COMPLEMENTATION OF BACTERIOPHAGE-LAMBDA INTEGRASE MUTANTS - EVIDENCE FOR AN INTERSUBUNIT ACTIVE-SITE

Site-specific recombination of bacteriophage lambda starts with the fo rmation of higher-order protein - DNA complexes, called 'intasomes', a nd is followed by a series of steps, including the initial DNA cleavag e, top-strand exchange, branch migration and bottom-strand exchange, t o produce recombinant products. One of the intasomes formed during exc isive recombination (the attL complex) is composed of the phage-encode d integrase (Int), integration host factor (IHF) and one of the recomb ination substrates, attL DNA. Int is the catalytic recombinase and has two different DNA binding domains. When IHF is present, Int binds to two types of sites in attL DNA, the three arm-type sites (P'123) and t he core-type sites (B and C') where the reciprocal strand exchange tak es place. The Tyr342 residue of Int serves as a nucleophile during str and cleavage and covalently attaches to the DNA through a phosphotyros yl bond. In vitro complementation assays have been performed for stran d cleavage using attL suicide substrates and mutant proteins containin g amino acid substitutions at residues conserved in the integrase fami ly of recombinases. We demonstrate that at least two Int monomers are required to form the catalytically-competent species that performs cle avage at the B site. It is likely that the active site is formed by tw o Int monomers.