SUNY at Albany
June 19-23, 2001
Electron and Scanning Force Microscopy of Supercoiled DNA Molecules
Supercoiled structure of DNA plays an important role in the regulation of various cellular events via specific and non-specific DNA/protein interactions by formation of DNA/protein complexes. Usually natural negative supercoiling modulates these interactions and hence the efficiency/specificity/stability of the complexes. Formation of the complexes, their structure and localization can be analyzed by means of microscopy techniques. However, twist and writhe of supercoiled molecules are very sensitive to the environment conditions such as temperature, and the nature and concentrations of mono and divalent cations. Another difficulty for the analysis stems from the fact that microscopy studies imply that DNA molecules have to be transferred from 3D state in the solution into 2D state onto the solid surface. Together, these factors complicate the interpretation of microscopy data of scDNA molecules and their complexes with various specific/unspecific bound ligands.
We carried out systematic study of scDNA molecules with negative and positive supercoiling under various salt conditions and showed that electron microscopy allows the characterization of the superhelical state of the molecules similar to that of determined by gel electrophoresis. It was found that decrease in concentration of monovalent cations from 50 - 100 mM to ~1 mM resulted in a significant change of apparent conformation of negatively supercoiled DNA from a plectonemic form with virtually ~15 nodes (an expected value for the molecules of ~3000 bp) to 1-2 nodes. The effect did not depend on the nature of monovalent cation nor the nature of support used for EM imaging. At very low salt concentration a single denatured region of several hundred bp in length often occurred.
Positively supercoiled molecules behaved in a different manner than their negative counterparts when the ion concentration was varied. For these molecules an increase in salt concentration resulted in apparent relaxation (as expected); however, a decrease in salt concentration also led to an apparent relaxation manifested by a slight decrease of the number of nodes.
Scanning force microscopy imaging of negatively supercolied DNA molecules deposited onto mica surface under various salt conditions also revealed an apparent relaxation of scDNA molecules. However, due to weak interactions with mica surface in the presence of mixture mono/divalent cations, the effect occurred under conditions differing from those used for electron microscopy.
References and Footnotes
Dmitry I. Cherny and Thomas M. Jovin
Dept./Mol. Biol., Max Planck Inst./Biophysical Chem., Am Fassberg 11, D-37077 Göttingen, Germany email: firstname.lastname@example.org Ph: (+) 49 551 201 1383; F: (+) 49 551 201 1467