Raman spectroscopy of supercoiled and nicked ColE1 plasmid.

Abstract

Native supercoiled and nicked ColE1 DNA were examined using laser Raman spectroscopy. ColE1 contains 6646 base pairs (bp) and, when supercoiled, approximately 47 negative supercoils. An analytical buoyant density gradient centrifugation technique developed by Burke and Bauer was scaled to preparative quantities, and used to isolate the supercoiled plasmid fraction from its nicked counterpart. This procedure allowed enriched fractions of the supercoiled plasmid to be extracted without the use of the optical contaminant ethidium bromide. The intensities of several Raman bands were altered between the spectra of the two topological forms. Notably absent were any changes in bands arising from cytosine and guanine vibrations. The observed changes are interpreted in terms of the polymorphic structures which have been observed in many DNA structural studies. The results of this study suggest that accommodation of supercoiling takes place chiefly in A-T base pairs and backbone moieties, without substantial modification of G-C base-pair structure. Premelting effects may account for the observed changes, including a slight shift to lower frequency of a band known to be responsive to base-pair disruption. Heteronomous ribose sugar pucker is evident in both supercoiled and nicked plasmid species. No gross conformational transitions were detected for native supercoiled DNA, and consequently, subtle rearrangements appear sufficient to absorb the supercoiling deformations.