HAIRPINS IN A DNA SITE FOR TOPOISOMERASE-II STUDIED BY H-1-NMR AND P-31-NMR

H-1- and P-31-NMR and UV-absorption studies were carried out with the oligonucleotide strands d(AGCT-TATC-ATC-GATAAGCT) (-ATC-) and d(AGCTTATC-GAT-GATAAGCT) (-GAT-) contained in the strongest and salt resistant cleavage site for topoisomerase II in pBR322 DNA. We found that the two oligonucleotides were stabilized under a hairpin structure characterized by a eight base pair stem and a three base loop at low DNA and salt concentrations. In such experimental conditions, only the -GAT- oligonucleotide displayed a partial homoduplex structure in slow equilibrium with its folded structure. Temperature dependencies of imino protons showed that the partial homoduplex of -GAT- melted at a lower temperature than the hairpin structure. It was suggested that the appearance of the partial homoduplex in -GAT- is related to the formation of two stabilizing (G-T) mismatched base pairs in the central loop of this structure. Finally, it was inferred from the dispersion of chemical shifts in the P-31-NMR spectra that the distortions affecting the backbone of the hairpin loop are larger in the case of -ATC-compared with -GAT-. At the same time NOEs proved that the base stacking was stronger within the loop of the -ATC-hairpin.