Topological measurement of an A-tract bend angle: effect of magnesium

Sequences of four to six adenine residues, termed A-tracts, have been shown to produce curvature in the DNA double helix. A-tracts have been used extensively as reference standards to quantify bending induced by other sequences as well as by DNA binding proteins when they bind to their sites. However, the ability of an A-tract to serve as such a standard is hampered by the wide variation of values reported for the amount of bend conferred by an A-tract. One experimental condition that differs in these studies is the presence of divalent cation. To evaluate this effect, a new application of a topological method, termed rotational variant analysis, is used here to measure for the first time the effect of the presence of magnesium ion on the bend angle conferred by an A-tract. This method, which has the unique ability to measure a bend angle in the presence or absence of magnesium ion, demonstrates that magnesium ion markedly increases the bend angle. For example, when measured in a commonly used gel electrophoretic buffer, the bend angle conferred by a tract of six adenine residues increases from about 7 degrees in the absence of magnesium ion to 19 degrees in the presence of 3.9 mM magnesium ion. This quantitative demonstration of substantial magnesium ion dependence has several important implications. First, it explains discrepancies among bend values reported in various previous studies, particularly those employing gel electrophoretic versus other solution methods. In addition, these findings necessitate substantial revisions of the conclusions in a large number of studies that have used A-tract DNA as the bend angle reference standard in comparison measurements. Finally, any such future studies employing this comparison methodology will need to use the same sequence analyzed in the original measurements as well as replicate the original measurement conditions (e.g. ionic composition and temperature).