Topological Measurement of an A-tract Bend Angle: Comparison of the Bent and Straightened States

It is well established that an A-tract imparts curvature to the DNA double helix. Constructs of such A-tracts have been used as bend standards in a large number of both structural and functional studies, and A-tracts can confer significant activation in transcription. An accurate value for the bend angle induced by an A-tract is centrally important to all such studies, but the estimates reported for the bend angle of an A-tract differ by greater than threefold. To address this problem, we have used the rotational variant method to measure the angle of DNA curvature conferred by a tract of six adenine bases (A6 tract). The original version of the method measured a protein-induced bend angle independent of external standards. It compared the effect of bent and straight forms of the sequence on the topology of a DNA plasmid in which the sequence is cloned as a series of tandem repeats. To adapt the approach to the measurement of an intrinsic bend, high temperature was used to generate the straightened reference state, with the required topological relaxation being performed by a hyperthermophile topoisomerase. Appropriate plasmids containing tandem repeats of A-tracts were constructed and topologically analyzed in this manner. The bend value measured at 4 °C was 26(±2), and decreased linearly to 17(±2) at 37 °C. The relationship to other estimates and the application of these values are discussed.