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

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 that flank a branch point. In the presence of Mg2+, the four arms are kno wn to stack in pairs forming two helical domains whose orientations are ant iparallel but twisted by about 60 degrees. The basis for the antiparallel o rientation of the domains could be either junction structure or the effect of electrostatic repulsion between domains. To discriminate between these t wo possibilities, we have constructed and characterized an analogue, called a bowtie junction, in which one strand contains a 3',3' linkage at the bra nch point, the strand opposite it contains a 5',5' linkage, and the other t wo strands contain conventional 3',5' linkages. Electrostatic effects are e xpected to lead to an antiparallel structure in this system. We have charac terized the molecule in comparison with a conventional immobile branched ju nction 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 linkag es occur at the branch point and that the arms stack to form the same domai ns as the conventional junction. Cooper-Hagerman gel mobility analyses have been used to determine the relative orientations of the helical domains. R emarkably, we find them to be closer to parallel than to antiparallel, sugg esting that the preferred structure of the branch point dominates over elec trostatic repulsion. We have controlled for the number of available bonds i n the branch point, for gel concentration, and for the role of divalent cat ions. This finding suggests that control of branch point structure alone ca n lead to parallel domains, which are generally consistent with recombinati on models derived from genetic data.