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. 2004 Jul 1;18(13):1618-29.
doi: 10.1101/gad.1200804.

Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA

Michelle L Duquette  1 , Priya Handa, Jack A Vincent, Andrew F Taylor, Nancy Maizels
Affiliations

Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA

Michelle L Duquette et al. Genes Dev. .

Abstract

We show that intracellular transcription of G-rich regions produces novel DNA structures, visible by electron microscopy as large (150-500 bp) loops. These G-loops are formed cotranscriptionally, and they contain G4 DNA on one strand and a stable RNA/DNA hybrid on the other. G-loop formation requires a G-rich nontemplate strand and reflects the unusual stability of the rG/dC base pair. G-loops and G4 DNA form efficiently within plasmid genomes transcribed in vitro or in Escherichia coli. These results establish that G4 DNA can form in vivo, a finding with implications for stability and maintenance of all G-rich genomic regions.

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Figures

Figure 1.
Figure 1.
G4 DNA and G-quartets. (A) Four guanines assemble in a planar ring to form a G-quartet. (B) G4 DNA is a stable structure composed of stacked G-quartets (shaded squares). (C) Diagram of predicted structure formed upon transcription of G-rich DNA. The G-rich strand contains G4 DNA, stabilized by G-quartets; and the C-rich template strand is hybridized to the RNA transcript (gray). For simplicity, only two regions of G4 DNA structure are diagrammed, although loops formed upon transcription of a long G-rich region could, in principle, contain many structured regions.
Figure 2.
Figure 2.
Loops form in transcribed G-rich DNAs. (A) Map of plasmid templates, which carry G-rich sequences downstream of a T7 promoter. (B) G-rich regions within the plasmid templates analyzed by EM. (C) Examples of loops formed upon transcription of pRX15F (Sμ repeat; left), pPH600 (Sγ3; middle), and pHumtel (telomeric repeat, right). DNA was transcribed in vitro, linearized with AflIII, and visualized by EM. Bar, 500 nm. (D) Maps of loops visualized in 15 randomly selected pRX15F (left), pPH600 (middle), and pHumtel (right) templates. Maps are drawn to scale.
Figure 3.
Figure 3.
Loops contain an RNA/DNA hybrid formed cotranscriptionally. (A) Examples of loops formed in pHumtel transcribed with Dig-UTP and visualized with anti-Dig/gold beads. Bar, 200 nm. (B) Loops are destroyed by RNase H. Transcribed pRX15F before (left) and after (right) RNase H treatment. No loops were identified among 134 molecules visualized following treatment with RNase H. Bar, 500 nm. (C) Loops form cotranscriptionally. Treatment of pPH600 templates with 20 μg/mL RNase A during (example on left) or after (example on right) transcription did not alter loop formation; loops were evident in 58% (53/90) and 60% (60/99) of molecules, respectively. Bar, 500 nm.
Figure 4.
Figure 4.
Loops contain G4 DNA. (A) Examples of GQN1 cleavage of loops in transcribed pRX15F (Sμ; left), pPH600 (Sγ3; middle), and pHumtel (telomeric repeat, right). Arrows mark cleaved G-loops. Bar, 200 nm. (B) Effect of GQN1 treatment on transcribed pRX15F, pPH600, and pHumtel templates. DNAs were visualized by EM, and loops were scored as cleaved if they were opened and contained a clearly visible arm or ends, as in Figure 4A. (n) Number of molecules scored. (C) Effect of competitor G4 DNA (10-fold excess) or single-stranded DNA (1000-fold excess) on GQN1 cleavage of G-loops in transcribed plasmids, normalized to 100% cleavage in the absence of competitor. (D) G4 DNA recognized by recombinant biotinylated Nucleolin-428/streptavidin gold beads. Arrows indicate beads bound at loops. Bar, 200 nm. (E) DMS footprinting analysis verifies the presence of G-quartets. (Left) Example of a DMS footprint of pRG4F (left, mock transcribed; right, transcribed). (Right) compiled results of six independent footprinting experiments showing ratio of DMS accessibility of each G-run in the transcribed sample relative to the untranscribed control. Diagram indicates positions of G-runs (boxes) and single Gs (lines), and direction of transcription (arrows). Bars, S.D.
Figure 5.
Figure 5.
Loops containing G4 DNA form in plasmids transcribed in E. coli. (A) Examples of loops formed in pPH600 (Sγ3) transcribed in E. coli strain NM 256 [AB1157 (λDE3) rnh::cat recQ::kan]. Bar, 200 nm. (B) Examples of GQN1 cleavage of loops formed in pPH600 (Sγ3) transcribed in E. coli NM 256. Arrows indicate broken loops. Bar, 200 nm. (C) Sensitivity of loops formed in E. coli to GQN1 cleavage. Loops were analyzed by EM and were scored as cleaved if they were opened and contained clearly visible DNA ends. (n) Number of molecules scored.
Figure 6.
Figure 6.
Effect of topology on G-loop formation. (A) Examples of loops formed on supercoiled, relaxed, and linearized pPH600 templates. Bar, 500 nm. (B) Effect of plasmid topology on efficiency of loop formation. Loops were visualized by EM, and at least 400 molecules of each topoisomer were scored. (C) Effect of plasmid topology on G4 DNA formation. Templates of each topoisomer were treated with GQN1 and visualized by EM to assay cleavage; 200 GQN1-treated and 200-untreated molecules of each topoisomer were visualized. (D) Effect of plasmid topology on loop size. Loop size was measured following transcription of supercoiled, relaxed, or linear pPH600 templates, by EM visualization of 35 molecules of each topoisomer.
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