Determination of the DNA Helical Repeat and of the Structure of Supercoiled DNA by Cryo-Electron Microscopy

Cryo-electron microscopy provides an unique possibility to directly observe shape of individual DNA molecules freely suspended in cryo-vitrifled liquid media. We used this technique to characterize the superhelical trajectory adopted by linear DNA molecules composed of directly repeated intrinsically bent DNA segments which have 10 or 11 base pair each. Although the DNA helix is not directly discerned by this method, the measured values of diameter, pitch and handedness of the formed superhelices allow to determine the chirality and the number of base-pairs per turn of the constituting DNA. We also used cryo-electron microscopy to study the response of supercoiled DNA molecules to increasing counterions’ concentration. We observed that upon substantial neutralization of the negative charge of the DNA, the supercoiled molecules have a tendency to adopt a so called “tight” configuration, whereby the opposing segments of interwound molecules directly approach each other. Metropolis Monte Carlo simulations of shapes of supercoiled DNA molecules revealed that some short range attractive interactions between DNA segments would be needed to compensate for the entropy loss during transition from a loose toward a “tight” configuration of supercoiled DNA molecules. Since earlier studies of DNA helical repeat and DNA supercoiling are not familiar to non-biologists we included in to this chapter a short chronological overview of these studies.