Receipt of the C-terminal Tail from a Neighboring λ Int Protomer Allosterically Stimulates Holliday Junction Resolution

Abstract

Bacteriophage λ integrase (Int) catalyzes the integration and excision of the phage λ chromosome into and out of the Esherichia coli host chromosome. The seven carboxy-terminal residues (Cterminal tail) of Int comprise a context-sensitive regulatory element that links catalytic function with protein multimerization and also coordinates Int functions within the multimeric recombinogenic complex. The experiments reported here show that the β5-strand of Int is not simply a placeholder for the C-terminal tail but rather exerts its own allosteric effects on Int function in response to the incoming tail. Using a mutant integrase in which the C-terminal tail has been deleted (W350ter), we demonstrate that the C-terminal tail is required for efficient and accurate resolution of Holliday junctions by tetrameric Int. Addition of a free heptameric peptide of the same sequence as the Cterminal tail partially reverses the W350ter defects by stimulating Holliday junction resolution. The peptide also stimulates the topoisomerase function of monomeric W350ter. Single residue alterations in the peptide sequence and a mutant of the β5 strand indicate that the observed stimulation arises from specific contacts with the β5 strand (residues 239-243). The peptide does not stimulate binding of W350ter to its cognate DNA sites and therefore appears to recapitulate the effects of the normal C-terminal tail intermolecular contacts in wild-type Int. Models for the allosteric stimulation of Int activity by β5 strand contacts are discussed. Keywords bacteriophage lambda; integrase; Holliday junction; site-specific recombination; peptide Bacteriophage lambda integrase (Int) is a tyrosine site-specific recombinase that functions as a higher-order tetrameric complex to catalyze the integration and excision of the viral chromosome into and out of the Escherichia coli chromosome by means of a highly coordinated sequence of cleavage and ligation reactions at specific loci (att sites, see Figure 1(a)). 1 Previous work has established that the seven carboxy-terminal residues (C-terminal tail) of Int are integral to regulatory mechanisms that link catalytic function with protein multi-merization and also coordinate Int functions within the multimeric recombinogenic complex. 2-6 Here we extend these results by demonstrating that the β5 strand of Int is not simply a placeholder for the multi-positional C-terminal tail but rather exerts its own allosteric effects on Int function in response to the incoming tail.Int is a member of the tyrosine recombinase family, whose hallmark is an ordered pair of transesterification reactions that first generate and then resolve a Holliday junction recombination intermediate (Figure 1(a)). 7-9 The transesterification reactions are mediated by transient covalent 3′ phospho-tyrosine bonds that are formed at each of the four "core-type"

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

“…The Flp‐related tyrosine recombinases Cre, lambda Int and XerC/XerD resemble vaccinia topoisomerases in that each houses its active site within a protein monomer. Nevertheless, the full chemical competence and functional regulation of the cis active site may require trans allosteric activation by a neighbouring monomer (Guo et al , 1997; Hallet et al , 1999; Biswas et al , 2005; Hazelbaker et al , 2005). It would be interesting to test whether Cre, Int and XerC/XerD are more Flp‐like or topoisomerase‐like in regards to the reaction steps that are prone to hydrolysis and whether they use phosphate electrostatics or active site electrostatics to deal with this encumbrance.…”

Mechanisms of Site-Specific Recombination

“…NMR data found that the tail is flexible and is not altered by the addition of a single Int monomer-binding site (65). However, activity of the wild-type monomer can be restored by adding an excess of a peptide that mimics the C-terminal tail (66). Such negative regulation may be particularly important for Int, which binds crossover site DNA with relatively weak sequence specificity.…”

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

“…27 We expressed and purified four different protein constructs comprising fragments of the enzyme involved in core site binding and cleavage: (1) Int CB , a protein construct containing the core-binding domain of λ-Int, (2) Int CB+Cat , containing both the central and catalytic domains, (3) Int Cat , the isolated catalytic domain, and (4) Int CatΔ349C197S , a fragment of the catalytic domain bearing a Cys-Ser substitution at position 197 to improve sample behavior and lacking the last seven residues from the C terminus; these residues have been implicated in mediating inter-protomer interactions important for coordinated cleavage. 23,24,28,29 All constructs containing the catalytic domain, (i.e. Int CB+Cat , Int Cat and Int CatΔ349C197S ) were competent for site-specific cleavage of DNA half-site substrates, 6,26 with the Int CatΔ349C197S construct exhibiting slightly increased activity 24 and improved stability compared to Int Cat .…”