The Holliday junction is a prominent intermediate in genetic recombination that consists of four double helical arms of DNA flanking a branch point. Under many conditions, the Holliday junction arranges its arms into two stacked domains that can be oriented so that genetic markers are parallel or antiparallel. In this arrangement, two strands retain a helical conformation, and the other two strands effect the crossover between helical domains. The products of recombination are altered by a crossover isomerization event, which switches the strands fulfilling these two roles. It appears that effecting this switch from the parallel conformation by the simplest mechanism results in braiding the crossover strands at the branch point. In previous work we showed by topological means that a short, parallel, DNA double crossover molecule with closed ends did not braid its branch point; however, that molecule was too short to adopt the necessary positively supercoiled topology. Here, we have addressed the same problem using a larger molecule of the same type. We have constructed a parallel DNA double crossover molecule with closed ends, containing 14 double helical turns in each helix between its crossover points. We have prepared this molecule in a relaxed form by simple ligation and in a positively supercoiled form by ligation in the presence of netropsin. The positively supercoiled molecule is of the right topology to accommodate braiding. We have compared the relaxed and supercoiled versions for their responses to probes that include hydroxyl radicals, KMnO4, the junction resolvases endonuclease VII and RuvC, and RuvC activation of KMNO4 sensitivity. In no case did we find evidence for a braid at the crossover point. We conclude that Holliday junctions do not braid at their branch points, and that the topological problem created by crossover isomerization in the parallel conformation is likely to be solved by distributing the stress over the helices that flank the branch point.
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