Electron and scanning force microscopy studies of alterations in supercoiled DNA tertiary structure

The configuration of supercoiled DNA (scDNA) was investigated by electron microscopy and scanning force microscopy. Changes in configuration were induced by varying monovalent/divalent salt concentrations and manifested by variation in the number of nodes (crossings of double helical segments). It was found that a decrease in the concentration of monovalent cations from 50 mM to ~1 mM resulted in a significant change of apparent configuration of negatively scDNA from a plectonemic form with virtually ~15 nodes (the value expected for molecules of ~3000 bp) to 1-2 nodes. This result was in good agreement with values calculated using an elastic rod model of DNA and salt concentration in the range of 5-50 mM. The effect did not depend on the identity of the monovalent cation (Na+, K+) or the nature of the support used for EM imaging (glow-discharged carbon film, polylysine film). At very low salt concentration, a single denatured region several hundred bp in length was often detected. Similarly, at low concentrations of divalent cations (Mg2+, Ca2+, Zn2+ ) scDNA was apparently relaxed, although the effect was slightly dependent on the nature of the cation. Positively scDNA behaved in a different manner than its negative counterpart when the ion concentration was varied. As expected for these molecules an increase in salt concentration resulted in an apparent relaxation; however, a decrease in salt concentration also led to an apparent relaxation manifested by a slight decrease in the number of nodes. Scanning force microscopy imaging of negatively supercoiled DNA molecules deposited onto mica surface under various salt conditions also revealed an apparent relaxation of scDNA molecules. However, due to weak interactions with the mica surface in the presence of a mixture mono/divalent cations, the effect occurred under conditions differing from those used for electron microscopy. We conclude that the observed changes in scDNA configuration are inherent to the DNA structure and do not reflect artifacts arising from the method(s) of sample preparation