DETERMINATION OF THE NUMBER AND LOCATION OF THE MANGANESE BINDING-SITES OF DNA QUADRUPLEXES IN SOLUTION BY EPR AND NMR IN THE PRESENCE AND ABSENCE OF THROMBIN

The interaction of a DNA quadruplex with thrombin has been studied by first determining the sites of manganese binding to the quadruplex in the absence of thrombin, This has been followed by determining if the interactions with thrombin displace the bound manganese. A different D NA quadruplex has also been studied as a control. The refined solution structures of two DNA quadruplexes have been used to predict the elec trostatic potentials of these DNAs. The calculated electrostatic poten tials have been used to predict the locations of the binding sites of the paramagnetic ion manganese to these DNAs. The enhanced relaxation of DNA protons due to the binding of the paramagnetic metal ion Mn2+ h as been used to experimentally determine the! locations of the binding sites. The NMR results and the predictions based on tile electrostati c potentials both place the binding sites of the manganese in the narr ow grooves of these quadruplex DNAs. The predicted locations are spati ally close to those experimentally observed, and the predicted and exp erimental locations also have similar electrostatic potential energy, These results have allowed a validation of the predictions of electros tatic potentials from structure. The 15mer quadruplex has two strong M n2+ binding sites with one in each narrow groove, Both Mn2+ are releas ed when the 15mer is complexed with thrombin, indicating that both nar row grooves are involved in the 15mer-thrombin interactions. The dimer quadruplex has a different structural motif than the 15mer and the pr esence of thrombin does not appreciably affect its interactions with M n2+. (C) 1996 Academic Press Limited