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Methods in molecular biology (Clifton, N.J.)

Analysis of DNA supercoiling induced by DNA-protein interactions.


PMID 19378184

Abstract

Certain DNA-interacting proteins induce a pronounced bending in the double helix and cause topological stresses that are compensated by the formation of supercoils in DNA. Such supercoils, when forming on a circular plasmid, give rise to a series of topoisomers that run at different speeds during electrophoresis. The number of supercoils introduced in the plasmid can provide information on the protein; it can for example help determine the number of nucleosomes that are assembled on the plasmid or indicate whether the DNA-bending activity of a transcription factor is important enough to cause a topological stress. Because a DNA-protein activity can lead to either an overwinding or an underwinding of the helix, supercoiling can occur in either direction. Determining whether a plasmid contains positively or negatively supercoiled DNA is possible, thanks to an agarose gel containing an intercalating agent known to positively supercoil DNA, such as chloroquine. The speed of migration of the topoisomers varies in a characteristic way in the presence and absence of the agent. Topoisomer standards can furthermore be generated to allow the easy evaluation of the number of supercoils induced in a plasmid by a DNA-protein interaction.

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