Determination of the DNA bend angle induced by the restriction endonuclease EcoRV in the presence of Mg2

Abstract We have used the method of Zinkel and Crothers (Zinkel, S.S., and Crothers, D.M. (1990) Biopolymers 29, 29-38) to determine the degree of bending induced by the binding of the restriction endonuclease EcoRV to its recognition sequence (-GATATC-). A set of four calibration DNA fragments 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 inactive EcoRV mutant that still binds DNA specifically in the presence of Mg2+. In the absence of Mg2+, which is necessary for specific binding, there is no difference in the mobilities of the fragments with a peripheral or a central EcoRV site complexed with EcoRV, indicating that nonspecific binding on average does not lead to measurable DNA bending.