title: 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.
authors: Zentrum Biochemie, Medizinische Hochschule Hannover, Germany.
authors: T
authors: T
authors: Stöver
authors: E
authors: E
authors: Köhler
authors: U
authors: U
authors: Fagin
authors: W
authors: W
authors: Wende
authors: H
authors: H
authors: Wolfes
authors: A
authors: A
authors: Pingoud
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation
chemical_list: D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D008274:Magnesium