Characterization of a mutation of bacteriophage lambda integrase. Putative role in core binding and strand exchange for a conserved residue

Site-specific recombination is involved in processes ranging from resolution of bacterial chromosome dimers to adeno-associated viral integration and is a versatile tool for mammalian genetics. The bacteriophage lambda-encoded site-specific recombinase integrase (Int) is one of the best studied site-specific recombinases and mediates recombination via four distinct pathways. We have characterized a mutant version of lambda Int, IntT236I; this mutant can perform the bent-L pathway only, whereas the corresponding IntT236A mutant can perform bent-L, excision and integration pathways. Experiments with both IntT236I and IntT236A show that the hydroxyl group of threonine is necessary for wild-type recombination. Substitution of the threonine by serine leads to nearly complete rescue of the mutant phenotypes. In addition, our data show that the IntT236I mutant is defective partially due to obstructive steric interactions. Comparisons of crystal structures reveal that the threonine at residue 236 may play an important role in stabilizing recombination intermediates through solvent-mediated protein-DNA interactions at the core-binding sites and that the hydroxyl group is important for effective cleavage and Holliday junction formation. Our data also indicate that Int contacts the core sites differently in intermediates assembled in excisive versus bent-L recombination.     

Complementation of bacteriophage lambda integrase mutants: evidence for an intersubunit active site.

Site-specific recombination of bacteriophage lambda starts with the formation of higher-order protein--DNA complexes, called 'intasomes', and is followed by a series of steps, including the initial DNA cleavage, top-strand exchange, branch migration and bottom-strand exchange, to produce recombinant products. One of the intasomes formed during excisive recombination (the attL complex) is composed of the phage-encoded integrase (Int), integration host factor (IHF) and one of the recombination substrates, attL DNA. Int is the catalytic recombinase and has two different DNA binding domains. When IHF is present, Int binds to two types of sites in attL DNA, the three arm-type sites (P'123) and the core-type sites (B and C') where the reciprocal strand exchange takes place. The Tyr342 residue of Int serves as a nucleophile during strand cleavage and covalently attaches to the DNA through a phosphotyrosyl bond. In vitro complementation assays have been performed for strand cleavage using attL suicide substrates and mutant proteins containing amino acid substitutions at residues conserved in the integrase family of recombinases. We demonstrate that at least two Int monomers are required to form the catalytically-competent species that performs cleavage at the B site. It is likely that the active site is formed by two Int monomers.     

Half-att site substrates reveal the homology independence and minimal protein requirements for productive synapsis in lambda excisive recombination

The early events in site-specific excisive recombination were studied with phage lambda half-att sites that have no DNA to one side of the strand exchange region; they carry a single core-type integrase binding site and either P or P' arm flanking DNA. These half-attR and half-attL sites exhibit normal properties for the initial (covalent) top-strand transfer and form stable intermediates independent of later steps in the reaction. With these novel substrates we show that Xis specifically promotes the first strand exchange and that attL enhances Int cleavage at the top-strand site of attR. It is also shown that synapsis and initial strand transfers do not require DNA-DNA pairing but are mediated by protein-protein and protein-DNA interactions. These involve the two top-strand Int binding sites (required for the first strand exchange) and, in addition, one of the two bottom-strand sites (C') responsible for the second strand exchange.     

Control and regulation of KplE1 prophage site-specific recombination: a new recombination module analyzed

KplE1 is one of the 10 prophage regions of Escherichia coli K12, located at 2464 kb on the chromosome. KplE1 is defective for lysis, but it is fully competent for excisive recombination. In this study, we have mapped the binding sites of the recombination proteins, namely IntS, TorI, and IHF on attL and attR, and the organization of these sites suggests that the intasome is architecturally different from the lambda canonical form. We also measured the relative contribution of these proteins to both excisive and integrative recombination by using a quantitative in vitro assay. These experiments show a requirement of the TorI excisionase for excisive recombination and of the IntS integrase for both integration and excision. Moreover, we observed a strong influence of the supercoiled state of the substrates. The KplE1 recombination module, composed of the integrase and excisionase genes together with the attL and attR DNA regions, is highly similar to that of several phages infecting various E. coli strains as well as Shigella flexneri and Shigella sonnei. The in vitro recombination data reveal that HK620 and KplE1 att sequences are exchangeable. This study thus defines a new site-specific recombination module, and implications for the mechanism and regulation of recombination are discussed.     

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.     

Site-specific photo-cross-linking between lambda integrase and its DNA recombination target

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to "arm-type" sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the "core-type" sequences where DNA cleavage and ligation are executed. We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position. When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically. The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of approximately 20%. The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly. Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the lambda Int family.     

Mutational analysis of integrase arm-type binding sites of bacteriophage lambda. Integration and excision involve distinct interactions of integrase with arm-type sites

Integrative recombination between specific attachment (att) regions of the bacteriophage lambda genome (attP) and the Escherichia coli genome (attB) results in a prophage flanked by the hybrid recombinant sites attL and attR. Each att site contains sequences to which proteins involved in recombination bind. Using site-directed mutagenesis, we have constructed a related set of point mutations within each of the five Int "arm-type" binding sites located within attP, attL and attR. Footprint analyses of binding demonstrate that mutating the arm-type sites significantly disrupts the binding of Int. Recombination analyses of mutant att sites in vivo and in vitro demonstrate that only three wild-type arm-type sites within attP are required for efficient integrative recombination. Similar analyses demonstrate that efficient excision can occur with two other different sets of wild-type arm-type sites in attL and attR. These results demonstrate that integrative and excisive recombination may involve interactions of Int with distinct and different subsets of arm-type sites.     

The amino terminus of bacteriophage lambda integrase is involved in protein-protein interactions during recombination

Bacteriophage lambda integrase (Int) catalyzes at least four site-specific recombination pathways between pairs of attachment (att) sites. Protein-protein contacts between monomers of Int are presumed to be important for these site-specific recombination events for several reasons: Int binds to the att sites cooperatively, catalytic Int mutants can complement each other for strand cleavage, and crystal structures for two other recombinases in the Int family (Cre from phage P1 and Int from Haemophilus influenzae phage HP1) show extensive protein-protein contacts between monomers. We have begun to investigate interactions between Int monomers by three approaches. First, using a genetic assay, we show that regions of protein-protein interactions occur throughout Int, including in the amino-terminal domain. This domain was previously thought to be important only for high-affinity protein-DNA interactions. Second, we have found that an amino-terminal His tag reduces cooperative binding to DNA. This disruption in cooperativity decreases the stable interaction of Int with core sites, where catalysis occurs. Third, using protein-protein cross-linking to investigate the multimerization of Int during recombination, we show that Int predominantly forms dimers, trimers, and tetramers. Moreover, we show that the cysteine at position 25 is present at or near the interface between monomers that is involved in the formation of dimers and tetramers. Our evidence indicates that the amino-terminal domain of Int is involved in protein-protein interactions that are likely to be important for 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.     

Mutations in the amino-terminal domain of lambda-integrase have differential effects on integrative and excisive recombination

Lambda integrase (Int) forms higher-order protein-DNA complexes necessary for site-specific recombination. The carboxy-terminal domain of Int (75-356) is responsible for catalysis at specific core-type binding sites whereas the amino-terminal domain (1-70) is responsible for cooperative arm-type DNA binding. Alanine scanning mutagenesis of residues 64-70, within full-length integrase, has revealed differential effects on cooperative arm binding interactions that are required for integrative and excisive recombination. Interestingly, while these residues are required for cooperative arm-type binding on both P'1,2 and P'2,3 substrates, cooperative binding at the arm-type sites P'2,3 was more severely compromised than binding at arm-type sites P'1,2 for L64A. Concomitantly, L64A had a much stronger effect on integrative than on excisive recombination. The arm-binding properties of Int appear to be intrinsic to the amino-terminal domain because the phenotype of L64A was the same in an amino-terminal fragment (Int 1-75) as it was in the full-length protein.     

Site-specific recombination Xis-independent excisive recombination of bacteriophage lambda

The integration of bacteriophage lambda into the Escherichia coli chromosome depends on the phage-encoded Int protein; prophage excision depends on Int and a second phage function, Xis. Limited excisive recombination has been observed in vivo with certain xis mutants, suggesting that Int may be able to carry out excision without Xis.We report here that purified Int protein carries out lambda site-specific excisive recombination in vitro in the absence of Xis. This reaction requires host factors derived from a non-lysogenic E. coli strain and is influenced strongly by ionic strength. Excision in the absence of Xis occurs slowly at low salt concentrations (40 mm-NaCl) and very little excision occurs at high salt concentrations (100 mm-NaCl). In the presence of Xis, excisive recombination proceeds rapidly at both low and high ionic strengths. These observations are consistent with previous experiments that suggested the partial dispensability of Xis for excision.     

Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli

The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element ( att  P) and the chromosomal ( att  B) sites. These regions share an identity segment of 58 bp extending from the anti-codon loop through the 3' end of a tRNA^Pro gene. To facilitate the study of the att  B site, the int and xis genes, expressed from an inducible promoter, and att  P from pSAM2 were cloned on plasmids in Escherichia coli . Compatible plasmids carrying the different att  B regions to be tested were introduced in these E . coli strains. Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision. This experimental system was used to study the sequences required in att  B for efficient site-specific recombination. A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of att  B; shorter sequences allowed integration with lower efficiencies. By comparing the 26-bp-long att  B with att  P, according to the Lambda model, we propose that B and B', C and C' core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.     

Interactions between lambda Int molecules bound to sites in the region of strand exchange are required for efficient Holliday junction resolution

lambda Site-specific recombination proceeds via two sequential single-strand exchanges that first generate and then resolve a Holliday recombination intermediate. The resolution of artificial Holliday junctions (chi-forms) is well suited to studying the mechanisms involved in reciprocal strand exchange because the linear products of this reaction are stable and easily quantitated. To study the interactions between Int molecules bound at the sites of strand exchange, artificial Holliday junctions containing only the seven base-pair overlap region and the four core-type Int binding sites were used as a model system. In vitro resolution of these structures yields products of both top- and bottom-strand exchange. An abortive product resulting from simultaneous cleavage of the top and bottom strands also occurs at low frequency. Inactivation of one of the four Int binding sites by multiple base substitutions does not significantly affect the efficiency of resolution but has a dramatic effect on the directionality, i.e. the choice of top- or bottom-strand exchange. When any two of the four core-type sites are similarly inactivated, strand exchange is very inefficient and the amount of aberrant cleavage is somewhat greater than for the Holliday junction with four intact Int binding sites. Analysis of the resolution products of Holliday junctions with various combinations of defective Int binding sites leads to the following conclusions: (1) three functional core-type Int binding sites are necessary and sufficient for a strand exchange; (2) the Int molecules that are partners in a strand exchange interact with Int bound to a "cross-core" site that is not directly involved in carrying out the reaction; (3) Int molecules bound to the core-type sites interact in a way that reduces the occurrence of abortive double-strand cleavage events.     

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.     

A biotin interference assay highlights two different asymmetric interaction profiles for lambda integrase arm-type binding sites in integrative versus excisive recombination

The site-specific recombinase integrase encoded by bacteriophage lambda promotes integration and excision of the viral chromosome into and out of its Escherichia coli host chromosome through a Holliday junction recombination intermediate. This intermediate contains an integrase tetramer bound via its catalytic carboxyl-terminal domains to the four "core-type" sites of the Holliday junction DNA and via its amino-terminal domains to distal "arm-type" sites. The two classes of integrase binding sites are brought into close proximity by an ensemble of accessory proteins that bind and bend the intervening DNA. We have used a biotin interference assay that probes the requirement for major groove protein binding at specified DNA loci in conjunction with DNA protection, gel mobility shift, and genetic experiments to test several predictions of the models derived from the x-ray crystal structures of minimized and symmetrized surrogates of recombination intermediates lacking the accessory proteins and their cognate DNA targets. Our data do not support the predictions of "non-canonical" DNA targets for the N-domain of integrase, and they indicate that the complexes used for x-ray crystallography are more appropriate for modeling excisive rather than integrative recombination intermediates. We suggest that the difference in the asymmetric interaction profiles of the N-domains and arm-type sites in integrative versus excisive recombinogenic complexes reflects the regulation of recombination, whereas the asymmetry of these patterns within each reaction contributes to directionality.     

CTnDOT Integrase Interactions with Attachment Site DNA and Control of Directionality of the Recombination Reaction

IntDOT is a tyrosine recombinase encoded by the conjugative transposon CTnDOT. The core binding (CB) and catalytic (CAT) domains of IntDOT interact with core-type sites adjacent to the regions of strand exchange, while the N-terminal arm binding (N) domain interacts with arm-type sites distal to the core. Previous footprinting experiments identified five arm-type sites, but how the arm-type sites participate in the integration and excision of CTnDOT was not known. In vitro integration assays with substrates containing arm-type site mutants demonstrated that attDOT sequences containing mutations in the L1 arm-type site or in the R1 and R2 or R1 and R2′ arm-type sites were dramatically defective in integration. Substrates containing mutations in the L1 and R1 arm-type sites showed a 10- to 20-fold decrease in detectable in vitro excision, but introduction of multiple arm-type site mutations in attR did not have an effect on the excision frequency. A sixth arm-type site, the R1′ site, was also identified and shown to be required for integration and important for efficient excision. These results suggest that intramolecular IntDOT interactions are required for integration, while the actions of accessory factors are more important for excision. Gel shift assays performed in the presence of core- and arm-type site DNAs showed that IntDOT affinity for the attDOT core was enhanced when IntDOT was simultaneously bound to arm-type site DNA.Conjugative transposons (CTns), also known as integrative and conjugative elements (ICEs), are mobile genetic elements that are widespread in Bacteroides spp. and are implicated in the spread of antibiotic resistance. These elements are normally integrated into the host chromosome but can excise, replicate, and transfer to a recipient cell by conjugation (34). Since CTns commonly carry antibiotic resistance genes, it is likely that the increase in antibiotic-resistant Bacteroides strains has been mediated through the lateral transfer of these elements (36). One of the best-studied ICEs in Bacteroides is the conjugative transposon CTnDOT. CTnDOT is 65 kb in size and carries genes encoding resistance to tetracycline and erythromycin. Over the past 30 years, the incidence of tetracycline resistance has increased to 80% of Bacteroides isolates due to the presence of CTnDOT-type elements (36).Integration and excision of CTnDOT results from site-specific recombination between regions of DNA known as attachment (att) sites. During integration, the joined ends of the closed circular intermediate (attDOT) recombine with the bacterial target sequence (attB) to form the recombinant sites (attL and attR). The integration reaction requires IntDOT, a CTnDOT-encoded protein that has been identified as a member of the tyrosine recombinase family, as well as a host factor encoded by Bacteroides (8, 21). Site-specific recombination between the attL and attR attachment sites results in excision of CTnDOT from the host chromosome. IntDOT is also required for excision, as are three element-encoded proteins: Orf2c, Orf2d, and Exc, as well a Bacteroides host factor (8, 38). The roles of these accessory proteins are not well understood, although Orf2c and Orf2d have been shown to bind DNA (unpublished results).One of the best-studied tyrosine recombinases is the integrase (Int) of the lambda system. The C terminus of Int includes the core binding (CB) and catalytic (CAT) domains that bind to core-type sites, which flank the sites of cleavage and strand exchange (2, 24). The N-terminal arm-binding (N) domain binds to arm-type sites that are distal to the core-type sites. In the presence of the appropriate host and accessory factors, Int binding to arm-type sites is required for the formation of higher-order protein/DNA complexes known as intasomes, which are required for integration and excision (15, 18, 22). Int is capable of making intramolecular interactions (interactions between Int monomers on the same attachment site) and intermolecular interactions (interactions between Int monomers on different attachment sites) during recombination (15, 16). In the lambda system, the directionality of the reaction is regulated by Int interactions with arm-type sites in conjunction with the integration host factor (IHF) during the formation of an integrative intasome, or IHF, Xis, and FIS during the formation of the two excisive intasomes (1, 4, 42).Presumably, IntDOT occupancy of specific arm-type sites in conjunction with interactions of accessory factors with att sites leads to the assembly of integrative or excisive intasomes and thus contributes to the directionality of IntDOT-mediated recombination. Previous DNase I footprinting experiments identified five arm-type binding sites on attDOT (11). In this study, mutations were constructed in the five sites to determine their roles in the integration and excision of CTnDOT. In addition, a sixth arm-type site was discovered that is important for both integrative and excisive recombination. The results of gel shift assays also show that the interaction of IntDOT with core-type sites and arm-type sites involves cooperative interactions.     

Regulation of site-specific recombination by the C-terminus of lambda integrase

Site-specific recombination catalyzed by bacteriophage λ integrase (Int) is essential for establishment and termination of the viral lysogenic life cycle. Int is the archetype of the tyrosine recombinase family whose members are responsible for DNA rearrangement in prokaryotes, eukaryotes and viruses. The mechanism regulating catalytic activity during recombination is incompletely understood. Studies of tyrosine recombinases bound to their target substrates suggest that the C-termini of the proteins are involved in protein–protein contacts that control the timing of DNA cleavage events during recombination. We investigated an Int truncation mutant (W350) that possesses enhanced topoisomerase activity but greater than 100-fold reduced recombination activity. Alanine scanning mutagenesis of the C-terminus indicates that two mutants, W350A and I353A, cannot perform site-specific recombination although their DNA binding, cleavage and ligation activities are at wild-type levels. Two other mutants, R346A and R348A, are deficient solely in the ability to cleave DNA. To explain these results, we have constructed a homology-threaded model of the Int structure using a Cre crystal structure. We propose that residues R346 and R348 are involved in orientation of the catalytic tyrosine that cleaves DNA, whereas W350 and I353 control and make intermolecular contacts with other Int proteins in the higher order recombination structures known as intasomes. These results suggest that Int and the other tyrosine recombinases have evolved regulatory contacts that coordinate site-specific recombination at the C-terminus.     

Human genomic site-specific recombination catalyzed by coliphage HK022 integrase

It has been previously demonstrated that the wild type integrase (Int) protein of coliphage HK022 can catalyze site-specific recombination in human cells between attachment (att) sites that were placed on extrachromosomal plasmids. In the present report it is shown that Int can catalyze the site-specific recombination reactions in a human cell culture on the chromosomal level. These include integrative (attP x attB) as well as excisive (attL x attR) reactions each in two configurations. In the cis configuration both sites are on the same chromosome, in the trans configuration one site is on a chromosome and the other on an episome. The reactions in cis were observed without any selection force, using the green fluorescent protein (GFP) as a reporter. The reactions in trans could be detected only when a selection force was applied, using the hygromycin-resistant (Hyg(R)) phenotype as a selective marker. All reactions were catalyzed without the need to supply any of the accessory proteins that are required by Int in its Escherichia coli host. The versatility of the att sites may be an advantage in the utilization of Int to integrate plasmid DNA into the genome, followed by a partial exclusion of the integrated plasmid.     

A comparison of the effects of single-base and triple-base changes in the integrase arm-type binding sites on the site-specific recombination of bacteriophage lambda.

Triple-base changes were made in each of the five Integrase (Int) arm-type binding sites of bacteriophage lambda. These triple changes, called ten mutants, were compared with single-base changes (hen mutants) for their effects on integrative and excisive recombination. The presence of ten or hen mutations in the P1, P'2, or P'3 sites inhibited integration, but the ten P'3 mutant was 10-fold more defective than the analogous hen mutant. The results with these mutants suggest that the P1, P'2, P'3, and possibly the P'1 sites are required for integration. In wild-type E. coli, the ten P'1 mutant reduced the frequency of excision 5-fold, whereas the hen P'1 mutant had no effect. The presence of ten mutations in the P2, P'1, or P'2 sites inhibited lambda excision in an E. coli strain deficient in the production of FIS, while hen mutations in the P2 and P'2 sites had little or no effect. The results with the ten mutants suggest that the P2, P'1, and P'2 sites are required for excision. The differences in the severity of the effects between the ten and hen mutations may be due to the inability of cooperative interactions among Int, IHF, Xis, and FIS to overcome the disruption of Int binding to sites with triple-base changes compared to sites with single-base changes.     

Mapping of a higher order protein-DNA complex: two kinds of long-range interactions in lambda attL

To map the protein-protein and protein-DNA interactions involved in lambda site-specific recombination, Int cleavage assays with suicide substrates, nuclease protection patterns, gel retardation experiments, and quantitative Western blotting were applied to wild-type attL and attL mutants. The results lead to a model in which one IHF molecule bends the attL DNA and forms a higher order complex with the three bivalent Int molecules required for excisive recombination. It is proposed that each of the Int molecules binds in a unique manner: one bridges two DNA binding sites in cis, one is held via its high affinity amino-terminal DNA binding domain, and the third depends upon protein-protein interactions in addition to its low affinity carboxy-terminal DNA binding domain. This protein-DNA complex contains two unsatisfied DNA binding domains, each with a different sequence specificity, and is well suited to specific interactions with an appropriate recombination partner.