Attenuating functions of the C terminus of λ integrase

The tyrosine family site-specific recombinases, in contrast to the related type I topoisomerases, which act as monomers on a single DNA molecule, rely on multi-protein complexes to synapse partner DNAs and coordinate two sequential strand exchanges involving four nicking-closing reactions. Here, we analyze three mutants of the catalytic domain of λ integrase (Int), A241V, I353M and W350ter that are defective for normal recombination, but possess increased topoisomerase activity. The mutant enzymes can carry out individual DNA strand exchanges using truncated substrates or Holliday junctions, and they show more DNA-cleavage activity than wild-type Int on isolated att sites. Structural modeling predicts that the substituted residues may destabilize interactions between the C-terminal β-strand (β7) of Int and the core of the protein. The cleavage-competent state of Int requires the repositioning of the nucleophile (Y342) located on β6 and the catalyst K235 located on the flexible β2-β3 loop, relative to their positions in a crystal structure of the inactive conformation. We propose that the anchoring of β7 against the protein core restrains the movement of Tyr342 and/or Lys235, causing an attenuation of cleavage activity in most contexts. Within a bona fide recombination complex, the release of strand β7 would allow Tyr342 and Lys235 to assume catalytically active conformations in coordination with other Int protomers in the complex. The loss of β7 packing by misalignment or truncation in the mutant proteins described here causes a loss of regulated activity, thereby favoring DNA cleavage activity in monomeric complexes and forfeiting the coordination of strand-exchange necessary for efficient recombination.