The DNA architectural protein Xis regulates the construction of higher-order nucleoprotein intasomes that integrate and excise the genome of phage lambda from the Escherichia coli chromosome. Xis modulates the directionality of site-specific recombination by stimulating phage excision 10 6 -fold, while simultaneously inhibiting phage reintegration. Control is exerted by cooperatively assembling onto a Ϸ35-bp DNA regulatory element, which it distorts to preferentially stabilize an excisive intasome. Here, we report the 2.6-Å crystal structure of the complex between three cooperatively bound Xis proteins and a 33-bp DNA containing the regulatory element. Xis binds DNA in a head-to-tail orientation to generate a micronucleoprotein filament. Although each protomer is anchored to the duplex by a similar set of nonbase specific contacts, malleable protein-DNA interactions enable binding to sites that differ in nucleotide sequence. Proteins at the ends of the duplex sequence specifically recognize similar binding sites and participate in cooperative binding via protein-protein interactions with a bridging Xis protomer that is bound in a less specific manner. Formation of this polymer introduces Ϸ72°of curvature into the DNA with slight positive writhe, which functions to connect disparate segments of DNA bridged by integrase within the excisive intasome.DNA bending ͉ recombination directionality factors ͉ site-specific DNA recombination ͉ x-ray structure M obile genetic elements such as bacteriophages, conjugative transposons, and pathogenicity islands promote the lateral exchange of foreign DNA, enabling bacteria to acquire metabolic, pathogenic, and antibiotic resistance determinants. To prevent potentially catastrophic changes in the genome, these DNA rearrangements are often tightly controlled by regulatory factors that function together with the recombinase. The integration and excision reactions of phage , which are controlled by the phageencoded Xis protein, serve as a paradigm for studies of regulated site-specific recombination (1). Upon infection, specific DNA attachment sites located on the circularized phage genome (attP) and bacterial chromosome (attB) recombine to generate the integrated prophage with flanking hybrid sites (attL and attR) (Fig. 1A). Cellular DNA damage initiates a series of events that result in prophage excision to regenerate attP on the episomal phage genome and attB on the chromosome. Although the DNA strand transfer steps in each reaction are catalyzed by the phage-encoded tyrosine recombinase integrase (Int) protein, and are mechanistically similar, directionality control is achieved by guiding the assembly of distinct higher-order nucleoprotein structures called intasomes. Viral integration occurs within an integrative intasome containing Int and the Escherichia coli-encoded integration host factor (IHF) (2, 3), whereas excision is performed within an alternative excisive intasome complex containing Int, Xis, IHF, and the factor for inversion stimulation (4-7).Xis is the master regu...

SUMMARYThe phage-encoded Xis protein is the major determinant controlling the direction of recombination in phage lambda. Xis is a winged-helix DNA binding protein that cooperatively binds to the attR recombination site to generate a curved microfilament, which promotes assembly of the excisive intasome but inhibits formation of an integrative intasome. We find that lambda synthesizes surprisingly high levels of Xis immediately upon prophage induction when excision rates are maximal. However, because of its low sequence-specific binding activity, exemplified by a 1.9 Å co-crystal structure of a nonspecifically bound DNA complex, Xis is relatively ineffective at promoting excision in vivo in the absence of the host Fis protein. Fis binds to a segment in attR that almost entirely overlaps one of the Xis binding sites. Instead of sterically excluding Xis binding from this site, as has been previously believed, we show that Fis enhances binding of all three Xis protomers to generate the microfilament. A specific Fis-Xis interface is supported by the effects of mutations within each protein, and relaxed, but not completely sequence-neutral, binding by the central Xis protomer is supported by the effects of DNA mutations. We present a structural model for the 50 bp curved Fis-Xis cooperative complex that is assembled between the arm and Holliday junction Int binding sites whose trajectory places constraints on models for the excisive intasome structure.

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...

No abstract

This paper describes the crystallization, dehydration and preliminary X-ray data analysis of a complex containing several bacteriophage lambda excisionase (Xis) [Bushman et al. (1984). Cell, 39, 699-706] proteins cooperatively bound to a 33-mer DNA duplex (Xis-DNA(X1-X2)). Xis is expected to recognize this regulatory element in a novel manner by cooperatively binding and distorting multiple head-to-tail orientated DNA-binding sites. Crystals of this complex belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 107.7, c = 73.5 angstroms, alpha = beta = 90, gamma = 120 degrees. Based on the unit-cell parameters for the asymmetric unit, V(M) is 3.0 A(3) Da(-1), which corresponds to a solvent content of approximately 59%. The approaches used to crystallize the unusually long DNA fragment in the complex and the dehydration technique applied that dramatically improved the diffraction of the crystals from 10 to 2.6 angstroms are discussed.