Holliday junctions are intermediates in genetic recombination. They consist of four strands of DNA that flank a branch point. In natural systems, their sequences have 2-fold (homologous) sequence symmetry. This symmetry enables the molecules to undergo an isomerization, known as branch migration, that relocates the site of the branch point. Branch migration leads to polydispersity, which makes it difficult to characterize the physical properties of the junction and the effects of the sequence context flanking the branch point. Previous studies have reported two symmetric junctions that do not branch migrate: one that is immobilized by coupling to an asymmetric junction in a double crossover context, and a second that is based on molecules containing 5′,5′ and 3′,3′ linkages. Both are flawed by distorting the structure of the symmetric junction from its natural conformation. Here, we report an undistorted symmetric immobile junction based on the use of DNA parallelogram structures. We have used a series of these junctions to characterize the junction resolution reaction catalyzed by vaccinia virus DNA topoisomerase. The resolution reaction entails cleavage and rejoining at CCCTT↓N recognition sites arrayed on opposing sides of the four-arm junction. We find that resolution is optimal when the scissile phosphodiester (Tp↓N) is located two nucleotides 5′ to the branch point on the helical strand. Covalent topoisomerase-DNA adducts are precursors to recombinant strands in all reactions, as expected. Kinetic analysis suggests a rate limiting step after the first-strand cleavage.
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