λ Integrase Complementation at the Level of DNA Binding and Complex Formation

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Abstract
Site-specific recombinases of the λ Int family carry out two single-strand exchanges by binding as head-to-head dimers on inverted core-type DNA sites. Each protomer may cleave its own site as a monomer incis(as for Cre recombinase), or it may recruit the tyrosine from its partner intransto form a composite active site (as for Flp recombinase). The crystal structure of the λ Int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on λ integrase (Int) generated data that were not in agreement. Support forcisandtranscleavage came from assays with bispecific DNA substrates for λ and HK022 Ints and from functional complementation between recombination-deficient mutants, respectively. The data presented here do not provide new evidence forciscleavage, but they strongly suggest that the previously described complementation results cannot be used in support of atrans-cleavage mechanism. We show here that IntR212Q retains some residual catalytic function but is impaired in binding to core-type DNA on linear substrates and in forming higher-orderattL intasome structures. The binding-proficient mutant IntY342F can stabilize IntR212Q binding to core-type DNA through protein-protein interactions. Similarly, the formation of higher-order Int complexes with arm- and core-type DNA is boosted with both mutants present. This complementation precedes cleavage and thus precludes any conclusions about the mechanism of catalysis. Cross-core stimulation of wild-type HK022-Int cleavage on its cognate site (incis) by mutant λ Ints on bispecific core DNA suicide substrates is shown to be independent of the catalytic tyrosine but appears to be proportional to the respective core-binding affinities of the λ Int mutants.