Identification of the λ integrase surface that interacts with Xis reveals a residue that is also critical for Int dimer formation

Abstract

Lambda integrase (Int) is a heterobivalent DNA-binding protein that together with the accessory DNA-bending proteins IHF, Fis, and Xis, forms the higher-order protein-DNA complexes that execute integrative and excisive recombination at specific loci on the chromosomes of phage and its Escherichia coli host. The large carboxyl-terminal domain of Int is responsible for binding to core-type DNA sites and catalysis of DNA cleavage and ligation reactions. The small amino-terminal domain (residues 1-70), which specifies binding to arm-type DNA sites distant from the regions of strand exchange, consists of a three-stranded ␤-sheet, proposed to recognize the cognate DNA site, and an ␣-helix. We report here that a site on this ␣-helix is critical for both homomeric interactions between Int protomers and heteromeric interactions with Xis. The mutant E47A, which was identified by alanine-scanning mutagenesis, abolishes interactions between Int and Xis bound at adjacent binding sites and reduces interactions between Int protomers bound at adjacent arm-type sites. Concomitantly, this residue is essential for excisive recombination and contributes to the efficiency of the integrative reaction. NMR titration data with a peptide corresponding to Xis residues 57-69 strongly suggest that the carboxyl-terminal tail of Xis and the ␣-helix of the aminoterminal domain of Int comprise the primary interaction surface for these two proteins. The use of a common site on Int for both homotypic and heterotypic interactions fits well with the complex regulatory patterns associated with this site-specific recombination reaction.T he bacteriophage -encoded integrase (Int) belongs to a subgroup of the tyrosine recombinase family known as the heterobivalent recombinases (1, 2). These recombinases catalyze reactions that are distinguished from other pathways of sitespecific rearrangement and movement of DNA by their directionality. This feature is built on the ability of these recombinases to simultaneously bind and bridge two distinct and well separated DNA-binding sites (3-5). Int binds with high affinity to arm-type DNA sites by means of a small amino-terminal domain (residues 1-70) whereas it binds with lower affinity to core-type sites by means of a larger carboxyl-terminal domain (residues 75-356), which also executes DNA strand cleavage and ligation. Bridging between arm and core sites is facilitated by DNA bends induced by accessory proteins (IHF, Xis, and Fis) bound to DNA sites located between the arm-and core-type sequences (5-9). This ensemble of DNA-bending-and DNA-bridging-proteins forms the large recombinogenic complexes that confer directionality on the recombination reaction. In this article we show that formation of both the Int-Xis and Int-Int complexes, key elements in the structure and function of the higher-order recombinogenic structures, depends on a common site in the amino-terminal domain of Int.The Int recombinase catalyzes the integration and excision of viral DNA into and out of the chromosome of its Escherichia coli...

Pathogenicity Island Cross Talk Mediated by Recombination Directionality Factors Facilitates Excision from the Chromosome

“…The importance of integrase stability has also been demonstrated in the excision of a mycophage (which does not appear to require an RDF for excision) (54). The majority of RDFs that have been described are from prophages and were shown to control directionality of recombination by playing an architectural role by binding at the att sites, to each other, and/or to the integrase (20,21,24,(27)(28)(29)55). We determined that VefA and VefB bound the att sites of both islands, although VefB bound both sites at a higher affinity, causing a shift with four times less protein than VefA.…”

“…In TR integrase-encoding prophages, it was demonstrated that the excision event also requires an additional protein, an excisionase or recombination directionality factor (RDF) (24)(25)(26)(27)(28). In fact, it is believed that all TR integrases require an RDF to perform excision (20,29).…”

Structure of the cooperative Xis–DNA complex reveals a micronucleoprotein filament that regulates phage lambda intasome assembly

“…His Int ) that contains the DNA-binding and Xis cooperativity determinants (15)(16)(17). As shown in lane 2 of Fig.…”

Purification and Characterization of Bacteriophage P22 Xis Protein

“…Xis bends DNA approximately 140 degrees (32) and can function as a multiprotein complex with the FIS (factor for inversion stimulation) protein when the concentration of Xis is limiting (6, 33). The Xis is the best characterized of the Xis proteins because its residues involved in DNA binding (8,27) and in cooperative interactions with Int (7,9,24,31,34) have been identified, its structure has been solved by nuclear magnetic resonance spectroscopy (28), and a DNA-Xis cocrystal structure has been solved (26,27). Bacteriophage L5 Xis and P2 Xis (11,22,23,37) and the HP1 Cox protein (13-15) have also been studied.…”