Detection of G-quadruplex DNA in mammalian cells

Henderson, Alexander; Wu, Yuliang; Huang, Yu Chuan; Chavez, Elizabeth A.; Platt, Jesse M.; Johnson, F. Brad; Brosh, Robert M.; Sen, Dipankar; Lansdorp, Peter M.

doi:10.1093/nar/gkt957

Cited by 438 publications

(357 citation statements)

References 60 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: 91%

“…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: 91%

Section: Methodsmentioning

confidence: 99%

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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“…To date, the strongest arguments have been provided by quadruplex-specific antibodies, 7-9 including BG4 8 and 1H6. 9 However, an important limitation of immunodetection is its restriction to fixed and permeabilized cells (to allow entry of cell-impermeable antibodies), therefore limiting the detection of quadruplexes to cells whose morphological integrity is impaired. Rodriguez et al reported on an elegant way to tackle this issue, performing cell-based chemical labeling of a quadruplex ligand (PDS-α) bound to its genomic targets prior to cell fixing, thus enabling the identification of quadruplexes in functioning cells.…”

Section: Introductionmentioning

confidence: 99%

Visualization of RNA-Quadruplexes in Live Cells

et al. 2015

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Visualization of DNA and RNA quadruplex formation in human cells was demonstrated recently with different quadruplex-specific antibodies. Despite the significant interest in these immunodetection approaches, dynamic detection of quadruplex in live cells remains elusive. Here, we report on NaphthoTASQ (N-TASQ), a next-generation quadruplex ligand that acts as a multiphoton turn-on fluorescent probe. Single-step incubation of human and mouse cells with N-TASQ enables the direct detection of RNA-quadruplexes in untreated cells (no fixation, permeabilization or mounting steps), thus offering a unique, unbiased visualization of quadruplexes in live cells.

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“…G4 structures are known to be stabilized by large monovalent cations, preferably potassium 3 . G4-DNA structures were recently detected in cells 4,5 . Computational analysis showed that G4-DNA sequences are predominantly located within telomeric regions and promoter regions (within 1kb upstream of the transcription start site, TSS) 6,7 , indicating strongly that G4 structures have roles in the regulation of gene expression.…”

mentioning

confidence: 99%