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  • Title: DNA secondary structure and Raman markers of supercoiling in Escherichia coli plasmid pUC19.
    Author: Serban D, Benevides JM, Thomas GJ.
    Journal: Biochemistry; 2002 Jan 22; 41(3):847-53. PubMed ID: 11790106.
    Abstract:
    Negative supercoiling in the 2686 bp Escherichia coli plasmid pUC19 is comparable in linking number (Lk(0) = 258) and superhelical density (sigma = -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 degrees 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 alpha (O3'-P->-O5'-C5') and zeta (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 introduces small redistributions of Raman intensity within the 1460-1490 and 1660-1670 cm(-1) intervals, consistent with small structural perturbations. Importantly, no Raman markers of Watson-Crick base pairing, base stacking, or C2'-endo/anti deoxynucleoside conformations are perturbed significantly by supercoiling of pUC19, indicating that the B-DNA structure is largely conserved under moderate superhelical stress. Peak and trough features at 814 and 783 cm(-1), and at 1462 and 1489 cm(-1), respectively, in the Raman difference spectrum between superhelical and relaxed DNA are proposed as markers of moderate negative supercoiling. We also show that in Tris-buffered solutions the Raman signature of supercoiled DNA can be obscured by Raman bands of Tris counterions. The subtle structural perturbations to B-DNA induced by moderate supercoiling are consistent with proposed mechanisms of transcriptional activation.
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