A conformational switch controls the DNA cleavage activity of lambda integrase.

The bacteriophage lambda integrase protein (lambda Int) belongs to a family of tyrosine recombinases that catalyze DNA rearrangements. We have determined a crystal structure of lambda Int complexed with a cleaved DNA substrate through a covalent phosphotyrosine bond. In comparison to an earlier unliganded structure, we observe a drastic conformational change in DNA-bound lambda Int that brings Tyr342 into the active site for cleavage of the DNA in cis. A flexible linker connects the central and the catalytic domains, allowing the protein to encircle the DNA. Binding specificity is achieved through direct interactions with the DNA and indirect readout of the flexibility of the att site. The conformational switch that activates lambda Int for DNA cleavage exposes the C-terminal 8 residues for interactions with a neighboring Int molecule. The protein interactions mediated by lambda Int's C-terminal tail offer a mechanism for the allosteric control of cleavage activity in higher order lambda Int complexes.

Dynamics and DNA substrate recognition by the catalytic domain of lambda integrase.

Bacteriophage lambda integrase (lambda-Int) is the prototypical member of a large family of enzymes that catalyze site-specific DNA recombination via the formation of a Holliday junction intermediate. DNA strand cleavage by lambda-Int is mediated by nucleophilic attack on the scissile phosphate by a conserved tyrosine residue, forming an intermediate with the enzyme covalently attached to the 3'-end of the cleaved strand via a phosphotyrosine linkage. The crystal structure of the catalytic domain of lambda-Int (C170) obtained in the absence of DNA revealed the tyrosine nucleophile at the protein's C terminus to be located on a beta-hairpin far from the other conserved catalytic residues and adjacent to a disordered loop. This observation suggested that a conformational change in the C terminus of the protein was required to generate the active site in cis, or alternatively, that the active site could be completed in trans by donation of the tyrosine nucleophile from a neighboring molecule in the recombining synapse. We used NMR spectroscopy together with limited proteolysis to examine the dynamics of the lambda-Int catalytic domain in the presence and absence of DNA half-site substrates with the goal of characterizing the expected conformational change. Although the C terminus is indeed flexible in the absence of DNA, we find that conformational changes in the tyrosine-containing beta-hairpin are not coupled to DNA binding. To gain structural insights into C170/DNA complexes, we took advantage of mechanistic conservation with Cre and Flp recombinases to model C170 in half-site and tetrameric Holliday junction complexes. Although the models do not reveal the nature of the conformational change required for cis cleavage, they are consistent with much of the available experimental data and provide new insights into the how trans complementation could be accommodated.

Attenuating functions of the C terminus of lambda 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 lambda 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 beta-strand (beta7) of Int and the core of the protein. The cleavage-competent state of Int requires the repositioning of the nucleophile (Y342) located on beta6 and the catalyst K235 located on the flexible beta2-beta3 loop, relative to their positions in a crystal structure of the inactive conformation. We propose that the anchoring of beta7 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 beta7 would allow Tyr342 and Lys235 to assume catalytically active conformations in coordination with other Int protomers in the complex. The loss of beta7 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.

Differential affinity and cooperativity functions of the amino-terminal 70 residues of lambda integrase.

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination.

Gamma integrase complementation at the level of DNA binding and complex formation.

Site-specific recombinases of the gamma 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 in cis (as for Cre recombinase), or it may recruit the tyrosine from its partner in trans to form a composite active site (as for Flp recombinase). The crystal structure of the gamma Int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on gamma integrase (Int) generated data that were not in agreement. Support for cis and trans cleavage came from assays with bispecific DNA substrates for gamma and HK022 Ints and from functional complementation between recombination-deficient mutants, respectively. The data presented here do not provide new evidence for cis cleavage, but they strongly suggest that the previously described complementation results cannot be used in support of a trans-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-order attL 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 (in cis) by mutant gamma 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 gamma Int mutants.