Four-way branched DNA structures (Holliday junctions) are the key intermediates formed during genetic recombination between two duplex DNA molecules. The outcome of recombination at specific DNA sequences may depend on the three-dimensional structure at the junction site, We have used time-resolved fluorescence resonance energy transfer methods to measure interarm distance distributions in a series of related four-way DNA junctions. The aim of these studies is to establish how the global structure and flexibility of the junction are influenced by the base sequence flanking the branch point. Energy transfer donor and acceptor dyes were conjugated to the 5' termini of the DNA strands via a C{sub}6 linker. Six pairwise combinations of labeled arms have been examined for each junction, yielding six interarm distance distributions. All the junctions we examined formed "X" shape structures by pair-wise stacking of duplex arms, although there were significant differences in the range of interarm distances, depending on the junction sequence. Certain sequences appear to produce a mixture of the two possible stacking arrangements. These studies help to define the molecular interactions that dictate the crossover bias at a specific junction sequence.
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