ACTIVE-SITE ASSEMBLY AND MODE OF DNA CLEAVAGE BY FLP RECOMBINASE DURING FULL-SITE RECOMBINATION

A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revea led the following features of the half-site recombination reaction. (i ) The Flp active site is assembled by sharing of catalytic residues fr om at least two monomers of the protein. (ii) A Flp monomer does not c leave the half site to which it is bound (DNA cleavage in cis); rather , it cleaves a half site bound by a second Flp monomer (DNA cleavage i n trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shar ed active site and of trans DNA cleavage. However, reactions with line ar suicide attB substrates and synthetic Holliday junctions are more c ompatible with cis than with trans DNA cleavage. These Int results eit her argue against a common mode of active-site assembly within the Int family or challenge the validity of Pip half sites as mimics of the n ormal full-site substrates. We devised a strategy to assay catalytic c omplementation between Flp monomers in full sites. We found that the f ull-site reaction follows the shared active-site paradigm and the tran s mode of DNA cleavage. These results suggest that within the Int fami ly, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.