Quantitative visualization of DNA G-quadruplex structures in human cells

Biffi, Giulia; Tannahill, David; McCafferty, John; Balasubramanian, Shankar

doi:10.1038/nchem.1548

Cited by 1,883 publications

(1,877 citation statements)

References 24 publications

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“…Binding of 1H6 antibodies to G-rich oligonucleotides [(TTAGGG) 4 ] was also observed. Marginal binding to the singlestranded DNA (ssDNA) was recorded, as previously reported (41,42). These results confirm that under our conditions, both the telomeric and myc oligonucleotides fold into G-quadruplexes.…”

Section: Resultssupporting

confidence: 73%

“…The pSANG10-3F-BG4 plasmid was a gift from the laboratories of S. Balasubramanian and J. McCafferty (Addgene plasmid number 55756) (41). The single-chain Flag-BG4 antibody was expressed in Escherichia coli and purified by affinity chromatography as described previously (41).…”

Section: Methodsmentioning

confidence: 99%

“…The ELISA was performed with the BG4 or 1H6 antibodies that bind specifically to the G-quadruplex structures (41,42). Biotinylated oligonucleotides of the Myc promoter (5=-Biosg/ACTACT ACTGGGGAGGGTGGGGAGGGTGGGGAAGG-3=) and telomeric repeats (5=-Biosg/ACTACTACTGGTTAGGGT TAGGGTTAGGGTTAGGGTTAG-3=), which fold in G-quadruplex, and ssDNA (5=-Biosg/ACTACTACTGGCAT AGTGCGTGGGCG-3=), which supports formation of G-quadruplexes, were purchased from IDT (Coralville, Iowa, USA).…”

Section: Methodsmentioning

confidence: 99%

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Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Gilbert-Girard

Gravel

Artusi

et al. 2017

J Virol

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Human herpesviruses 6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes using a mechanism that remains poorly understood. To achieve a better understanding of the HHV-6A/B integration mechanism, we made use of BRACO-19, a compound that stabilizes G-quadruplex secondary structures and prevents telomere elongation by the telomerase complex. First, we analyzed the folding of telomeric sequences into G-quadruplex structures and their binding to BRACO-19 using G-quadruplex-specific antibodies and surface plasmon resonance. Circular dichroism studies indicate that BRACO-19 modifies the conformation and greatly stabilizes the G-quadruplexes formed in G-rich telomeric DNA. Subsequently we assessed the effects of BRACO-19 on the HHV-6A initial phase of infection. Our results indicate that BRACO-19 does not affect entry of HHV-6A DNA into cells. We next investigated if stabilization of G-quadruplexes by BRACO-19 affected HHV-6A's ability to integrate its genome into host chromosomes. Incubation of telomerase-expressing cells with BRACO-19, such as HeLa and MCF-7, caused a significant reduction in the HHV-6A integration frequency (P Ͻ 0.002); in contrast, BRACO-19 had no effect on HHV-6 integration frequency in U2OS cells that lack telomerase activity and elongate their telomeres through alternative lengthening mechanisms. Our data suggest that the fluidity of telomeres is important for efficient chromosomal integration of HHV-6A and that interference with telomerase activity negatively affects the generation of cellular clones containing integrated HHV-6A.IMPORTANCE HHV-6A/B can integrate their genomes into the telomeres of infected cells. Telomeres consist of repeated hexanucleotides (TTAGGG) of various lengths (up to several kilobases) and end with a single-stranded 3= extension. To avoid recognition and induce a DNA damage response, the single-stranded overhang folds back on itself and forms a telomeric loop (T-loop) or adopts a tertiary structure, referred to as a G-quadruplex. In the current study, we have examined the effects of a G-quadruplex binding and stabilizing agent, BRACO-19, on HHV-6A chromosomal integration. By stabilizing G-quadruplex structures, BRACO-19 affects the ability of the telomerase complex to elongate telomeres. Our results indicate that BRACO-19 reduces the number of clones harboring integrated HHV-6A. This study is the first of its kind and suggests that telomerase activity is essential to restore a functional telomere of adequate length following HHV-6A integration.

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Section: Resultssupporting

confidence: 73%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Gilbert-Girard

Gravel

Artusi

et al. 2017

J Virol

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“…Interest in the more general therapeutic significance of G4 has expanded during the past decade including G4 structures in the promoters of a wide range of genes important in cell signaling, recognized as hallmarks of cancer. More interestingly, G4 DNA structures have been now visualized in human cells, corroborating the concept of G4 ligands as therapeutically susceptible hotspots (6,7).…”

Section: Introductionmentioning

confidence: 56%

“…G-quadruplex (G4) ligands are small molecules able to bind to and stabilize G4 structures widely described at the telomeric ends of chromosomes (1)(2)(3)(4)(5)(6). Interest in the more general therapeutic significance of G4 has expanded during the past decade including G4 structures in the promoters of a wide range of genes important in cell signaling, recognized as hallmarks of cancer.…”

Section: Introductionmentioning

confidence: 99%

Targeting G-Quadruplex DNA Structures by EMICORON Has a Strong Antitumor Efficacy against Advanced Models of Human Colon Cancer

Porru¹,

Artuso²,

Salvati³

et al. 2015

Molecular Cancer Therapeutics

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We previously identified EMICORON as a novel G-quadruplex (G4) ligand showing high selectivity for G4 structures over the duplex DNA, causing telomere damage and inhibition of cell proliferation in transformed and tumor cells. Here, we evaluated the antitumoral effect of EMICORON on advanced models of human colon cancer that could adequately predict human clinical outcomes. Our results showed that EMICORON was well tolerated in mice, as no adverse effects were reported, and a low ratio of sensitivity across human and mouse bone marrow cells was observed, indicating a good potential for reaching similar blood levels in humans. Moreover, EMICORON showed a marked therapeutic efficacy, as it inhibited the growth of patient-derived xenografts (PDX) and orthotopic colon cancer and strongly reduced the dissemination of tumor cells to lymph nodes, intestine, stomach, and liver. Finally, activation of DNA damage and impairment of proliferation and angiogenesis are proved to be key determinants of EMICORON antitumoral activity. Altogether, our results, performed on advanced experimental models of human colon cancer that bridge the translational gap between preclinical and clinical studies, demonstrated that EMICORON had an unprecedented antitumor activity warranting further studies of EMI-CORON-based combination treatments.

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DNA G‐quadruplexes show strong interaction with DNA methyltransferases in vitro

et al. 2016

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The DNA methyltransferase enzymes (DNMTs) catalyzing cytosine methylation do so at specific locations of the genome, although with some level of redundancy. The de novo methyltransferases DNMT3A and 3B play a vital role in methylating the genome of the developing embryo in regions devoid of methylation marks. The ability of DNMTs to colocalize at sites of DNA damage is suggestive that recognition of mispaired bases and unusual structures is inherent to the function of these proteins. We provide evidence for G-quadruplex formation within imprinted gene promoters, and report highaffinity binding of recombinant human DNMTs to such DNA G-quadruplexes in vitro. These observations suggest a potential interaction of G-quadruplexes with the DNA methylation machinery, which may be of epigenetic and biological significance.

show abstract

Quantitative visualization of DNA G-quadruplex structures in human cells

Cited by 1,883 publications

References 24 publications

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

Targeting G-Quadruplex DNA Structures by EMICORON Has a Strong Antitumor Efficacy against Advanced Models of Human Colon Cancer

DNA G‐quadruplexes show strong interaction with DNA methyltransferases in vitro

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