Electrophoretic analysis of protein-induced DNA bending and twist changes

DNA fragments with a varied phasing of two intrinsic bends at their ends and a tet operator in-between were constructed to determine Tet repressor-induced twist changes in the operator DNA. These distance variants show a sinusoidal dependence of their electrophoretic mobilities on the phasing of their bends in polyacrylamide gels. Complex formation with Tet repressor leads to a displacement of the first minimum, indicating an unwinding of the tet operator DNA. Model calculations were performed to assess the contribution of Tet repressor-induced DNA bending to this result. They revealed that the amplitude of the electrophoretic mobilities of the distance variants may be used as a parameter to separate the effects of Tet repressor-induced twist change and bending. The systematic analysis presented here may help to improve the quantitative interpretation of gel shifts and promote the use of this highly sensitive method to gain structural information about protein-DNA complexes.