Structural Characterization of Torsional Destabilization in DNA

Abstract

Within cells, supercoiled DNA is inherently subjected to various degrees of bending and twisting, and many DNA-binding proteins are now known to be sensitive to these mechanical features of DNA. Under significant levels of bending and/or torsional stress, DNA has been shown to depart from native B-form structure and adopt a number of more energetically favorable alternate conformations. However, very little is known about the structural details of these stress-induced structures. Recently, the DNA nuclease BAL 31 was used to assay for helical destabilizations in small DNA minicircles sustaining fixed amounts of bending and torsional stresses (Nucl. Acids Res., 36(4), 2008). Here, we seek to determine if the helical destabilizations that accompany elevated levels of negative torsional stress in tightly looped minicircles produce localized structures that confer enhanced bending flexibility to the DNA structure, such as would occur in kinked DNA. Towards this end, we synthesized untwisted, 100-bp minicircles that are recognized and digested by BAL 31 and overtwisted 106-bp minicircles that are resistant to degradation by BAL 31. Using cryo-electron microscopy, we then generated three-dimensional image reconstructions of these two minicircle species. From these quantitative descriptions of the minicircle geometries, we observe no evidence of DNA kinking in either the BAL 31-sensitive, 100-bp minicircles or the BAL 31-insensitive, 106-bp minicircles. Since the torsional destabilizations that are recognized by BAL 31 were not observed to confer significant enhancements in bending flexibility, we propose that the torsional destabilizations appearing with the 100-bp minicircle relieve negative torsional stress through the formation of a left-handed dinucleotide stack with no interruption in base-stacking at the right-to-left transition, thus minimally affecting the local bending stiffness. Our observations are consistent with the formation of Z(WC)-DNA, which has been previously theorized to be the predominate form of left-handed DNA appearing in nature.