Two structural features of λ integrase that are critical DNA cleavege by multimers but not by monomers

Despite many years of genetic and biochemical studies on the λ integrase (Int) recombination system, it is still not known whether the Int protein is competent for DNA cleavage as a monomer. We have addressed this question, as part of a larger study of Int functions critical for the formation of higher-order complexes, by isolating "multimer-specific" mutants. We identify a pair of oppositely charged residues, E153 and R169, that comprise an intermolecular salt bridge within a functional Int multimer. Mutation of either of these residues significantly reduces both the cleavage of full-att sites and the resolution of Holliday junctions without compromising the cleavage of half-att site substrates. Allele-specific suppressor mutations were generated at these residues. Their interaction with wild-type Int on preformed Holliday junctions indicates that the mutated residues comprise an intermolecular salt bridge. We have also shown that the most C-terminal seven residues of Int, which comprise another previously identified subunit interface, inhibit DNA cleavage by monomeric but not multimeric Int. Taken together, our results lead us to conclude that Int can cleave DNA as a monomer. We also identify and discuss unique structural features of Int that act negatively to reduce its activity as a monomer and other features that act positively to enhance its activity as a multimer.