NEAR-SIMULTANEOUS DNA CLEAVAGE BY THE SUBUNITS OF THE JUNCTION-RESOLVING ENZYME T4 ENDONUCLEASE-VII

In common with a number of other DNA junction-resolving enzymes, endon uclease VII of bacteriophage T4 binds to a four-way DNA junction as a dimer, and cleaves two strands of the junction. We have used a superco il-stabilized cruciform substrate to probe the simultaneity of cleavag e at the two sites. Active endonuclease VII converts the supercoiled c ircular DNA directly into linear product, indicating that the two clea vage reactions must occur within the lifetime of the protein-junction complex. By contrast, a hetero-dimer of active enzyme and an inactive mutant endonuclease VII leads to the formation of nicked circular prod uct, showing that the subunits operate fully independently.