Crossed-stranded DNA structures for investigating the molecular dynamics of the Holliday junction

We have developed a simple and rapid procedure for the synthesis and isolation of figure-eight DNA molecules (figure-8s) beginning with phagemid vectors. The figure-8 molecules generated contain a Holliday junction within 2.9 kb (1 kb = 10(3) bases or base-pairs) of continuous homology connecting the two monomer duplexes. The structure of these molecules was verified by restriction endonuclease analysis, two-dimensional agarose gel electrophoresis, and electron microscopy. Digestion of the figure-8 molecules with a restriction enzyme that cleaves only within the region of DNA sequence homology converted them to X-forms that could dissociate to yield linear monomers. The X-form molecules were stable under physiological conditions, a finding contradicting the notion that spontaneous branch migration is fast.