DETERMINATION OF THE DNA BEND ANGLE INDUCED BY THE RESTRICTION ENDONUCLEASE ECORV IN THE PRESENCE OF MG2+

We have used the method of Zinkel and Crothers (Zinkel, S. S., and Cro thers, D. M. (1990) Biopolymers 29, 29-38) to determine the degree of bending induced by the binding of the restriction endonuclease EcoRV t o its recognition sequence (-GATATC-). A set of four calibration DNA f ragments was constructed that contained zero, two, four, or six phased A-tracts in their centers and an EcoRV site at the 5'-end to account for the electrophoretic influence of the bound protein. The mobilities of these calibration molecules complexed with EcoRV were compared to that of a test DNA containing a central EcoRV site also complexed with EcoRV. The EcoRV-induced bend angle was found to be 44-degrees +/- 4- degrees. These experiments were performed with a catalytically inactiv e EcoRV mutant that still binds DNA specifically in the presence of Mg 2+. In the absence of Mg2+, which is necessary for specific binding, t here is no difference in the mobilities of the fragments with a periph eral or a central EcoRV site complexed with EcoRV, indicating that non specific binding on average does not lead to measurable DNA bending.