Influence of Global DNA Topology on Cruciform Formation in Supercoiled DNA

DNA supercoiling plays an important role in many genetic processes such as replication, transcription, and recombination. Supercoiling provides energy for helix unpairing and drives the formation of alternative DNA structural transitions, like cruciforms. Supercoiling also allows distant DNA regions to be brought into close proximity through the formation of interwound supercoils. Recently, we showed that the inverted repeat-to-cruciform transition acts as a molecular switch, influencing the global topology of a topological plasmid domain. As alternative DNA structures can affect global topology, a corollary hypothesis might be that the localization of a specific DNA sequence within a topological domain may affect the energetics required for formation of an alternative DNA structure. Here, we test this hypothesis and show that the localization of an inverted repeat to an apical position increases the rate of cruciform formation and reduces the superhelical energy required to drive the transition. For this, we created a series of plasmids containing an inverted repeat and an A-tract bent DNA sequence. The A-tract forms a permanent 180° bend irrespective of DNA topology. The inverted repeat and the bent sequence were placed either at six oʹclock or nine oʹclock positions with respect to each other. Using 2D agarose gel electrophoresis, we show that the six oʹclock construct extrudes the cruciform at a lower superhelical density than a control plasmid without the bend. Atomic force microscopy shows that the nine oʹclock construct has the propensity to form branched molecules with the cruciform at the end of one branch. These results demonstrate that the localization of sequences within specific regions of a topological domain can affect the energetics of structural transitions as well as the branching structure of the domain. As structural transitions can be involved in biological processes, localization of alternative conformation-forming sequences to specific locations within a domain provides an additional means for gene regulation.