DNA Secondary Structure and Raman Markers of Supercoiling in Escherichia coli Plasmid pUC19

Negative supercoiling in the 2686 bp Escherichia coli plasmid pUC19 is comparable in linking number (Lk0 = 258) and superhelical density (σ = −0.05) to the moderate supercoiling exhibited by many eukaryotic chromosomal DNAs in vivo. Supercoiled and relaxed forms of purified pUC19 in aqueous solution (0.1 M NaCl, pH 8.3, 20 °C) have been investigated by Raman spectroscopy to assess changes in B-DNA secondary structure induced by superhelical stress and to identify putative Raman markers of DNA supercoiling. We find that supercoiling leads to small but significant changes to the B-form Raman signature of linear DNA. Spectral band shifts in the 780−850 cm-1 interval are interpreted as resulting from a small net change in the average phosphodiester torsions α (O3‘−P−〉−O5‘−C5‘) and ζ (C3‘−O3‘−〉−P−O5‘) from the gauche-/gauche- range to the gauche-/trans range with supercoiling. The magnitude of the spectral intensity change implies that approximately 5% of the nucleotide moieties are affected. Supercoiling also...