanian 1,2. 1 Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK 2 Cambridge Institute, Cancer Research UK, Li Ka

    Pierre Murat 1,2 Shankar Balasubramanian 1,2.
    1 Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK 2 Cambridge Institute, Cancer Research UK, Li Ka Shing Center, Cambridge CB2 0RE, UK

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.

The self-assembly of guanylic acid derivatives has been known for more than a century [1 ] and the structural basis for this phenomenon was elucidated in the 1960s [2 ]. Guanine-tetrad formation (Figure 1 a) drives the assembly of four-stranded helixes by guanine-rich oligonucleotides (G-quadruplexes, Figure 1 b). Seminal studies by Sen and Gilbert, and by others [3. 4. 5. 6 and 7 ], showed that these cation-dependent, G-quadruplex structures are thermodynamically stable under physiological conditions, and subsequently such structures were proposed to be involved in telomere association, recombination and replication. Biophysical methods have provided extensive in vitro data on the structure(s) and thermodynamics of DNA G-quadruplexes formed from oligonucleotides derived from genomic sequences that have included the human telomere (Figure 1 c) [8 ] and promoter regions of oncogenes (e.g. MYC ) [9 ]. Structural data has facilitated the design and synthesis of G-quadruplex-specific small molecules [10 ] (see Figure 1 d for example of G-quadruplex ligands), several of which trigger cellular mechanisms proposed to be linked with G-quadruplexes. Notable examples of chemical biological studies include the small molecule inhibition of telomerase action via G-quadruplex stabilization [11 ], and also transcriptional suppression of MYC by a G-quadruplex ligand [12 ]. There are indeed many more examples in the literature of cell-based studies that provide supportive correlations. However, some such studies have not addressed whether the key G-quadruplex in question actually exists in the genomic DNA and if so whether it is responsible for causation of the observed effects.

Figure 1. (a) Structure of guanine tetrads formed by the coplanar arrangement of four guanines held by Hoogsteen bonds and stabilised by monovalent cations (usually K + ). (b) Schematic representation of a quadruplex motif formed by guanine rich DNA sequences and stabilized by the stacking of guanine tetrads. (c) Side and top view of the crystal structure of the human telomeric quadruplex (PDB: 1KF1 ). (d) Small molecule G-quadruplex ligands used to study G-quadruplex DNA in human cells and yeast.

Biophysical studies on G-quadruplex structures formed by oligonucleotides in vitro have allowed the formulation of quadruplex-prediction algorithms on the basis of sequence motifs such as G≥3 NX G≥3 NX G≥3 NX G≥3. The use of such algorithms, for example, QuadParser [13 ] and G4P Calculator [14 ], has shown that quadruplex motifs prevalent in genomes are enriched within regions associated with regulation including promoters, introns and UTRs [13. 14. 15 and 16 ] (Figure 2 a). The great potential for G-quadruplex formation in cellular genomic DNA has stimulated the need to experimentally confirm the presence of these structures in cells.

Figure 2. (a) G-quadruplex frequency in a generic human RefSeq gene (extracted from Ref [16 ]). (b) Top: Immunostaining of a replicating macronucleus of Stylonychia lemnae using the single chain antibody, Sty49, directed against telomeric quadruplex. Bottom: hybridization of an FITC-labeled telomeric probe to a replicating macronucleus; arrow points to the replication band (extracted from Ref [17 ]). (c) Immunofluorescence for BG4 on metaphase chromosomes isolated from Hela cervical cancer cells. Discrete BG4 foci (red) were observed both within the non-telomeric regions (top) and at the telomeres (bottom) (extracted from Ref [20 •• ]). (d) Top: Quantification of BG4 foci per nucleus for staining of MCF-7 cells. Bottom: Quantification of BG4 foci number per nucleus (U2OS cells) with or without PDS treatment.

G-quadruplex DNA-recognizing antibodies have been exploited to visualize these structures within genomic DNA. In a landmark paper, Schaffitzel et al. described use of high-affinity single-chain antibodies, generated by ribosome display, to visualize quadruplex structures at the telomeres of the ciliate Stylonychia lemnae [17 ]. Immunofluorescence studies show that one of the selected antibodies, Sty49, reacts specifically with the macronucleus but not the micronucleus of the ciliate (Figure 2 b). Of particular note is the observation that, the replication band is not stained suggesting that G-quadruplex DNA is resolved during replication in ciliates. Using the same antibody, Paeschke et al. showed that the telomere end-bing proteins (TEBPα and TEBPβ) co-operate to control the formation of anti-parallel G-quadruplex structures at telomeres in vivo in S. lemnae [18 ] via a mechanism biochemically linked to a cell cycle-dependent phosphorylation of TEBPβ [19 ]. Recent work reported by Biffi et al. described a monoclonal single chain antibody, BG4, generated by phage display with high affinity and specificity for intramolecular G-quadruplex structures [20 •• ]. Immunostaining of a range of human cells shows the presence of G-quadruplex structures in cellular genomic DNA. Interestingly, positional analysis of foci either by metaphase chromosome spreads or by analysis of co-localization with antibodies to the telomere binding protein, TRF2, indicated quadruplex formation in telomeres and outside telomeres with a higher proportion at non-telomeric sites (Figure 2 c). Quantitation of the immunofluorescent foci in synchronized cells showed that: (a) some quadruplex formation was evident during all phases of the cell cycle; and (b) that overall quadruplex levels are modulated during cell-cycle progression with a maximal number of foci observed during the S phase, consistent with replication-dependent formation of G-quadruplex structures (Figure 2 d) [20 •• ]. Treatment of live cells with the G-quadruplex-trapping small molecule pyridostatin (PDS), before immunostaining, increases the number of foci, providing substantive evidence that a small molecule can trap quadruplex structures in cellular DNA (Figure 2 d). Indeed, complementary studies previously carried out using the radioactively labeled G-quadruplex ligand [ 3 H]-360A, showed selective binding at the telomeres of chromosomes of both human normal (peripheral blood lymphocytes) and tumor (T98G and CEM1301) cells [21 ]. Collectively, such studies have provided insights into the formation of G-quadruplex structures in the DNA in a cellular context. It cannot be ruled out that the process of fixing cells or the binding probe influences the formation of G-quadruplex-structures. However, the dynamic changes in apparent quadruplex sites coupled to changes in cellular states suggest the observations reflect structures that are intrinsic to genomic DNA.

The emergence of practical high-throughput DNA sequencing has transformed our ability to characterize genomic features at scale with precision [22 ]. Earlier this year, Lam et al. reported the use of another single-chain G-quadruplex specific antibody, hf2, to enrich for genomic DNA fragments containing folded G-quadruplex structures from mechanically fragmented DNA derived from MCF7 breast cancer cells [23 ]. Deep sequencing of libraries generated from the enriched DNA was used to identify technically reproducible peaks that correlated with computationally predicted G-quadruplex motifs. Stable quadruplex structures were experimentally mapped in regions that included sub-telomeres, gene bodies and gene regulatory sites. This approach allowed the identification of several genes with associated promoter G-quadruplexes, including PVT1 and STARD8. whose expression could be modulated by addition of the quadruplex ligand PDS to cells [23 ]. Rodriguez et al. used deep sequencing to map the sites of the DNA damage marker γH2AX induced by the treatment of human cancer cells with the quadruplex binding small molecule PDS [24 •• ]. Chromatin immunoprecipitation with an antibody against the DNA damage marker γH2AX followed by sequencing of the enriched DNA (ChIP-Seq) identified regions that were enriched for computationally predicted G-quadruplex motifs. Cell cycle analysis and the use of chemical inhibitors confirmed that PDS induces double strand breaks which are replication and transcription dependent.

Natural G-quadruplex binding proteins have provided important insights into the location of G-quadruplex structures in genomic DNA. For example, the binding sites of the Saccharomyces cerevisiae Pif1 DNA helicase, a potent unwinder of G-quadruplex structures in vitro. were mapped by ChIP-Seq [25 • ] to G-quadruplex motifs in a significant subset of the high-confidence Pif1-binding sites. Again consistent with an association in replication, Pif1 was more strongly associated with G-quadruplex motifs in late S phase and DNA Pol2 levels are higher at G-quadruplex sites in the absence of Pif1, suggestive of pausing. This approach experimentally identified 138 (of the 558 predicted) quadruplex motifs in the genome of S. cerevisiae. These observations are complemented by studies that employed super-resolution microscopy with fluorescent tagging of a PDS derivative that showed significant co-localization with Pif1 foci in human U2OS cells [24 •• ].

Both visualization and mapping experiments [ 17. 20••. 24•• and 25• ] suggest that G-quadruplex DNA formation is associated with replication. It is worth noting that the creation of single-strand gaps on the lagging strand at replication forks may create a context particularly prone to G-quadruplex formation. Thus any biological events that need DNA in single stranded form may be affected by the formation of metastable secondary structures such as G-quadruplex DNA.

We now focus on recent articles that describe biological consequences that are linked to quadruplex DNA. Many natural proteins have been identified that interact with quadruplex-DNA and Table 1 illustrates a range of protein activities that support the relevance of G-quadruplex DNA to replication and transcription.

Table 1. Selection of proteins that have been identified to interact with G-quadruplex DNA in vitro and that have been associated to G-quadruplex biology.






Photogallery Quadruplex in:


Higher Order G-Quadruplex Architectures | Memorial Sloan Kettering ...


In silico identification of novel ligands for G-quadruplex in the ...


Enantioselective DielsAlder Reactions with G-Quadruplex DNA-Based ...


preview_1258754.jpg


The role of supercoiling in transcriptional control of MYC and its ...


IJMS | Free Full-Text | DNA and RNA Quadruplex-Binding Proteins | HTML


ESI Mass Spectrometric Exploration of Selective Recognition of G ...


Higher Order G-Quadruplex Architectures | Memorial Sloan Kettering ...


Two graphs (left) showing changes with time of the 2GKU quadruplex ...


IJMS | Free Full-Text | DNA and RNA Quadruplex-Binding Proteins | HTML


Dr Julian Huppert - Research


G-quadruplexes regulate Epstein-Barr virusencoded nuclear antigen ...


G-quadruplexes regulate Epstein-Barr virusencoded nuclear antigen ...


Publications | Department of Chemistry


Biovaria:Technologies Details


Helquats, c-myc downregulation small molecules - IOCB TTO


A simple, label-free optical method for studies on the G ...


Telomere- and telomerase-interacting protein that unfolds telomere ...


Detection of G-Quadruplexes in Cells and Investigation of G ...