The effects of DNA supercoiling on G-quadruplex formation

Abstract

Guanine-rich DNAs can fold into four-stranded structures that contain stacks of G-quartets. Bioinformatics studies have revealed that G-rich sequences with the potential to adopt these structures are unevenly distributed throughout genomes, and are especially found in gene promoter regions. With the exception of the single-stranded telomeric DNA, all genomic G-rich sequences will always be present along with their C-rich complements, and quadruplex formation will be in competition with the corresponding Watson-Crick duplex. Quadruplex formation must therefore first require local dissociation (melting) of the duplex strands. Since negative supercoiling is known to facilitate the formation of alternative DNA structures, we have investigated G-quadruplex formation within negatively supercoiled DNA plasmids. Plasmids containing multiple copies of (G3T)n and (G3T4)n repeats, were probed with dimethylsulphate, potassium permanganate and S1 nuclease. While dimethylsulphate footprinting revealed some evidence for G-quadruplex formation in (G3T)n sequences, this was not affected by supercoiling, and permanganate failed to detect exposed thymines in the loop regions. (G3T4)n sequences were not protected from DMS and showed no reaction with permanganate. Similarly, both S1 nuclease and 2D gel electrophoresis of DNA topoisomers did not detect any supercoil-dependent structural transitions. These results suggest that negative supercoiling alone is not sufficient to drive G-quadruplex formation.

Figure 1.

Reaction of the (G₃T)_n-containing plasmid inserts with dimethylsulphate (DMS). Supercoiled (SC) and linearised (LIN) plasmids were modified with DMS, in the presence (A, C, E, G) or absence (B, D, F, G) of 100 mM KCl. Lanes labelled 1 and 3 are untreated DNA, while lanes 2 and 4 correspond to reaction with dimethylsulphate. The final panel (I) shows the reaction of DMS with the single stranded oligonucleotide [(G₃T)₅]₂ (labelled at the 5′-end) in the absence (-) and presence of 100 mM KCl. The plasmids were incubated in buffer overnight before adding DMS. Tracks labelled GA correspond to Maxam-Gilbert markers specific for purines. The locations of the G₃ blocks are indicated by the filled boxes. For the dimeric inserts the central GATC is indicated by a bracket.

Figure 2.

Densitometric scans of the reaction of dimethylsulphate with the (G₃T)₄ and (G₃T)₅ plasmid inserts in the presence (left) and absence (right) of 100 mM KCl. The sequences run from 5′-3′- left to right and the locations of the G₃ tracts are indicated by the filled bars. SC, supercoiled DNA; LIN linear DNA.

Figure 3.

Reaction of the (G₃T₄)_n-containing plasmid inserts with dimethylsulphate (DMS). Supercoiled (SC) and linearised (LIN) plasmids were modified with DMS in the presence of 100 mM KCl. Lanes labelled 1 and 3 are untreated DNA, while lanes 2 and 4 correspond to reaction with DMS. The plasmids were incubated in buffer overnight before adding DMS. Tracks labelled GA correspond to Maxam-Gilbert markers specific for purines. The locations of the G₃ blocks are indicated by the filled boxes. For the dimeric inserts (second and fourth panels) the central GATC is indicated by a bracket.

Figure 4.

Densitometric scans of the reaction of dimethylsulphate with the (G₃T₄)₄ and (G₃T₄)₅ plasmid inserts in the presence (left) and absence (right) of 100 mM KCl. The sequence runs from 5′-3′- left to right and the locations of the G₃ tracts are indicated by the filled bars. SC, supercoiled DNA; LIN linear DNA.

Figure 5.

Reaction of the (G₃T)_n- and (G₃T₄)_n-containing plasmid inserts with potassium permanganate. Supercoiled (SC) and linearized (LIN) plasmids were modified with permanganate in the presence of 100 mM KCl. Lanes labelled 1 and 3 are untreated DNA, while lanes 2 and 4 correspond to reaction with permanganate. The plasmids were incubated in buffer overnight before adding permanganate. The final panel shows the reaction of permanganate with the single stranded oligonucleotide [(G₃T)₅]₂ (labelled at the 5′-end) in the absence (–) and presence (+) of 100 mM KCl. Tracks labelled GA correspond to Maxam-Gilbert markers specific for purines. The locations of the G₃ blocks are indicated by the filled boxes. For the dimeric inserts the central GATC is indicated by a bracket.

Figure 6.

Mapping of S1 nuclease-sensitive sites in pUC19 and plasmids containing [(G₃T)₄]₂ and (G₃T₄)₄ and inserts. Lane 1, DNA size marker; lane 2, native supercoiled DNA; lane 3, cleavage with S1 nuclease; lane 4, digestion with S1 nuclease followed by ScaI; lane 5 digestion with ScaI; lane 6, digestion with Sca1 followed by S1 nuclease; lane 7, digestion with EcoRI; lane 8, digestion with EcoRI and ScaI. The products of S1 nuclease followed by ScaI digestion are indicated by the asterisks.