Trans Cooperativity by a Split DNA Recombinase: The Central and Catalytic Domains of Bacteriophage Lambda Integrase Cooperate in Cleaving DNA Substrates When the Two Domains Are not Covalently Linked

Abstract

Site-specific recombinases of the lambda-integrase family recognize and cleave their cognate DNA sites through cooperative binding to opposite sides of the DNA substrate by a C-terminal catalytic domain and a flexibly linked "core-binding" domain; regulation of this cleavage is achieved via the formation of higher-order complexes. We report that the core-binding domain of lambda-integrase is able to stimulate the activity of the catalytic domain even when the two domains are not linked. This trans stimulation is accomplished without significantly increasing the affinity of the catalytic domain for its DNA substrate. Moreover, we show that mutations in the DNA substrate can abrogate this effect while retaining specificity determinants for cleavage. Since the domains do not significantly interact directly, this finding implies that trans activation is achieved via the DNA substrate in a manner that may be mechanistically important in this and similar DNA binding and cleaving enzymes.

Crystallization and structure determination of the core-binding domain of bacteriophage lambda integrase

“…As a ligand, a 15-base-pair cognate DNA duplex, 5 0 -d(GCT CAA GTT AGT ACG)-3 0 , and its complement were used. Oligonucleotide preparation, protein expression and purification were performed using methods described previously (Subramaniam et al, 2003(Subramaniam et al, , 2007Kamadurai et al, 2003). The Int CB -DNA complex was assembled in 25 mM Tris pH 8.5 and 10 mM sodium chloride (assembly buffer) at a final protein concentration of 0.6 mM with a 10% stoichiometric excess of DNA or at an equimolar concentration of 1.75 mM.…”

“…These crystal structures show that Int CB and Int Cat bind to opposite faces of the recognition sequence by adopting a clamp-like architecture with an extended linker connecting the two domains. Experiments in our laboratory with isolated protein constructs of Int CB and Int Cat showed that Int CB contributes to an increase in DNA cleavage by Int Cat even when they are not covalently linked together (Subramaniam et al, 2007). We proposed that Int CB might render the substrate DNA more suitable for cleavage by Int Cat by inducing structural changes in the DNA.…”

“…Characterization of Int CB -DNA interactions in solution using biophysical techniques revealed that Int CB indeed induces structural changes in the DNA and concomitantly undergoes a transition from a molten globule-like structure to a well folded structure upon binding DNA (Kamadurai & Foster, 2007). In order to characterize the Int CBmediated DNA structural changes in more detail, we attempted to crystallize an Int CB -cognate DNA complex; fortuitously, we observed diffraction-quality crystals of Int CB that were free of DNA under certain crystallization conditions, although the presence of DNA in the mother liquor strongly promoted crystallization.…”

DNA Recognition via Mutual-Induced Fit by the Core-Binding Domain of Bacteriophage λ Integrase

“…This finding suggests that the role of Int CB is not limited to anchoring Int Cat near the site of cleavage, but also promotes DNA cleavage through some other mechanism. The observed boost in DNA cleavage in the presence of Int CB does not appear to be due to direct Int CB -Int Cat interactions since crystallographic studies with the Int CB+Cat construct do not reveal significant interdomain contacts (9), nor do NMR studies reveal any large perturbations in the conformation of Int Cat due to the presence of Int CB (14). Importantly, the stimulation of DNA cleavage by Int CB can be selectively abolished through changes in the substrate DNA sequence without affecting the basal DNA cleavage activity by Int Cat , suggesting a role for the substrate DNA in the Int CB -promoted DNA cleavage activity (14).…”

“…If the enhancement of Int Cat by Int CB were adequately described by the chelate effect, one would expect that the cooperative effects would be lost if the two domains were separated. However, DNA cleavage assays with isolated Int Cat and Int CB constructs show that Int CB is able to increase the cleavage activity of Int Cat even when they are not expressed together as a single protein constructsthat is, when provided in trans (14). This finding suggests that the role of Int CB is not limited to anchoring Int Cat near the site of cleavage, but also promotes DNA cleavage through some other mechanism.…”