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

The configuration of supercoiled DNA (scDNA) was investigated by electron m icroscopy and scanning force microscopy. Changes in configuration were indu ced by varying monovalent/divalent salt concentrations and manifested by va riation in the number of nodes (crossings of double helical segments). A de crease in the concentration of monovalent cations from 50 mM to similar to1 mM resulted in a significant change of apparent configuration of negativel y supercoiled DNA from a plectonemic form with virtually similar to 15 node s (the value expected for molecules of similar to 3000 bp) to one or two no des. This result was in good agreement with values calculated using an elas tic rod model of DNA and salt concentration in the range of 5-50 mM. The ef fect did not depend on the identity of the monovalent cation (Na+, K+) or t he nature of the support used for electron microscopy imaging (glow-dischar ged carbon film, polylysine film). At very low salt concentrations, a singl e denatured region several hundred base-pairs in length was often detected. Similarly, at low concentrations of divalent cations (Mg2+, Ca2+, Zn2+), s cDNA was apparently relaxed, although the effect was slightly dependent on the nature of the cation. Positively supercoiled DNA behaved in a manner di fferent from that of its negative counterpart when the ion concentration wa s varied. As expected for these molecules, an increase in salt concentratio n resulted in an apparent relaxation; however, a decrease in salt concentra tion also led to an apparent relaxation manifested by a slight decrease in the number of nodes. Scanning force microscopy imaging of negatively scDNA molecules deposited onto a mica surface under various salt conditions also revealed an apparent relaxation of scDNA molecules. However, due to weak in teractions with the mica surface in the presence of a mixture of mono/dival ent cations, the effect occurred under conditions differing from those used for electron microscopy. We conclude that the observed changes in scDNA co nfiguration are inherent to the DNA structure and do not reflect artifacts arising from the method(s) of sample preparation. (C) 2001 Academic Press.