Ethidium Bromide Binds to DNA

 
Last Monday's Molecule was ethidium, better known by the name of its common salt, ethidium bromide [Monday's Molecule #35]. Ethidium is a large planer molecule that binds tightly to DNA. It is often used in biochemistry laboratories to visualize fragments of DNA that have been separated on gels. The ethidium molecule is fluorescent—when illuminated with ultraviolet light it shines in the visible range. Here's a picture (below right) of DNA fragments that are illuminated by ethidium binding. It's from an old paper of mine (Moran et al. 1979)—these days you usually can't publish simple experiments like this.

Ethidium binds by inserting itself bewteen the stacked bases in double-stranded DNA. Note that the ring structure of ethidium is hydrophobic and resembles the rings of the bases in DNA. Ethidium is capable of forming close van der Walls contacts with the base pairs and that's why it binds to the hydrophobic interior of the DNA molecule.

Molecules that bind in this manner are called intercalating agents because they intercalate into the compact array of stacked bases. In doing so, they distort the double helix and interfere with DNA replication, transcription, DNA repair, and recombination. This is why intercalating agents are often potent mutagens.

The cartoon below shows the distortion of the sugar-phosphate backbone when an intercalating agent bind and it also shows that the DNA is lengthened when intercalating agents bind. This changes the properties of DNA considerably. One of the tricks in separating closed circular molecules of DNA from linear fragments (such as genomic DNA) is to treat the DNA with ethidium bromide. The intercalating agent doesn't bind to closed circular molecules because they can't be lengthened enough to allow insertion of the chemical between the bases. The normal circular plasmid DNA can then be separated from linear DNA with bound ethidium because binding of ethidium changes the overall density of DNA.



The structure shown above (right) is from Reha et al. (2002). It shows a molecule of ethidium lying between two A/T base pairs.

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Moran,L., Mirault, M-E., Tissières, A., Lis, J., Schedl, P., Artavanis-Tsakonas, S., and Gehring, W. (1979) Physical Map of Two D. melanogaster DNA Segments Containing Sequences Coding for the 70,000 Dalton Heat Shock Protein. Cell 17:1-8.

Reha, D., Kabelác, M., Ryjácek, F., Sponer, J., Sponer, J.E., Elstner, M., Suhai, S., and Hobza, P. (2003) Intercalators. 1. Nature of stacking interactions between intercalators (ethidium, daunomycin, ellipticine, and 4',6-diaminide-2-phenylindole) and DNA base pairs. Ab initio quantum chemical, density functional theory, and empirical potential study. J. Am. Chem. Soc. 124:3366-76.