Parallel helical domains in DNA branched junctions containing 5',5' and 3',3' linkages

The Holliday junction is a central intermediate in genetic recombination. It contains four strands of DNA that are paired into four double helical arms that flank a branch point. In the presence of Mg2+, the four arms are known to stack in pairs forming two helical domains whose orientations are antiparallel but twisted by about 60 degrees. The basis for the antiparallel orientation of the domains could be either junction structure or the effect of electrostatic repulsion between domains. To discriminate between these two possibilities, we have constructed and characterized an analogue, called a bowtie junction, in which one strand contains a 3',3' linkage at the branch point, the strand opposite it contains a 5',5' linkage, and the other two strands contain conventional 3',5' linkages. Electrostatic effects are expected to lead to an antiparallel structure in this system. We have characterized the molecule in comparison with a conventional immobile branched junction by Ferguson analysis and by observing its thermal transition profile; the two molecules behave virtually identically in these assays. Hydroxyl radical autofootprinting has been used to establish that the unusual linkages occur at the branch point and that the arms stack to form the same domains as the conventional junction. Cooper-Hagerman gel mobility analyses have been used to determine the relative orientations of the helical domains. Remarkably, we find them to be closer to parallel than to antiparallel, suggesting that the preferred structure of the branch point dominates over electrostatic repulsion. We have controlled for the number of available bonds in the branch point, for gel concentration, and for the role of divalent cations. This finding suggests that control of branch point structure alone can lead to parallel domains, which are generally consistent with recombination models derived from genetic data.