CLEAVAGE OF DOUBLE-CROSSOVER MOLECULES BY T4 ENDONUCLEASE-VII

DNA double-crossover molecules containing two Holliday junctions have been prepared and treated with endonuclease VII, the resolvase from ba cteriophage T4. One molecule contains antiparallel double-helical doma ins, and the other molecule contains parallel domains. The parallel do uble-crossover model system has been made tractable by closing the fre e ends of the molecule, to convert it to a catenane. The products resu lting from the two substrates differ substantially. The molecule conta ining antiparallel helical domains is cleaved three nucleotides 3' to the crossover points, in a fashion similar to single Holliday junction analogs. The molecule containing parallel helical domains is cleaved, but the major points of scission are five nucleotides 5' to a branch point on the crossover strands and six nucleotides 3' to the same bran ch point on the non-crossover strands. The major sites of scission ref lect features of molecular symmetry in each case, suggesting that the resolvase recognizes structural features. The cleavage results suggest that the antiparallel structure is the natural substrate, if the Holl iday junction is unconstrained within the cell. It is straightforward to reconcile antiparallel Holliday junctions with the conventional par allel paradigm of recombination. Nevertheless, the cleavage of the par allel molecule shows that a parallel substrate could also be cleaved s ymmetrically by endonuclease VII (but with different products) if the molecule were constrained to assume that conformation within the cell.