Triplet anisotropy decay measurements of DNA internal motion.

Triplet anisotropy decay techniques have been used to measure the internal flexibility and overall rotational motions of DNA over a time range of 15 ns to 200 microseconds. Nearly monodisperse DNA fragments with lengths varying from 65 to 600 base pairs were studied using the intercalating dye methylene blue as a triplet probe. The slow end-over-end tumbling of short DNA fragments (less than 165 base pairs) is as predicted for a rigid rod. As expected, a longer DNA fragment (600 base pairs) experiences slow segmental motions of its helix axis. At the earliest times, anisotropy decays more rapidly than expected for a rigid rod, suggesting that, when it is bound, methylene blue monitors fast internal motions of the helix. Since the rod-like end-over-end tumbling rules out fast bending motions (for short DNA fragments), the fast components of DNA anisotropy decay must be due to twisting motions of the helix, occurring with a time constant near 50 ns. The same techniques were used to measure the conformational flexibility of DNA in the nucleosome. It is concluded that, when the DNA helix is wrapped to form a nucleosome, it experiences substantial internal flexibility, occurring with a time constant near 30 ns. The amplitude and time-scale of this motion appear to be similar to that seen in the uncomplexed DNA helix.