Characterization of bacteriophage lambda excisionase mutants defective in DNA binding

The bacteriophage lambda excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797-3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the alpha-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.     

Characterization of the bacteriophage lambda excisionase (Xis) protein: the C-terminus is required for Xis-integrase cooperativity but not for DNA binding.

We have performed a mutational analysis of the xis gene of bacteriophage lambda. The Xis protein is 72 amino acids in length and required for excisive recombination. Twenty-six mutants of Xis were isolated that were impaired or deficient in lambda excision. Mutant proteins that contained amino acid substitutions in the N-terminal 49 amino acids of Xis were defective in excisive recombination and were unable to bind DNA. In contrast, one mutant protein containing a leucine to proline substitution at position 60 and two truncated proteins containing either the N-terminal 53 or 64 amino acids continued to bind lambda DNA, interact cooperatively with FIS and promote excision. However, these three mutants were unable to bind DNA cooperatively with Int. Cooperativity between wild-type Xis and Int required the presence of FIS, but not the Int core-type binding sites. This study shows that Xis has at least two functional domains and also demonstrates the importance of the cooperativity in DNA binding of FIS, Xis and Int in lambda excision.     

Fis targets assembly of the Xis nucleoprotein filament to promote excisive recombination by phage lambda

The 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 A co-crystal structure of a non-specifically 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 core Int binding sites whose trajectory places constraints on models for the excisive intasome structure.     

Crystal structure of the excisionase-DNA complex from bacteriophage lambda

The excisionase (Xis) protein from bacteriophage lambda is the best characterized member of a large family of recombination directionality factors that control integrase-mediated DNA rearrangements. It triggers phage excision by cooperatively binding to sites X1 and X2 within the phage, bending DNA significantly and recruiting the phage-encoded integrase (Int) protein to site P2. We have determined the co-crystal structure of Xis with its X2 DNA-binding site at 1.7A resolution. Xis forms a unique winged-helix motif that interacts with the major and minor grooves of its binding site using an alpha-helix and an ordered beta-hairpin (wing), respectively. Recognition is achieved through an elaborate water-mediated hydrogen-bonding network at the major groove interface, while the preformed hairpin forms largely non-specific interactions with the minor groove. The structure of the complex provides insights into how Xis recruits Int cooperatively, and suggests a plausible mechanism by which it may distort longer DNA fragments significantly. It reveals a surface on the protein that is likely to mediate Xis-Xis interactions required for its cooperative binding to DNA.     

The effect of mutations in the Xis-binding sites on site-specific recombination in coliphage HK022

Excisionase (Xis) is an accessory protein that is required for the excision of the related prophages lambda and HK022. Xis binds to two tandemly arranged binding sites (X1 and X2) on the P arm of the recombination sites attP and attR. Gel-retardation analyses and site-specific recombination assays were conducted on derivatives bearing site-directed mutations in the X1 and X2 sites of phage HK022. The results confirm the cooperative binding of Xis to its sites, showing that binding to X1 stimulates further binding to X2. The results also show that mutants affected in a single site are inactive in excision, whereas mutants affected in both sites, which show a complete absence of Xis binding, display significant excision activity. This restored activity is attributed to the interaction of Xis with Integrase, the protein that catalyzes the site-specific recombination reaction.     

Solution structure and stability of the full-length excisionase from bacteriophage HK022.

Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the lambda-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28-->Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined alpha-helices (residues 5-11 and 18-27) and five beta-strands (residues 2-4, 30-31, 35-36, 41-44 and 48-49). These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage lambda excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical 'winged-helix' DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51-72, which were not resolved in the lambda Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins.     

Regulation of directionality in bacteriophage lambda site-specific recombination: structure of the Xis protein

Upon induction of a bacteriophage lambda lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision. Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function. Xis adopts an unusual "winged"-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single alpha-helix and loop structure ("wing"), respectively. The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA. We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase-Xis-DNA complex required for phage excision.     

Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination

Excision of the lambda prophage from the chromosome of its Escherichia coli host requires the products of the two viral genes int and xis. This paper reports a purification of the lambda xis gene product using a complementation assay in which functional Xis must be added to purified Int and an E. coli-derived host factor extract. Excisive recombination between a left (attL) and right (attR) prophage attachment site cloned on the same plasmid DNA substrate occurred efficiently under these conditions. Purified Int and Xis together could not carry out excision in vitro unless an extract derived from the E. coli host was added; purified integration host factor satisfied this requirement. Xis appears to have a molecular weight of 8800 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It possesses no detectable endonuclease or topoisomerase activities, does not appear to bind DNA to filters, and does not increase the ability of Int to bind DNA. The addition of Xis not only stimulated excisive recombination in vitro but also inhibited integrative recombination. Xis protected Int protein from heat inactivation, suggesting a possible interaction between the two proteins. In light of these observations, possible roles for Xis in recombination are discussed.     

Purification and characterization of bacteriophage P22 Xis protein

The temperate bacteriophages λ and P22 share similarities in their site-specific recombination reactions. Both require phage-encoded integrase (Int) proteins for integrative recombination and excisionase (Xis) proteins for excision. These proteins bind to core-type, arm-type, and Xis binding sites to facilitate the reaction. λ and P22 Xis proteins are both small proteins (λ Xis, 72 amino acids; P22 Xis, 116 amino acids) and have basic isoelectric points (for P22 Xis, 9.42; for λ Xis, 11.16). However, the P22 Xis and λ Xis primary sequences lack significant similarity at the amino acid level, and the linear organizations of the P22 phage attachment site DNA-binding sites have differences that could be important in quaternary intasome structure. We purified P22 Xis and studied the protein in vitro by means of electrophoretic mobility shift assays and footprinting, cross-linking, gel filtration stoichiometry, and DNA bending assays. We identified one protected site that is bent approximately 137 degrees when bound by P22 Xis. The protein binds cooperatively and at high protein concentrations protects secondary sites that may be important for function. Finally, we aligned the attP arms containing the major Xis binding sites from bacteriophages λ, P22, L5, HP1, and P2 and the conjugative transposon Tn916. The similarity in alignments among the sites suggests that Xis-containing bacteriophage arms may form similar structures.     

Activity of coliphage HK022 excisionase (Xis) in the absence of DNA binding

A mutated excisionase (Xis) protein of coliphage HK022 whose single Cys residue was replaced by Ser does not bind to its two tandem binding sites (X1, X2) on the P arm of attR. Despite its DNA-binding inability the protein showed 30% excision activity of the wild type Xis both in vitro and in vivo. This partial activity is attributed to the interaction of Xis with integrase that is retained in the mutant protein. This protein-protein interaction occurs in the absence of DNA binding.     

The Holliday junction intermediates of lambda integrative and excisive recombination respond differently to the bending proteins integration host factor and excisionase.

In lambda site-specific recombination, the integrative and excisive reactions proceed via two different Holliday junction intermediates, both of which are generated and resolved by a pair of sequentially ordered single strand exchanges. Factors affecting the directionality and efficiency of the second pair of strand exchanges were examined using artificial Holliday junctions (chi-forms). The integrative and excisive recombination intermediates respond differently to the accessory DNA bending proteins integration host factor and excisionase (Xis). These differences between the two recombination intermediates result from a different interaction pattern between proteins binding to the left (P arm) and right (P' arm) of the crossover region. The effect of Xis protein on the directionality of resolution, i.e. the choice of which strands are exchanged, is consistent with a role in promoting the second strand exchange during excision. Proteins binding to the left of the crossover region (P arm) primarily influence the directionality of resolution, while proteins binding to the right (P' arm) have a greater effect on the overall efficiency of resolution. Together, the effect of proteins binding to sites in the P and P' arms is to greatly enhance resolution of the two different Holliday intermediates and to favor resolution in the 'forward' direction for both integrative and excisive recombination.     

Protein-protein interaction between monomers of coliphage HK022 excisionase

Excisionase (Xis) is an accessory protein that is required for the site-specific excision reaction of the coliphages HK022 and lambda. Xis binds in a strong cooperative manner to two tandem binding sites (X1 and X2) located on the P arm of the attachment (att) sites on the phage genome. As a result of crosslinking experiments in vivo and in vitro of Xis-overexpressing cells, by gel filtration of purified Xis and by FRET analyses we show that Xis monomers of HK022 interact and form dimers that are not dependent on the single Cys residue of the protein and on the presence of DNA. The formation of the dimers may explain the strong binding cooperativity of Xis to its sites on DNA.     

The Structure of Xis Reveals the Basis for Filament Formation and Insight into DNA Bending within a Mycobacteriophage Intasome

The recombination directionality factor, Xis, is a DNA bending protein that determines the outcome of integrase-mediated site-specific recombination by redesign of higher-order protein–DNA architectures. Although the attachment site DNA of mycobacteriophage Pukovnik is likely to contain four sites for Xis binding, Xis crystals contain five subunits in the asymmetric unit, four of which align into a Xis filament and a fifth that is generated by an unusual domain swap. Extensive intersubunit contacts stabilize a bent filament-like arrangement with Xis monomers aligned head to tail. The structure implies a DNA bend of ~ 120°, which is in agreement with DNA bending measured in vitro. Formation of attR-containing intasomes requires only Int and Xis, distinguishing Pukovnik from lambda. Therefore, we conclude that, in Pukovnik, Xis-induced DNA bending is sufficient to promote intramolecular Int-mediated bridges during intasome formation.     

The structure of the excisionase (Xis) protein from conjugative transposon Tn916 provides insights into the regulation of heterobivalent tyrosine recombinases

Heterobivalent tyrosine recombinases play a prominent role in numerous bacteriophage and transposon recombination systems. Their enzymatic activities are frequently regulated at a structural level by excisionase factors, which alter the ability of the recombinase to assemble into higher-order recombinogenic nucleoprotein structures. The Tn916 conjugative transposon spreads antibiotic resistance in pathogenic bacteria and is mobilized by a heterobivalent recombinase (Tn916Int), whose activity is regulated by an excisionase factor (Tn916Xis). Unlike the well-characterized (lambda)Xis excisionase from bacteriophage lambda, Tn916Xis stimulates excision in vitro and in Escherichia coli only modestly. To gain insights into this functional difference, we have performed in vitro DNA-binding studies of Tn916Xis and Tn916Int, and we have solved the solution structure of Tn916Xis. We show that the heterobivalent Tn916Int protein is capable of bridging the DR2-type and core-type sites on the left arm of the tranpsoson. Consistent with the notion that Tn916Int is regulated only loosely, we find that Tn916Xis binding does not alter the stability of DR2-Tn916Int-core bridges or the ability of Tn916Int to recognize the arms of the transposon in vitro. Despite a high degree of divergence at the primary sequence level, we show that Tn916Xis and (lambda)Xis adopt related prokaryotic winged-helix structures. However, they differ at their C termini, with Tn916Xis replacing the flexible integrase contacting tail found in (lambda)Xis with a positively charged alpha-helix. This difference provides a structural explanation for why Tn916Xis does not interact cooperatively with its cognate integrase in vitro, and reveals how subtle changes in the winged-helix fold can modulate the functional properties of excisionase factors.     

The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth

We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site-specific recombinational switches. Furthermore, we show that induction of a Mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucts62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage lambda.     

The molecular basis of co-operative DNA binding between lambda integrase and excisionase

Higher-order nucleoprotein complexes often stabilize catalytic proteins in appropriate conformations for optimal activity and contribute to regulation during reactions requiring association of proteins and DNA. Formation of such complexes, known as intasomes, is required for site-specific recombination catalysed by bacteriophage Lambda Integrase protein (Int). Int-catalysed recombination is regulated by a second bacteriophage-encoded protein, Excisionase (Xis), which both stimulates excision and inhibits integration. To exert its effect, Xis binds co-operatively with Int, thereby inducing and stabilizing a DNA bend that alters the intasome structures formed during recombination. A rare int mutant, int 2268 ts, was reported (Enquist, L.W. and Weisberg, R.A. (1984) Mol Gen Genet 195: 62-69) to be more defective for excision than integration. Here, we have determined that this mutant Int protein contains an E47K substitution, and that the resultant excision-specific defect is due, at least in part, to destabilized interactions between Int and Xis. Analysis of several engineered substitutions at Int position 47 showed that a negatively charged residue is required for co-operative DNA binding between Int and Xis, and suggest that the Int-E47 residue may contact Xis directly. Substitutions at Int position 47 also affect co-operative binding among Int proteins at arm-type DNA sites, and thereby reduce the efficiency of both integration and excision. Collectively, these results suggest that a single surface of the Int amino-terminal domain mediates two alternate types of co-operative binding interactions.     

Escherichia coli plasmid vectors for high-level regulated expression of the bacteriophage lambda xis gene product

The bacteriophage lambda Xis protein is one of the proteins required for site-specific excisive recombination by which the lambda prophage is excised from the Escherichia coli bacterial chromosome. We cloned the lambda xis gene under the control of several prokaryotic promoters to obtain a sufficient source of the protein for biochemical studies. Our results demonstrate that E. coli lac promoter and lambda pL promoter fusions to the xis gene produce high levels of Xis protein. Induction of the expression vectors results in a 10- to 50-fold increase in Xis activity. In addition, one of these plasmids allows the control of xis expression in vivo.     

Xis protein binding to the left arm stimulates excision of conjugative transposon Tn916

Tn916 and related conjugative transposons are clinically significant vectors for the transfer of antibiotic resistance among human pathogens, and they excise from their donor organisms using the transposon-encoded integrase ((Tn916)Int) and excisionase ((Tn916)Xis) proteins. In this study, we have investigated the role of the (Tn916)Xis protein in stimulating excisive recombination. The functional relevance of (Tn916)Xis binding sites on the arms of the transposon has been assessed in vivo using a transposon excision assay. Our results indicate that in Escherichia coli the stimulatory effect of the (Tn916)Xis protein is mediated by sequence-specific binding to either of its two binding sites on the left arm of the transposon. These sites lie in between the core and arm sites recognized by (Tn916)Int, suggesting that the (Tn916)Xis protein enhances excision in a manner similar to the excisionase protein of bacteriophage lambda, serving an architectural role in the stabilization of protein-nucleic acid structures required for strand synapsis. However, our finding that excision in E. coli is significantly enhanced by the host factor HU, but does not depend on the integration host factor or the factor for inversion stimulation, defines clear mechanistic differences between Tn916 and bacteriophage lambda recombination.     

DnaJ (Hsp40 protein) binding to folded substrate impacts KplE1 prophage excision efficiency

Temperate phages mediate gene transfer and can modify the properties of their host organisms through the acquisition of novel genes, a process called lysogeny. The KplE1 prophage is one of the 10 prophage regions in Escherichia coli K12 MG1655. KplE1 is defective for lysis but fully competent for site-specific recombination. The TorI recombination directionality factor is strictly required for prophage excision from the host genome. We have previously shown that DnaJ promotes KplE1 excision by increasing the affinity of TorI for its site-specific recombination DNA target. Here, we provide evidence of a direct association between TorI and DnaJ using in vitro cross-linking assays and limited proteolysis experiments that show that this interaction allows both proteins to be transiently protected from trypsin digestion. Interestingly, NMR titration experiments showed that binding of DnaJ involves specific regions of the TorI structure. These regions, mainly composed of α-helices, are located on a surface opposite the DNA-binding site. Taken together, we propose that DnaJ, without the aid of DnaK/GrpE, is capable of increasing the efficiency of KplE1 excision by causing a conformational stabilization that allows TorI to adopt a more favorable conformation for binding to its specific DNA target.