Direct evidence for spontaneous branch migration in antiparallel DNA Holliday junctions

The Holliday junction is a central intermediate in genetic recombination. I t contains four strands of DNA that are paired into four double helical arm s flanking a branch point. In naturally occurring Holliday junctions, the s equence flanking the branch point contains 2-fold (homologous) symmetry. As a consequence of this symmetry, the junction can undergo a conformational isomerization known as branch migration, which relocates the site of branch ing. In the absence of proteins and in the presence of Mg2+ the four arms a re known to stack in pairs, forming two helical domains whose orientations are antiparallel. Nevertheless, the mechanistic models proposed for branch migration are all predicated on a parallel alignment of helical domains. He re, we have used antiparallel DNA double crossover molecules to demonstrate that branch migration can occur in antiparallel Holliday junctions. We hav e constructed a DNA double crossover molecule with three crossover points. Two adjacent branch points in this molecule are flanked by symmetric sequen ces. The symmetric crossover points are held immobile by the third crossove r point, which is flanked by asymmetric sequences. Restriction of the helic es that connect the immobile junction to the symmetric junctions releases t his constraint. The restricted molecule undergoes branch migration, even th ough it is constrained to an antiparallel conformation.