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 two-dimensional 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.

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ORCID for Keith R. Fox: orcid.org/0000-0002-2925-7315

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