FOLDING OF AN IMMOBILE DNA BRANCHED JUNCTION

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The Holliday junction is a four-stranded DNA molecule, in which the four strands pair to form four double helical arms that flank a branch point, that is a central intermediate in the process of genetic recombination. The site of the branch point in naturally occurring Holiday junctions is unstable, because it is flanked by twofold (homologous) sequence symmetry that permits the junction to relocate through an isomerization known as branch migration. In the presence of Mg cations, the junction folds into a structure in which the four arms stack on each other pairwise, to form two double helical domains; Htwo strands adopt a roughly helical structure, and the other two Hstrands cross over between the two domains. In the absence of Mg cations, the four arms adopt a fourfold symmetric arrangement. The hydroxyl radical cleavage pattern is sensitive to the folding of the junction in the presence of Mg. Relative to their pattern in linear duplex DNA, the two crossover strands are protected from attack in the Htwo-domain structure. Likewise, four nucleotides 3' to the branch Hpoint, the helical strands demonstrate protection, because they Hocclude each other there. Both protections appear to be lessened in Hthe absence of magnesium. Hence, this is a system whereby the Hkinetics of the folding of this key intermediate is measurable through Hthese reporter protection differences. Dose response experiments at Hthe synchrotron have been completed and footprinting conditions Hrequired 100 milliseconds of exposure for a 42 base target sequence.