M.S. - Biomedical Sciences (Biophysics)
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ItemESR-Spin label studies of intercalation mechanisms in DNA( 1975)A spin label technique has been developed for the study of intercalation mechanisms in DNA utilizing a modified ethidium bromide as a model ligand, and specifically synthesized spin labels of carcinogenic aromatic amines and a series of nitrobenzene chromophores. The technique takes advantage of the characteristic ESR anisotropy of the corresponding spin labels complexed with DNA. Analysis of the orientation dependent information thus obtained for the spin labels of ethidium bromide, 2-aminofluorene, 2-aminoanthracene and 6-aminochrysene have clearly shown that the ligand moieties in all four respective complexes are accommodated in adjacent base pair layers of the DNA with the nitroxide reporter oriented in one preferential direction. The geometry of the nitroxide rings with respect to their respective constraining ligands in a particular complex appeared to be largely dependent upon the size of the ligand moiety and the position of the reporter attachment on the ligand molecule. A coplanar configuration is most favored for the spin labels in which the ligand moiety per se is a three fused ring with the nitroxide substituted at a position which is on or close to the longitudinal end of the ligand molecule. In the case of nitrobenzene spin labels, the 2,4-dinitrobenzene analogues with amine linkage to the nitroxide reporter demonstrated the strongest binding with DNA by intercalation and the reporter nitroxide is oriented 45 degrees to the plane of the benzene ring and is due primarily to the steric hindrance of the 2-nitrosubstituent. This binding is found to be largely dependent upon the number of nitro-substituents, their relative position on the benzene ring, and the type of linkage between the ligand and the nitroxide reporter, suggesting that polirization bonding is a major driving force in their complex formation with DNA. Temperature studies in which the heat-induced release of the bound label is monitored by ESR gives an accurate measure of the complex dissociation characteristics which parallel exactly the helical-coil cooperative melting transition monitored by optical absorption measurements. The extent of dissociation of the bound spin labels parallels almost exactly the melting, i.e. unzipping of the DNA double helix suggesting that the spin labels are immobilized by intercalation at the site on the DNA which is directly involved in the maintenance of the double helical structure. Analysis of the ESR spectra of the complexes in terms of the extent of immobilization indicates that for aromatic amine spin labels, two different binding species exist, one strongly immobilized presumably at a G-C pair region and the other partially immobilized in a region other than G-C. This is quite different from those complexes formed with ethidium bromide and nitrobenzene in which no more than one species of immobilized spin was observed. Competitive binding of the labels with respect to their parent ligand and binding isotherm results show that nitroxide substitution does not effect the type of binding but does effect the extent of binding, i.e. binding constant with the labeled compound having a much lower affinity.