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FIGURE 6-27 Separation of relaxed and supercoiled DNA by gel electrophoresis!.

Relaxed and supercoiled ONA topoisomers are resolved by gel eleclropftoresis The speed with which the DNA molecules migrate increases as the number of superheitcd turns increases. (Source: Courtesy ot J. C. Wang)

FIGURE 6-27 Separation of relaxed and supercoiled DNA by gel electrophoresis!.

Relaxed and supercoiled ONA topoisomers are resolved by gel eleclropftoresis The speed with which the DNA molecules migrate increases as the number of superheitcd turns increases. (Source: Courtesy ot J. C. Wang)

{Figure 6-28), Because it fluoresces when exposed to ultraviolet light, and because its fluorescence increases dramatically after intercalation, ethidium is used as a stain to visualize DNA.

When an ethidium ion intercalates between two base pairs, it causes the DNA to unwind by 20°, reducing the normal rotation per base pair from -36c' to ~10c. In other words, ethidium decreases the twist of DNA. imagine the extreme case of a DNA molecule that has an ethidium ion between every base pair. Instead of 10 base pairs per turn it would have 36! When ethidium binds to linear DNA or to a nicked circle, it simply causes the helical pitch to increase. But consider what happens when ethidium binds to covalently closed, circular DNA. The linking number of the cccDNA does not change {no covalent bonds are broken and reseated), but the twist decreases by 26° for each molecule of ethidium that has bound to the DNA. Because Lk = Tw + Wr, this decrease in Tw must be compensated for by a corresponding increase in Wr. If the circular DNA is initially negatively supercoiled (as is normally the case for circular DNAs isolated from cells), then the addition of ethidium will increase Wr. In other words, the addition of ethidium will relax the DNA. If enough ethidium is added, the negative supercoiling will be brought to zero, and if even more ethidium is added, Wr will increase above zero and the DNA will become positively supercoiled.

FIGURE 6-28 Intercalation of ethtfium into DNA, Shidium increases the spacing of successive base pairs, distorts tbe regular sugar-pliosphate backbone, and decreases the twist of the lielix.

nucleotide backbone

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