1 2012 Vol: 13(11):770-780. DOI: 10.1038/nrg3296

DNA secondary structures: stability and function of G-quadruplex structures

In addition to the canonical double helix, DNA can fold into various other inter- and intramolecular secondary structures. Although many such structures were long thought to be in vitro artefacts, bioinformatics demonstrates that DNA sequences capable of forming these structures are conserved throughout evolution, suggesting the existence of non-B-form DNA in vivo. In addition, genes whose products promote formation or resolution of these structures are found in diverse organisms, and a growing body of work suggests that the resolution of DNA secondary structures is critical for genome integrity. This Review focuses on emerging evidence relating to the characteristics of G-quadruplex structures and the possible influence of such structures on genomic stability and cellular processes, such as transcription.

Mentions
Figures
Figure 1: G-quadruplex DNA.a | An illustration of the interactions in a G-quartet. This quartet is represented schematically as a square in the other panels of this figure. M+ denotes a monovalent cation. b | Schematic diagrams of intramolecular (left) and intermolecular (right) G-quadruplex (G4) DNA structures. The arrowheads indicate the direction of the DNA strands. The intermolecular structures shown have two (upper) or four (lower) strands. Figure 2: Putative functional roles of G-quadruplex structures at telomeres.Telomeric sequences can fold into G-quadruplex (G4) structures in vitro. Currently, many groups are investigating the physiological relevance of this phenomenon. a | G4 structures may form at the telomeric 3′ overhang and have a role in protecting telomeres from degradation by nucleases (red) or other events. b | Work in ciliates shows that G4 structures do form at telomeres and have a role in telomere protection and tethering to the nuclear scaffold. Formation, stabilization and tethering is faciliated by G4-binding proteins (green). c | Ligands (blue) that bind to telomeric G4 structures are currently being analysed for their ability to influence telomere length by altering telomerase (yellow) activity. Figure 3: Putative functional roles of G-quadruplex structures during DNA replication.Computational studies show that in all tested organisms, many regions in the genome have the ability to form G-quadruplex (G4) structures. In vitro and in vivo studies indicate that unresolved G4 structures may influence DNA replication by slowing or stalling the replication fork machinery (replisome; blue). Figure 4: Putative functional roles of G-quadruplex structures during transcription.Genome-wide bioinformatic analyses identified loci with high potential to form G-quadruplex (G4) structures. Among these loci, the promoters, transcription factor binding sites and 5′UTR regions of mRNAs are highly enriched for G4 motifs. These analyses, together with protein–G4 interaction studies, provide insights into predicted functions of G4 structures during transcription. a | G4 structures are postulated to block transcription by inhibiting polymerase (purple). b | G4 structures are postulated to facilitate transcription by keeping the transcribed strand in the single-stranded conformation. c | G4 DNA may stimulate transcription by recruiting proteins (green) that recruit or stimulate polymerase. d | G4 structures are suggested to block transcription via the recruitment of G4 binding proteins (blue), which directly or indirectly (red) repress transcription. Figure 5: Putative roles for G-quadruplex structures in meiosis.a | It has been proposed that G-quadruplex (G4) structures might assist in the formation of the telomere-dependent bouquet structure during meiosis26. G4-promoting proteins (pink) could be involved in the formation of G4 structures and tethering of the telomeric bouquet to the nuclear scaffold. b | It has also been suggested that G4 structures could promote meiotic homologous recombination76, 117 if there is overlap between G4 motifs and preferred meiotic double-strand break sites15. Sites of homologous recombination are indicated by the dashed lines.
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References
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    • . . . The right-handed double helical structure of B-form DNA (B-DNA) has been known since 1953 (Ref. 1) . . .
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    • . . . These non-B-form secondary structures, which include G-quadruplex structures (G4 structures) (Fig. 1) as well as Z-DNA, cruciforms and triplexes (Box 1), were originally characterized in vitro using biophysical techniques (for example, circular dichroism2) . . .
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    • . . . G4 structures are stacked nucleic acid structures that can form within specific repetitive G-rich DNA or RNA sequences (reviewed in Ref. 3) . . .
    • . . . However, loops are usually small (1–7 nucleotides (nt)), and smaller loops result in more stable G4 structures, as do longer G-tracts3 . . .
    • . . . One of the best-studied systems for a role of G4 structures in transcription involves the mammalian MYC (also known as c-MYC) locus (reviewed in Refs 3,79), although findings similar to those discussed below have been reported for multiple loci80, 81, 82, 83, 84 . . .
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    • . . . In 1910, Bang4 was the first to report the fact that guanylic acid forms a gel at high concentrations, which suggested that G-rich sequences in DNA may form higher-order structures . . .
    • . . . There is good evidence that Neisseria gonorrhoeae, the bacterium that causes human gonorrhoea, uses a G4 based system to regulate expression of the genes that allow it to avoid the human immune system124 . . .
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    • . . . Fifty years later, Gellert and colleagues5 used X-ray diffraction to demonstrate that guanylic acids can assemble into tetrameric structures . . .
  6. Williamson, J. R., Raghuraman, M. K. & Cech, T. R. Monovalent cation-induced structure of telomeric DNA: the G-quartet model. Cell 59, 871-880 (1989). This study demonstrates that oligonucleotides composed of cilate telomeric repeat sequences form G-quartets in the presence of certain monovalent cations (for example, Na+ and K+). The authors also propose that G-quartets may form at telomeres in vivo and must be dealt with by the replication machinery , .
    • . . . This structure is stabilized by monovalent cations that occupy the central cavities between the stacks, neutralizing the electrostatic repulsion of inwardly pointing guanine oxygens6, 7, 8. . . .
    • . . . Regardless of the precise sequence of the telomere, the G-rich strand of various telomeric sequences can usually form stable G4 structures in vitro (Fig. 2); for example, in non-denaturing polyacrylamide gels, oligonucleotides corresponding to the telomeric G-rich strand display unexpected banding patterns that are due to the formation of G4 structures6, 24, 25, 26. . . .
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    • . . . This structure is stabilized by monovalent cations that occupy the central cavities between the stacks, neutralizing the electrostatic repulsion of inwardly pointing guanine oxygens6, 7, 8. . . .
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    • . . . This structure is stabilized by monovalent cations that occupy the central cavities between the stacks, neutralizing the electrostatic repulsion of inwardly pointing guanine oxygens6, 7, 8. . . .
    • . . . G4 RNA structures are reported to affect mRNA splicing, translation and degradation (reviewed in Refs 8,125,126) . . .
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    • . . . Furthermore, they can form within one strand (intramolecular) or from multiple strands (intermolecular), and various loop structures are also possible9, 10 . . .
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    • . . . Furthermore, they can form within one strand (intramolecular) or from multiple strands (intermolecular), and various loop structures are also possible9, 10 . . .
  11. Hardin, C. C., Perry, A. G. & White, K. Thermodynamic and kinetic characterization of the dissociation and assembly of quadruplex nucleic acids. Biopolymers 56, 147-194 (2000) , .
    • . . . G4 structures can be extremely stable, although the topology and stability of the G4 structure depends on many factors, including the length and sequence composition of the total G4 motif, the size of the loops between the guanines, strand stoichiometry and alignment11, 12, 13, and the nature of the binding cations14. . . .
  12. Guedin, A., Gros, J., Alberti, P. & Mergny, J. L. How long is too long? Effects of loop size on G-quadruplex stability. Nucleic Acids Res. 38, 7858-7868 (2010) , .
    • . . . G4 structures can be extremely stable, although the topology and stability of the G4 structure depends on many factors, including the length and sequence composition of the total G4 motif, the size of the loops between the guanines, strand stoichiometry and alignment11, 12, 13, and the nature of the binding cations14. . . .
  13. Bugaut, A. & Balasubramanian, S. A sequence-independent study of the influence of short loop lengths on the stability and topology of intramolecular DNA G-quadruplexes. Biochemistry 47, 689-697 (2008) , .
    • . . . G4 structures can be extremely stable, although the topology and stability of the G4 structure depends on many factors, including the length and sequence composition of the total G4 motif, the size of the loops between the guanines, strand stoichiometry and alignment11, 12, 13, and the nature of the binding cations14. . . .
  14. Patel, D. J., Phan, A. T. & Kuryavyi, V. Human telomere, oncogenic promoter and 5′-UTR G-quadruplexes: diverse higher order DNA and RNA targets for cancer therapeutics. Nucleic Acids Res. 35, 7429-7455 (2007) , .
    • . . . G4 structures can be extremely stable, although the topology and stability of the G4 structure depends on many factors, including the length and sequence composition of the total G4 motif, the size of the loops between the guanines, strand stoichiometry and alignment11, 12, 13, and the nature of the binding cations14. . . .
  15. Capra, J. A., Paeschke, K., Singh, M. & Zakian, V. A. G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae. PLoS Comput. Biol. 6, e1000861 (2010). This study reports a genome-wide computational analysis identifying the location and evolutionary conservation of G4 motifs in S. cerevisiae and related yeasts , .
  16. Todd, A. K., Johnston, M. & Neidle, S. Highly prevalent putative quadruplex sequence motifs in human DNA. Nucleic Acids Res. 33, 2901-2907 (2005) , .
    • . . . Computational analyses reveal that there are >375,000 G4 motifs in the human genome, whereas there are >1,400 G4 motifs in the Saccharomyces cerevisiae nuclear genome, including those in ribosomal and telomeric DNA, which are both particularly G4-rich15, 16, 17, 18 . . .
    • . . . In human, yeast and bacterial genomes, G4 motifs are similarly distributed and are over-represented in certain functional regions, such as promoters15, 16, 17, 18, 20 . . .
  17. Hershman, S. G. et al. Genomic distribution and functional analyses of potential G-quadruplex-forming sequences in Saccharomyces cerevisiae. Nucleic Acids Res. 36, 144-156 (2008) , .
    • . . . Computational analyses reveal that there are >375,000 G4 motifs in the human genome, whereas there are >1,400 G4 motifs in the Saccharomyces cerevisiae nuclear genome, including those in ribosomal and telomeric DNA, which are both particularly G4-rich15, 16, 17, 18 . . .
    • . . . In human, yeast and bacterial genomes, G4 motifs are similarly distributed and are over-represented in certain functional regions, such as promoters15, 16, 17, 18, 20 . . .
    • . . . In diverse organisms, G4 DNA motifs are also common in G-rich micro- and minisatellites, up- and downstream of TSSs (often near promoters), within the ribosomal DNA, near transcription factor binding sites, and at preferred mitotic and meiotic DSB sites15, 17, 18, 21, 22. . . .
    • . . . A similar enrichment of G4 motifs in promoter regions is found in other organisms, including yeast, plants and bacteria15, 17, 20, 73, 74 . . .
    • . . . In yeast, there is no distinct asymmetry in G4 motif location between the non-template and template strands, but there is a correlation between nucleosome-free regions and G4 motifs in promoters15, a finding that supports the prediction that G4 structures will form more easily in nucleosome-free regions17 . . .
    • . . . Similar studies have investigated the effects of mutations in helicases known to unwind G4 DNA on transcription genome wide17, 108 . . .
  18. Huppert, J. L. & Balasubramanian, S. Prevalence of quadruplexes in the human genome. Nucleic Acids Res. 33, 2908-2916 (2005). The authors performed a genome-wide computational analysis that identified all regions in the human genome with a high potential to form G4 structures , .
    • . . . Computational analyses reveal that there are >375,000 G4 motifs in the human genome, whereas there are >1,400 G4 motifs in the Saccharomyces cerevisiae nuclear genome, including those in ribosomal and telomeric DNA, which are both particularly G4-rich15, 16, 17, 18 . . .
    • . . . In human, yeast and bacterial genomes, G4 motifs are similarly distributed and are over-represented in certain functional regions, such as promoters15, 16, 17, 18, 20 . . .
  19. Huppert, J. L. Four-stranded nucleic acids: structure, function and targeting of G-quadruplexes. Chem. Soc. Rev. 37, 1375-1384 (2008) , .
    • . . . Computational studies in various organisms have revealed that G4 motifs are not randomly located within genomes, but rather they tend to cluster in particular genomic regions (reviewed in Ref. 19) . . .
  20. Rawal, P. et al. Genome-wide prediction of G4 DNA as regulatory motifs: role in Escherichia coli global regulation. Genome Res. 16, 644-655 (2006) , .
    • . . . In human, yeast and bacterial genomes, G4 motifs are similarly distributed and are over-represented in certain functional regions, such as promoters15, 16, 17, 18, 20 . . .
    • . . . A similar enrichment of G4 motifs in promoter regions is found in other organisms, including yeast, plants and bacteria15, 17, 20, 73, 74 . . .
  21. Nakken, S., Rognes, T. & Hovig, E. The disruptive positions in human G-quadruplex motifs are less polymorphic and more conserved than their neutral counterparts. Nucleic Acids Res. 37, 5749-5756 (2009) , .
    • . . . Furthermore, the locations and nucleotide compositions of G4 motifs are conserved in human populations and among related yeast species15, 21 . . .
    • . . . In diverse organisms, G4 DNA motifs are also common in G-rich micro- and minisatellites, up- and downstream of TSSs (often near promoters), within the ribosomal DNA, near transcription factor binding sites, and at preferred mitotic and meiotic DSB sites15, 17, 18, 21, 22. . . .
  22. Eddy, J. & Maizels, N. Gene function correlates with potential for G4 DNA formation in the human genome. Nucleic Acids Res. 34, 3887-3896 (2006) , .
    • . . . In diverse organisms, G4 DNA motifs are also common in G-rich micro- and minisatellites, up- and downstream of TSSs (often near promoters), within the ribosomal DNA, near transcription factor binding sites, and at preferred mitotic and meiotic DSB sites15, 17, 18, 21, 22. . . .
    • . . . Intriguingly, bioinformatics show that the promoters of human oncogenes and regulatory genes (for example, transcription factors) are more likely than the average gene to contain G4 motifs, whereas G4 motifs are under-represented in the promoters of housekeeping and tumour suppressor genes22, 72 . . .
  23. Zakian, V. A. Telomeres: the beginnings and ends of eukaryotic chromosomes. Exp. Cell Res. 318, 1456-1460 (2012) , .
    • . . . Telomeres are essential to protect chromosomes from degradation, end-to-end fusions, and being recognized as DSBs23 . . .
  24. Henderson, E. et al. Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs. Cell 51, 899-908 (1987) , .
    • . . . Regardless of the precise sequence of the telomere, the G-rich strand of various telomeric sequences can usually form stable G4 structures in vitro (Fig. 2); for example, in non-denaturing polyacrylamide gels, oligonucleotides corresponding to the telomeric G-rich strand display unexpected banding patterns that are due to the formation of G4 structures6, 24, 25, 26. . . .
  25. Sundquist, W. I. & Klug, A. Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops. Nature 342, 825-829 (1989). This in vitro analysis demonstrates that telomeric DNA can fold into G4 structures , .
    • . . . Regardless of the precise sequence of the telomere, the G-rich strand of various telomeric sequences can usually form stable G4 structures in vitro (Fig. 2); for example, in non-denaturing polyacrylamide gels, oligonucleotides corresponding to the telomeric G-rich strand display unexpected banding patterns that are due to the formation of G4 structures6, 24, 25, 26. . . .
    • . . . The possibility that G4 structures might form in vivo is demonstrated by in vitro experiments showing that telomere structural proteins, such as TEBPα and TEBPβ in ciliates and Rap1 in S. cerevisiae, can promote the formation of G4 DNA25, 27, 28, 29 . . .
  26. Sen, D. & Gilbert, W. Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis. Nature 334, 364-366 (1988) , .
  27. Fang, G. & Cech, T. R. The β subunit of Oxytricha telomere-binding protein promotes G-quartet formation by telomeric DNA. Cell 74, 875-885 (1993) , .
    • . . . The possibility that G4 structures might form in vivo is demonstrated by in vitro experiments showing that telomere structural proteins, such as TEBPα and TEBPβ in ciliates and Rap1 in S. cerevisiae, can promote the formation of G4 DNA25, 27, 28, 29 . . .
    • . . . TEBPα binds to the telomeric overhang and recruits TEBPβ, which is able to promote the formation of G4 structures with its highly charged carboxyl terminus, as shown in vitro27, 28. . . .
    • . . . However, the existence of chaperones (for example, TEBPβ and Rap1) that promote the formation of G4 DNA27, 28, 29 suggests that nature has evolved mechanisms to overcome this slow formation . . .
  28. Paeschke, K., Simonsson, T., Postberg, J., Rhodes, D. & Lipps, H. J. Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. Nature Struct. Mol. Biol. 12, 847-854 (2005). This study includes compelling evidence for the in vivo existence of G4 structures at telomeres. Telomere-binding proteins are shown to regulate the formation of such structures , .
    • . . . The possibility that G4 structures might form in vivo is demonstrated by in vitro experiments showing that telomere structural proteins, such as TEBPα and TEBPβ in ciliates and Rap1 in S. cerevisiae, can promote the formation of G4 DNA25, 27, 28, 29 . . .
    • . . . With these antibodies, it is possible to show that G4 structures exist in vivo at Stylonychia lemnae telomeres and to determine proteins that are required for their formation and unfolding28, 32, 33 . . .
    • . . . Second and third, immunofluorescence and gene knockdown analyses show that two enzymes, the telomerase holoenzyme and a RecQ family helicase, are recruited to telomeric G4 structures at the end of S phase and are essential for the unfolding of telomeric G4 structures28, 33, 34, 35 . . .
    • . . . However, the existence of chaperones (for example, TEBPβ and Rap1) that promote the formation of G4 DNA27, 28, 29 suggests that nature has evolved mechanisms to overcome this slow formation . . .
  29. Giraldo, R. & Rhodes, D. The yeast telomere-binding protein RAP1 binds to and promotes the formation of DNA quadruplexes in telomeric DNA. EMBO J. 13, 2411-2420 (1994) , .
    • . . . The possibility that G4 structures might form in vivo is demonstrated by in vitro experiments showing that telomere structural proteins, such as TEBPα and TEBPβ in ciliates and Rap1 in S. cerevisiae, can promote the formation of G4 DNA25, 27, 28, 29 . . .
    • . . . However, the existence of chaperones (for example, TEBPβ and Rap1) that promote the formation of G4 DNA27, 28, 29 suggests that nature has evolved mechanisms to overcome this slow formation . . .
  30. Zaug, A. J., Podell, E. R. & Cech, T. R. Human POT1 disrupts telomeric G-quadruplexes allowing telomerase extension in vitro. Proc. Natl Acad. Sci. USA 102, 10864-10869 (2005) , .
    • . . . By contrast, the human telomeric G-strand binding protein protection of telomeres protein 1 (POT1) promotes the unfolding of G4 structures in vitro30, 31 . . .
  31. Wang, H., Nora, G. J., Ghodke, H. & Opresko, P. L. Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding. J. Biol. Chem. 286, 7479-7489 (2011) , .
    • . . . By contrast, the human telomeric G-strand binding protein protection of telomeres protein 1 (POT1) promotes the unfolding of G4 structures in vitro30, 31 . . .
  32. Schaffitzel, C. et al. In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl Acad. Sci. 98, 8572-8577 (2001) , .
    • . . . With these antibodies, it is possible to show that G4 structures exist in vivo at Stylonychia lemnae telomeres and to determine proteins that are required for their formation and unfolding28, 32, 33 . . .
    • . . . Only the antibodies raised against antiparallel G4 structures bind to S. lemnae telomeres, indicating that antiparallel, and not parallel, G4 DNA is present in viv o32 . . .
    • . . . Accordingly, telomeric G4 structures, which are present during most of the S. lemnae cell cycle, are resolved during DNA replication32 . . .
  33. Paeschke, K. et al. Telomerase recruitment by the telomere end binding protein-β facilitates G-quadruplex DNA unfolding in ciliates. Nature Struct. Mol. Biol. 15, 598-604 (2008) , .
    • . . . With these antibodies, it is possible to show that G4 structures exist in vivo at Stylonychia lemnae telomeres and to determine proteins that are required for their formation and unfolding28, 32, 33 . . .
    • . . . Second and third, immunofluorescence and gene knockdown analyses show that two enzymes, the telomerase holoenzyme and a RecQ family helicase, are recruited to telomeric G4 structures at the end of S phase and are essential for the unfolding of telomeric G4 structures28, 33, 34, 35 . . .
  34. Postberg, J., Tsytlonok, M., Sparvoli, D., Rhodes, D. & Lipps, H. J. A telomerase-associated RecQ protein-like helicase resolves telomeric G-quadruplex structures during replication. Gene 497, 147-154 (2012) , .
    • . . . Second and third, immunofluorescence and gene knockdown analyses show that two enzymes, the telomerase holoenzyme and a RecQ family helicase, are recruited to telomeric G4 structures at the end of S phase and are essential for the unfolding of telomeric G4 structures28, 33, 34, 35 . . .
  35. Juranek, S. A. & Paeschke, K. Cell cycle regulation of G-quadruplex DNA structures at telomeres. Curr. Pharm. Des. 18, 1867-1872 (2012) , .
    • . . . Second and third, immunofluorescence and gene knockdown analyses show that two enzymes, the telomerase holoenzyme and a RecQ family helicase, are recruited to telomeric G4 structures at the end of S phase and are essential for the unfolding of telomeric G4 structures28, 33, 34, 35 . . .
  36. Paeschke, K., McDonald, K. R. & Zakian, V. A. Telomeres: structures in need of unwinding. FEBS Lett. 584, 3769-3772 (2010) , .
    • . . . However, RecQ helicases in other organisms, such as Sgs1 in S. cerevisiae and WRN and BLM in humans, also act on telomeres and can unwind G4 structures in vitro (reviewed in Ref. 36) . . .
  37. Yang, Q. et al. Verification of specific G-quadruplex structure by using a novel cyanine dye supramolecular assembly: I. recognizing mixed G-quadruplex in human telomeres. Chem. Commun. 9, 1103-1105 (2009) , .
    • . . . There is also evidence for G4 DNA at telomeres in human cultured cells: BMVC (3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide) is a fluorescent biomarker that binds and stabilizes G4 structures in vitro, and in vivo staining with BMVC marks the distal ends of metaphase chromosomes in human lung adenocarcinoma cells37, 38, suggesting telomeric binding . . .
  38. Chang, C. C. et al. A novel carbazole derivative, BMVC: a potential antitumor agent and fluorescence marker of cancer cells. Chem. Biodivers. 1, 1377-1384 (2004) , .
    • . . . There is also evidence for G4 DNA at telomeres in human cultured cells: BMVC (3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide) is a fluorescent biomarker that binds and stabilizes G4 structures in vitro, and in vivo staining with BMVC marks the distal ends of metaphase chromosomes in human lung adenocarcinoma cells37, 38, suggesting telomeric binding . . .
  39. Shay, J. W. & Wright, W. E. Role of telomeres and telomerase in cancer. Seminars Cancer Biol. 21, 349-353 (2011) , .
    • . . . Human telomerase is inactive in most somatic cells but is upregulated in most cancers, in which it is thought to promote the lifespan of malignant cells39 . . .
  40. Zahler, A. M., Williamson, J. R., Cech, T. R. & Prescott, D. M. Inhibition of telomerase by G-quartet DNA structures. Nature 350, 718-720 (1991). The authors report the first observation that telomerase action is influenced by G4 structures , .
    • . . . G4 structures influence telomerase activity: intramolecular antiparallel G4 structures block telomerase activity, whereas intermolecular parallel G4 DNA is permissive for extension by telomerase40, 41, 42. . . .
  41. Oganesian, L., Moon, I. K., Bryan, T. M. & Jarstfer, M. B. Extension of G-quadruplex DNA by ciliate telomerase. EMBO J. 25, 1148-1159 (2006) , .
    • . . . G4 structures influence telomerase activity: intramolecular antiparallel G4 structures block telomerase activity, whereas intermolecular parallel G4 DNA is permissive for extension by telomerase40, 41, 42. . . .
  42. Oganesian, L., Graham, M. E., Robinson, P. J. & Bryan, T. M. Telomerase recognizes G-quadruplex and linear DNA as distinct substrates. Biochemistry 46, 11279-11290 (2007) , .
    • . . . G4 structures influence telomerase activity: intramolecular antiparallel G4 structures block telomerase activity, whereas intermolecular parallel G4 DNA is permissive for extension by telomerase40, 41, 42. . . .
  43. Neidle, S. Human telomeric G-quadruplex: the current status of telomeric G-quadruplexes as therapeutic targets in human cancer. FEBS J. 277, 1118-1125 (2010) , .
    • . . . Because telomerase is active in most human cancers and this activity can be influenced by G4 structures, a variety of small molecule ligands with different specificities and target regions that bind and stabilize G4 structures are being tested in various assays43 . . .
  44. Rezler, E. M. et al. Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric G-quadruplex structure. J. Am. Chem. Soc. 127, 9439-9447 (2005) , .
    • . . . For example, telomestatin has nanomolar affinity for telomeric G4 structures (which is nearly two orders of magnitude lower than its affinity for double-stranded DNA) and stabilizes intramolecular antiparallel G4 structures in vitro44, 45 . . .
  45. Kim, M. Y., Vankayalapati, H., Shin-Ya, K., Wierzba, K. & Hurley, L. H. Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular G-quadruplex. J. Am. Chem. Soc. 124, 2098-2099 (2002) , .
    • . . . For example, telomestatin has nanomolar affinity for telomeric G4 structures (which is nearly two orders of magnitude lower than its affinity for double-stranded DNA) and stabilizes intramolecular antiparallel G4 structures in vitro44, 45 . . .
  46. De Cian, A. et al. Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action. Proc. Natl Acad. Sci. USA 104, 17347-17352 (2007) , .
    • . . . Moreover, telomestatin inhibits telomerase46 and causes gradual telomere shortening and growth arrest or apoptosis in human tissue culture cancer cells47, 48, 49, 50, 51, 52 . . .
  47. Gomez, D. et al. Telomerase downregulation induced by the G-quadruplex ligand 12459 in A549 cells is mediated by hTERT RNA alternative splicing. Nucleic Acids Res. 32, 371-379 (2004) , .
    • . . . Moreover, telomestatin inhibits telomerase46 and causes gradual telomere shortening and growth arrest or apoptosis in human tissue culture cancer cells47, 48, 49, 50, 51, 52 . . .
  48. Kim, M. Y., Gleason-Guzman, M., Izbicka, E., Nishioka, D. & Hurley, L. H. The different biological effects of telomestatin and TMPyP4 can be attributed to their selectivity for interaction with intramolecular or intermolecular G-quadruplex structures. Cancer Res. 63, 3247-3256 (2003) , .
    • . . . Moreover, telomestatin inhibits telomerase46 and causes gradual telomere shortening and growth arrest or apoptosis in human tissue culture cancer cells47, 48, 49, 50, 51, 52 . . .
  49. Shammas, M. A. et al. Telomerase inhibition and cell growth arrest after telomestatin treatment in multiple myeloma. Clin. Cancer Res. 10, 770-776 (2004) , .
    • . . . Moreover, telomestatin inhibits telomerase46 and causes gradual telomere shortening and growth arrest or apoptosis in human tissue culture cancer cells47, 48, 49, 50, 51, 52 . . .
  50. Tahara, H. et al. G-quadruplex stabilization by telomestatin induces TRF2 protein dissociation from telomeres and anaphase bridge formation accompanied by loss of the 3′ telomeric overhang in cancer cells. Oncogene 25, 1955-1966 (2006) , .
    • . . . Moreover, telomestatin inhibits telomerase46 and causes gradual telomere shortening and growth arrest or apoptosis in human tissue culture cancer cells47, 48, 49, 50, 51, 52 . . .
    • . . . However, telomeric DNA damage also increases in telomestatin-treated cells50, 53, 54 . . .
  51. Tauchi, T. et al. Activity of a novel G-quadruplex-interactive telomerase inhibitor, telomestatin (SOT-095), against human leukemia cells: involvement of ATM-dependent DNA damage response pathways. Oncogene 22, 5338-5347 (2003) , .
    • . . . Moreover, telomestatin inhibits telomerase46 and causes gradual telomere shortening and growth arrest or apoptosis in human tissue culture cancer cells47, 48, 49, 50, 51, 52 . . .
  52. Tauchi, T. et al. Telomerase inhibition with a novel G-quadruplex-interactive agent, telomestatin: in vitro and in vivo studies in acute leukemia. Oncogene 25, 5719-5725 (2006) , .
    • . . . Moreover, telomestatin inhibits telomerase46 and causes gradual telomere shortening and growth arrest or apoptosis in human tissue culture cancer cells47, 48, 49, 50, 51, 52 . . .
  53. Gomez, D. et al. Interaction of telomestatin with the telomeric single-strand overhang. J. Biol. Chem. 279, 41487-41494 (2004) , .
    • . . . However, telomeric DNA damage also increases in telomestatin-treated cells50, 53, 54 . . .
    • . . . In addition, telomestatin, a chemical ligand that is able to stabilize G4 structures in vitro53, 61, 62, causes impaired proliferation and increased apoptosis and DNA damage in FANCJ-deficient cells63 . . .
  54. Gomez, D. et al. The G-quadruplex ligand telomestatin inhibits POT1 binding to telomeric sequences in vitro and induces GFP-POT1 dissociation from telomeres in human cells. Cancer Res. 66, 6908-6912 (2006) , .
    • . . . However, telomeric DNA damage also increases in telomestatin-treated cells50, 53, 54 . . .
  55. Smith, J. S. et al. Rudimentary G-quadruplex-based telomere capping in Saccharomyces cerevisiae. Nature Struct. Mol. Biol. 18, 478-485 (2011) , .
    • . . . Indeed, in S. cerevisiae, G4 structures are thought to contribute to telomere capping when natural capping is impaired55 . . .
  56. London, T. B. et al. FANCJ is a structure-specific DNA helicase associated with the maintenance of genomic G/C tracts. J. Biol. Chem. 283, 36132-36139 (2008) , .
    • . . . Most of the human helicases that unwind G4 structures in vitro56, 57, 58, 59, 60 are associated with human diseases that cause genomic instability, including the RecQ helicases WRN (associated with premature ageing) and BLM (associated with increased cancer risk) as well as FANCJ (associated with increased cancer risk) and PIF1 (associated with increased cancer risk) . . .
    • . . . The best evidence that human disease is associated with loss of G4 unwinding comes from the finding that cell lines from human patients with Fanconi anaemia carrying FANCJ mutations display deletions that overlap G-rich regions with the potential to form G4 structures56 . . .
  57. Mohaghegh, P. et al. The Bloom's and Werner's syndrome proteins are DNA structure-specific helicases. Nucleic Acids Res. 29, 2843-2849 (2001) , .
    • . . . Most of the human helicases that unwind G4 structures in vitro56, 57, 58, 59, 60 are associated with human diseases that cause genomic instability, including the RecQ helicases WRN (associated with premature ageing) and BLM (associated with increased cancer risk) as well as FANCJ (associated with increased cancer risk) and PIF1 (associated with increased cancer risk) . . .
  58. Huber, M. D., Lee, D. C. & Maizels, N. G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific inhibition. Nucleic Acids Res. 30, 3954-3961 (2002) , .
    • . . . Most of the human helicases that unwind G4 structures in vitro56, 57, 58, 59, 60 are associated with human diseases that cause genomic instability, including the RecQ helicases WRN (associated with premature ageing) and BLM (associated with increased cancer risk) as well as FANCJ (associated with increased cancer risk) and PIF1 (associated with increased cancer risk) . . .
  59. Ribeyre, C. et al. The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming CEB1 sequences in vivo. PLoS Genet. 5, e1000475 (2009) , .
    • . . . Most of the human helicases that unwind G4 structures in vitro56, 57, 58, 59, 60 are associated with human diseases that cause genomic instability, including the RecQ helicases WRN (associated with premature ageing) and BLM (associated with increased cancer risk) as well as FANCJ (associated with increased cancer risk) and PIF1 (associated with increased cancer risk) . . .
    • . . . The S. cerevisiae Pif1 helicase acts at G4 motifs64, and members of the Pif1 DNA helicase family are particularly efficient in vitro unwinders of parallel intramolecular G4 substrates59 . . .
    • . . . Other studies also found instability of G4 motifs in pif1 cells59, 65 . . .
  60. Sanders, C. M. Human Pif1 helicase is a G-quadruplex DNA binding protein with G-quadruplex DNA unwinding activity. Biochem. J. 430, 119-128 (2010) , .
    • . . . Most of the human helicases that unwind G4 structures in vitro56, 57, 58, 59, 60 are associated with human diseases that cause genomic instability, including the RecQ helicases WRN (associated with premature ageing) and BLM (associated with increased cancer risk) as well as FANCJ (associated with increased cancer risk) and PIF1 (associated with increased cancer risk) . . .
  61. Arola, A. & Vilar, R. Stabilisation of G-quadruplex DNA by small molecules. Curr. Top. Med. Chem. 8, 1405-1415 (2008) , .
    • . . . In addition, telomestatin, a chemical ligand that is able to stabilize G4 structures in vitro53, 61, 62, causes impaired proliferation and increased apoptosis and DNA damage in FANCJ-deficient cells63 . . .
  62. Neidle, S. The structures of quadruplex nucleic acids and their drug complexes. Curr. Opin. Struct. Biol. 19, 239-250 (2009) , .
    • . . . In addition, telomestatin, a chemical ligand that is able to stabilize G4 structures in vitro53, 61, 62, causes impaired proliferation and increased apoptosis and DNA damage in FANCJ-deficient cells63 . . .
  63. Wu, Y., Shin-ya, K. & Brosh, R. M. Jr. FANCJ helicase defective in Fanconia anemia and breast cancer unwinds G-quadruplex DNA to defend genomic stability. Mol. Cell. Biol. 28, 4116-4128 (2008) , .
    • . . . In addition, telomestatin, a chemical ligand that is able to stabilize G4 structures in vitro53, 61, 62, causes impaired proliferation and increased apoptosis and DNA damage in FANCJ-deficient cells63 . . .
  64. Paeschke, K., Capra, J. A. & Zakian, V. A. DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase. Cell 145, 678-691 (2011). This paper demonstrates that the S. cerevisiae Pif1 helicase binds to G4 motifs genome-wide and is important for DNA replication and genome stability at such sites , .
    • . . . The S. cerevisiae Pif1 helicase acts at G4 motifs64, and members of the Pif1 DNA helicase family are particularly efficient in vitro unwinders of parallel intramolecular G4 substrates59 . . .
    • . . . Twenty-five per cent is likely to be an underestimate as, for technical reasons, this number excludes the large number of G4 motifs in ribosomal and telomeric DNA, both of which are strong Pif1 binding sites64 . . .
    • . . . Together, these data make a strong argument that G4 structures form in vivo and that their resolution by Pif1 suppresses genome instability64 . . .
    • . . . The frequent mutation of G4 motifs in pif1 mutant cells suggests the involvement of error-prone processes when G4 motifs are replicated and repaired in Pif1-deficient cells64 . . .
    • . . . Genetic experiments provide the most persuasive evidence to date for the in vivo existence of G4 structures during replication64, 68, 69 and transcription99 . . .
  65. Lopes, J. et al. G-quadruplex-induced instability during leading-strand replication. EMBO J. 30, 4033-4046 (2011) , .
    • . . . Other studies also found instability of G4 motifs in pif1 cells59, 65 . . .
  66. Sarkies, P., Reams, C., Simpson, L. J. & Sale, J. E. Epigenetic instability due to defective replication of structured DNA. Mol. Cell 40, 703-713 (2010). The authors show that failure to properly replicate through G4 motifs affects chromatin structure , .
    • . . . Indeed, in DT40 chicken cells, REV1, a translesion polymerase, is implicated in replication fork progression past G4 motifs on the leading strand66. . . .
    • . . . These stalled forks might be restarted with the aid of translesion polymerases, as suggested by data from DT40 chicken cells66, in which REV1, a Y family translesion polymerase113, is implicated in G4 lesion bypass . . .
    • . . . In the absence of REV1, DNA synthesis is uncoupled from histone recycling mechanisms, and transcriptional activation is blocked66 . . .
    • . . . The authors postulate that REV1 functions in replication at G4 motifs in order to preserve histone modifications66 . . .
  67. Rodriguez, R. et al. Small-molecule-induced DNA damage identifies alternative DNA structures in human genes. Nature Chem. Biol. 8, 301-310 (2012) , .
    • . . . One study used chromatin immunoprecipitation followed by sequencing (ChIP–seq) in combination with in vivo labelling with pyridostatin, a G4 binding molecule67 . . .
  68. Cheung, I., Schertzer, M., Rose, A. & Lansdorp, P. M. Disruption of dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA. Nature Genet. 31, 405-409 (2002). Genetic experiments reveal that deficiencies in the DOG-1 helicase lead to genome instabilty at G-rich sequences in C. elegans , .
    • . . . Similar to what is seen in cells from patients with Fanconi anaemia whose disease is due to mutations in the FANCJ helicase, mutations in the Caenorhabditis elegans DOG-1 helicase, which is distantly related to FANCJ, cause genome-wide deletions in G-rich sequences with the potential to form G4 structures68, 69 . . .
    • . . . The mutation rate in dog-1 mutants is very high (up to 4% per generation68) and increases with the length of the G-tract69 . . .
    • . . . Genetic experiments provide the most persuasive evidence to date for the in vivo existence of G4 structures during replication64, 68, 69 and transcription99 . . .
  69. Kruisselbrink, E. et al. Mutagenic capacity of endogenous G4 DNA underlies genome instability in FANCJ-defective C. elegans. Curr. Biol. 18, 900-905 (2008) , .
    • . . . Similar to what is seen in cells from patients with Fanconi anaemia whose disease is due to mutations in the FANCJ helicase, mutations in the Caenorhabditis elegans DOG-1 helicase, which is distantly related to FANCJ, cause genome-wide deletions in G-rich sequences with the potential to form G4 structures68, 69 . . .
    • . . . The mutation rate in dog-1 mutants is very high (up to 4% per generation68) and increases with the length of the G-tract69 . . .
    • . . . Genetic experiments provide the most persuasive evidence to date for the in vivo existence of G4 structures during replication64, 68, 69 and transcription99 . . .
  70. Vannier, J. B., Pavicic-Kaltenbrunner, V., Petalcorin, M. I., Ding, H. & Boulton, S. J. RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity. Cell 149, 795-806 (2012). This study indicates that G4 structures cause vertebrate telomere fragility, which can be counteracted by the RTEL1 helicase , .
    • . . . Recent data indicate that the human RTEL helicase helps to resolve G4 DNA at telomeres, perhaps in conjunction with BLM, to ensure telomere stability70 . . .
  71. Barber, L. J. et al. RTEL1 maintains genomic stability by suppressing homologous recombination. Cell 135, 261-271 (2008) , .
    • . . . For instance, C. elegans rtel-1 has high sequence similarity to dog-1, although G-rich sequences are not unstable in worms deficient for rtel-1 (Ref. 71) as they are in dog-1 mutant animals . . .
  72. Huppert, J. L. & Balasubramanian, S. G-quadruplexes in promoters throughout the human genome. Nucleic Acids Res. 35, 406-413 (2007). Computational analysis revealed that G4 motifs are significantly enriched at promoters in human DNA , .
    • . . . Indeed, one or more G4 motifs are found within 1,000 nt upstream of the TSS of 50% of human genes72 . . .
    • . . . Intriguingly, bioinformatics show that the promoters of human oncogenes and regulatory genes (for example, transcription factors) are more likely than the average gene to contain G4 motifs, whereas G4 motifs are under-represented in the promoters of housekeeping and tumour suppressor genes22, 72 . . .
  73. Yadav, V. K., Abraham, J. K., Mani, P., Kulshrestha, R. & Chowdhury, S. QuadBase: genome-wide database of G4 DNA-occurrence and conservation in human, chimpanzee, mouse and rat promoters and 146 microbes. Nucleic Acids Res. 36, D381-D385 (2008) , .
    • . . . A similar enrichment of G4 motifs in promoter regions is found in other organisms, including yeast, plants and bacteria15, 17, 20, 73, 74 . . .
  74. Mullen, M. A. et al. RNA G-quadruplexes in the model plant species Arabidopsis thaliana: prevalence and possible functional roles. Nucleic Acids Res. 38, 8149-8163 (2010) , .
    • . . . A similar enrichment of G4 motifs in promoter regions is found in other organisms, including yeast, plants and bacteria15, 17, 20, 73, 74 . . .
  75. Huppert, J. L., Bugaut, A., Kumari, S. & Balasubramanian, S. G-quadruplexes: the beginning and end of UTRs. Nucleic Acids Res. 36, 6260-6268 (2008) , .
    • . . . Those that are on the template strand tend to cluster at the 5′ end of the 5′UTR75 . . .
  76. Duquette, M. L., Handa, P., Vincent, J. A., Taylor, A. F. & Maizels, N. Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA. Genes Dev. 18, 1618-1629 (2004) , .
  77. Kouzine, F., Sanford, S., Elisha-Feil, Z. & Levens, D. The functional response of upstream DNA to dynamic supercoiling in vivo. Nature Struct. Mol. Biol. 15, 146-154 (2008) , .
    • . . . It is well known that supercoiling has both positive and negative effects on transcription77, and G4 structures are thought to form as a result of supercoiling-induced stress during transcription78 . . .
  78. Sun, D. & Hurley, L. H. The importance of negative superhelicity in inducing the formation of G-quadruplex and i-motif structures in the c-Myc promoter: implications for drug targeting and control of gene expression. J. Med. Chem. 52, 2863-2874 (2009) , .
    • . . . It is well known that supercoiling has both positive and negative effects on transcription77, and G4 structures are thought to form as a result of supercoiling-induced stress during transcription78 . . .
    • . . . In vitro studies show that the formation of G4 structures can compensate for the negative supercoiling78, 79 . . .
  79. Brooks, T. A., Kendrick, S. & Hurley, L. Making sense of G-quadruplex and i-motif functions in oncogene promoters. FEBS J. 277, 3459-3469 (2010) , .
    • . . . In vitro studies show that the formation of G4 structures can compensate for the negative supercoiling78, 79 . . .
    • . . . One of the best-studied systems for a role of G4 structures in transcription involves the mammalian MYC (also known as c-MYC) locus (reviewed in Refs 3,79), although findings similar to those discussed below have been reported for multiple loci80, 81, 82, 83, 84 . . .
    • . . . In addition to gene-specific approaches, results from genome-wide studies analysing the effects of drugs that stabilize and/or induce G4 formation have been used to argue that G4 structures affect transcription79, 107 . . .
  80. Qin, Y. & Hurley, L. H. Structures, folding patterns, and functions of intramolecular DNA G-quadruplexes found in eukaryotic promoter regions. Biochimie 90, 1149-1171 (2008) , .
    • . . . Second, proteins bound to the G4 structures (for example, transcriptional enhancers versus repressors) could also affect transcription (reviewed in Ref. 80). . . .
    • . . . One of the best-studied systems for a role of G4 structures in transcription involves the mammalian MYC (also known as c-MYC) locus (reviewed in Refs 3,79), although findings similar to those discussed below have been reported for multiple loci80, 81, 82, 83, 84 . . .
  81. Gunaratnam, M. et al. G-quadruplex compounds and cis-platin act synergistically to inhibit cancer cell growth in vitro and in vivo. Biochem. Pharmacol. 78, 115-122 (2009) , .
    • . . . One of the best-studied systems for a role of G4 structures in transcription involves the mammalian MYC (also known as c-MYC) locus (reviewed in Refs 3,79), although findings similar to those discussed below have been reported for multiple loci80, 81, 82, 83, 84 . . .
  82. Hsu, S. T. et al. A G-rich sequence within the c-kit oncogene promoter forms a parallel G-quadruplex having asymmetric G-tetrad dynamics. J. Am. Chem. Soc. 131, 13399-13409 (2009) , .
    • . . . One of the best-studied systems for a role of G4 structures in transcription involves the mammalian MYC (also known as c-MYC) locus (reviewed in Refs 3,79), although findings similar to those discussed below have been reported for multiple loci80, 81, 82, 83, 84 . . .
  83. Palumbo, S. L., Ebbinghaus, S. W. & Hurley, L. H. Formation of a unique end-to-end stacked pair of G-quadruplexes in the hTERT core promoter with implications for inhibition of telomerase by G-quadruplex-interactive ligands. J. Am. Chem. Soc. 131, 10878-10891 (2009) , .
    • . . . One of the best-studied systems for a role of G4 structures in transcription involves the mammalian MYC (also known as c-MYC) locus (reviewed in Refs 3,79), although findings similar to those discussed below have been reported for multiple loci80, 81, 82, 83, 84 . . .
  84. Bejugam, M. et al. Trisubstituted isoalloxazines as a new class of G-quadruplex binding ligands: small molecule regulation of c-kit oncogene expression. J. Am. Chem. Soc. 129, 12926-12927 (2007) , .
    • . . . One of the best-studied systems for a role of G4 structures in transcription involves the mammalian MYC (also known as c-MYC) locus (reviewed in Refs 3,79), although findings similar to those discussed below have been reported for multiple loci80, 81, 82, 83, 84 . . .
  85. Marcu, K. B., Bossone, S. A. & Patel, A. J. myc function and regulation. Annu. Rev. Biochem. 61, 809-860 (1992) , .
    • . . . Increased levels of MYC expression are observed in 80% of human cancer cells, and this increase promotes tumorigenesis85, 86, 87, 88, 89, 90 . . .
  86. D'Cruz, C. M. et al. c-MYC induces mammary tumorigenesis by means of a preferred pathway involving spontaneous Kras2 mutations. Nature Med. 7, 235-239 (2001) , .
    • . . . Increased levels of MYC expression are observed in 80% of human cancer cells, and this increase promotes tumorigenesis85, 86, 87, 88, 89, 90 . . .
  87. Strieder, V. & Lutz, W. Regulation of N-myc expression in development and disease. Cancer Lett. 180, 107-119 (2002) , .
    • . . . Increased levels of MYC expression are observed in 80% of human cancer cells, and this increase promotes tumorigenesis85, 86, 87, 88, 89, 90 . . .
  88. Lutz, W., Leon, J. & Eilers, M. Contributions of Myc to tumorigenesis. Biochim. Biophys. Acta 1602, 61-71 (2002) , .
    • . . . Increased levels of MYC expression are observed in 80% of human cancer cells, and this increase promotes tumorigenesis85, 86, 87, 88, 89, 90 . . .
  89. Pelengaris, S., Khan, M. & Evan, G. c-MYC: more than just a matter of life and death. Nature Rev. Cancer 2, 764-776 (2002) , .
    • . . . Increased levels of MYC expression are observed in 80% of human cancer cells, and this increase promotes tumorigenesis85, 86, 87, 88, 89, 90 . . .
  90. Pelengaris, S., Khan, M. & Evan, G. I. Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression. Cell 109, 321-334 (2002) , .
    • . . . Increased levels of MYC expression are observed in 80% of human cancer cells, and this increase promotes tumorigenesis85, 86, 87, 88, 89, 90 . . .
  91. Simonsson, T., Pecinka, P. & Kubista, M. DNA tetraplex formation in the control region of c-myc. Nucleic Acids Res. 26, 1167-1172 (1998) , .
    • . . . This element contains a G4 motif that forms a G4 structure in vitro91 . . .
  92. Siddiqui-Jain, A., Grand, C. L., Bearss, D. J. & Hurley, L. H. Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc. Natl Acad. Sci. USA 99, 11593-11598 (2002) , .
    • . . . Footprinting studies and luciferase reporter assays comparing the expression of a gene with a wild-type NHE III1 versus one with a mutated NHE III1 that cannot form a G4 structure demonstrate that the G4 motif in NHE III1 represses transcription92 . . .
    • . . . In another study, TMPyP4, a compound that binds to and stabilizes G4 structures (but also binds duplex DNA)93, 94, reduced MYC transcription in lymphoma cell lines and showed antitumour activity in mice92, 95 . . .
  93. Han, H., Langley, D. R., Rangan, A. & Hurley, L. H. Selective interactions of cationic porphyrins with G-quadruplex structures. J. Am. Chem. Soc. 123, 8902-8913 (2001) , .
    • . . . In another study, TMPyP4, a compound that binds to and stabilizes G4 structures (but also binds duplex DNA)93, 94, reduced MYC transcription in lymphoma cell lines and showed antitumour activity in mice92, 95 . . .
  94. Sun, D. et al. Inhibition of human telomerase by a G-quadruplex-interactive compound. J. Med. Chem. 40, 2113-2116 (1997) , .
    • . . . In another study, TMPyP4, a compound that binds to and stabilizes G4 structures (but also binds duplex DNA)93, 94, reduced MYC transcription in lymphoma cell lines and showed antitumour activity in mice92, 95 . . .
  95. Grand, C. L. et al. The cationic porphyrin TMPyP4 down-regulates c-MYC and human telomerase reverse transcriptase expression and inhibits tumor growth in vivo. Mol. Cancer Ther. 1, 565-573 (2002) , .
    • . . . In another study, TMPyP4, a compound that binds to and stabilizes G4 structures (but also binds duplex DNA)93, 94, reduced MYC transcription in lymphoma cell lines and showed antitumour activity in mice92, 95 . . .
  96. Brown, R. V., Danford, F. L., Gokhale, V., Hurley, L. H. & Brooks, T. A. Demonstration that drug-targeted down-regulation of MYC in non-Hodgkins lymphoma is directly mediated through the promoter G-quadruplex. J. Biol. Chem. 286, 41018-41027 (2011) , .
    • . . . GQC-05 binds the G4 structure in the NHE III1 region of MYC in vitro with high affinity and selectivity, and when added to Burkitt's lymphoma cell lines, GQC-05 results in reduced levels of transcribed MYC mRNA96 . . .
  97. Boddupally, P. V. et al. Anticancer activity and cellular repression of c-MYC by the G-quadruplex-stabilizing 11-piperazinylquindoline is not dependent on direct targeting of the G-quadruplex in the c-MYC promoter. J. Med. Chem. 55, 6076-6086 (2012) , .
    • . . . However, a recent publication found that 11 known G4 DNA ligands that affect MYC expression in cell-free assays do not interact directly with the MYC G4 structure in certain Burkitt's lymphoma cell lines97, clouding the interpretation of the GQC-05 results. . . .
  98. Gonzalez, V., Guo, K., Hurley, L. & Sun, D. Identification and characterization of nucleolin as a c-myc G-quadruplex-binding protein. J. Biol. Chem. 284, 23622-23635 (2009) , .
    • . . . This hypothesis is based on the in vivo binding of nucleolin to the MYC promoter in HeLa cells and the dose-dependent reduction in MYC transcription that occurs in nucleolin-treated cells98 . . .
    • . . . However, human nucleolin binds many G4 structures and can induce the formation of G4 DNA in vitro98, 100, 101, 102, 103 . . .
  99. Gonzalez, V. & Hurley, L. H. The c-MYC NHE III1: function and regulation. Annu. Rev. Pharmacol. Toxicol. 50, 111-129 (2010) , .
    • . . . One hypothesis is that nucleolin-mediated G4 formation in NHE III1 inhibits MYC transcription by masking binding sites for MYC transcriptional activators, such as the transcripton factor SP1 and cellular nucleic acid-binding protein (CNBP)99 . . .
    • . . . Genetic experiments provide the most persuasive evidence to date for the in vivo existence of G4 structures during replication64, 68, 69 and transcription99 . . .
  100. Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O. & Miller, D. M. Antiproliferative activity of G-rich oligonucleotides correlates with protein binding. J. Biol. Chem. 274, 26369-26377 (1999) , .
    • . . . However, human nucleolin binds many G4 structures and can induce the formation of G4 DNA in vitro98, 100, 101, 102, 103 . . .
  101. Brys, A. & Maizels, N. LR1 regulates c-myc transcription in B-cell lymphomas. Proc. Natl Acad. Sci. USA 91, 4915-4919 (1994) , .
    • . . . However, human nucleolin binds many G4 structures and can induce the formation of G4 DNA in vitro98, 100, 101, 102, 103 . . .
  102. Dempsey, L. A., Sun, H., Hanakahi, L. A. & Maizels, N. G4 DNA binding by LR1 and its subunits, nucleolin and hnRNP D, a role for G-G pairing in immunoglobulin switch recombination. J. Biol. Chem. 274, 1066-1071 (1999) , .
    • . . . However, human nucleolin binds many G4 structures and can induce the formation of G4 DNA in vitro98, 100, 101, 102, 103 . . .
  103. Gonzalez, V. & Hurley, L. H. The C-terminus of nucleolin promotes the formation of the c-MYC G-quadruplex and inhibits c-MYC promoter activity. Biochemistry 49, 9706-9714 (2010) , .
    • . . . However, human nucleolin binds many G4 structures and can induce the formation of G4 DNA in vitro98, 100, 101, 102, 103 . . .
  104. Wei, Q. & Paterson, B. M. Regulation of MyoD function in the dividing myoblast. FEBS Lett. 490, 171-178 (2001) , .
    • . . . For example, myosin D (MyoD) family proteins are transcription factors that bind to E-boxes in the promoters of several muscle-specific genes to regulate muscle development104 . . .
  105. Shklover, J., Weisman-Shomer, P., Yafe, A. & Fry, M. Quadruplex structures of muscle gene promoter sequences enhance in vivo MyoD-dependent gene expression. Nucleic Acids Res. 38, 2369-2377 (2010) , .
    • . . . In vitro, MyoD homodimers bind preferentially to G4 structures that are derived from the promoter sequences of muscle specific genes105 . . .
  106. Yafe, A., Shklover, J., Weisman-Shomer, P., Bengal, E. & Fry, M. Differential binding of quadruplex structures of muscle-specific genes regulatory sequences by MyoD, MRF4 and myogenin. Nucleic Acids Res. 36, 3916-3925 (2008) , .
    • . . . Consequently, MyoD–MyoE heterodimers, which cannot bind G4 structures, bind to the E-box instead and enhance gene transcription106 . . .
  107. Fernando, H. et al. Genome-wide analysis of a G-quadruplex-specific single-chain antibody that regulates gene expression. Nucleic Acids Res. 37, 6716-6722 (2009) , .
    • . . . In addition to gene-specific approaches, results from genome-wide studies analysing the effects of drugs that stabilize and/or induce G4 formation have been used to argue that G4 structures affect transcription79, 107 . . .
  108. Johnson, J. E., Cao, K., Ryvkin, P., Wang, L. S. & Johnson, F. B. Altered gene expression in the Werner and Bloom syndromes is associated with sequences having G-quadruplex forming potential. Nucleic Acids Res. 38, 1114-1122 (2010) , .
    • . . . Similar studies have investigated the effects of mutations in helicases known to unwind G4 DNA on transcription genome wide17, 108 . . .
    • . . . For instance, in human fibroblasts deficient for the WRN or BLM RecQ helicases, the transcription of genes that are predicted to form intramolecular G4 structures is significantly upregulated (P < 0.0001), and this upregulation correlates with the G4 motifs, not simple G-richness108 . . .
  109. Wyatt, J. R., Davis, P. W. & Freier, S. M. Kinetics of G-quartet-mediated tetramer formation. Biochemistry 35, 8002-8008 (1996) , .
    • . . . Indeed, it is well documented that intermolecular G4 DNA structures form and resolve slowly under physiological conditions109, 110 . . .
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    • . . . Indeed, it is well documented that intermolecular G4 DNA structures form and resolve slowly under physiological conditions109, 110 . . .
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