The N-terminus of IntDOT forms hydrophobic interactions during Holliday Junction resolution.

DOT Integrase (IntDOT) is a member of the tyrosine recombinase family. It catalyzes the integration and excision reactions of an integrative and conjugative element (ICE) called CTnDOT. Like other tyrosine recombinases, the integration reaction proceeds by two sets of strand exchanges between the attDOT site on CTnDOT and an attB site in the host chromosome. The strand exchanges occur seven bases apart and define an overlap region. After the first strand exchanges a Holliday Junction (HJ) intermediate is formed. Previous work showed that a valine (V95) in a predicted alpha helix in the N-terminus of IntDOT is required for resolution of HJs to substrates and products. We have identified two additional hydrophobic residues in the helix (A92 and F99) that are involved in resolution of HJs. IntDOT proteins with substitutions at these residues form aberrant complexes in an electrophoretic mobility shift assay. We propose that these three residues participate in hydrophobic interactions that are involved in forming higher-order complexes and resolution of HJs.

The Xis2d protein of CTnDOT binds to the intergenic region between the mob and tra operons.

CTnDOT is a 65kbp integrative and conjugative element (ICE) that carries genes encoding both tetracycline and erythromycin resistances. The excision operon of this element encodes Xis2c, Xis2d, and Exc proteins involved in the excision of CTnDOT from host chromosomes. These proteins are also required in the complex transcriptional regulation of the divergently transcribed transfer (tra) and mobilization (mob) operons of CTnDOT. Transcription of the tra operon is positively regulated by Xis2c and Xis2d, whereas, transcription of the mob operon is positively regulated by Xis2d and Exc. Xis2d is the only protein that is involved in the excision reaction, as well as the transcriptional regulation of both the mob and tra operons. This paper helps establish how Xis2d binds the DNA in the mob and tra region. Unlike other excisionase proteins, Xis2d binds a region of dyad symmetry. The binding site is located in the intergenic region between the mob and tra promoters, and once bound Xis2d induces a bend in the DNA. Xis2d binding to this region could be the preliminary step for the activation of both operons. Then the other proteins, like Exc, can interact with Xis2d and form higher order complexes.

The Integration and Excision of CTnDOT.

Bacteroides species are one of the most prevalent groups of bacteria present in the human colon. Many strains carry large, integrated elements including integrative and conjugative elements (ICEs). One such ICE is CTnDOT, which is 65 kb in size and encodes resistances to tetracycline and erythromycin. CTnDOT has been increasing in prevalence in Bacteroides spp., and is now found in greater than 80% of natural isolates. In recent years, CTnDOT has been implicated in the spread of antibiotic resistance among gut microbiota. Interestingly, the excision and transfer of CTnDOT is stimulated in the presence of tetracycline. The tyrosine recombinase IntDOT catalyzes the integration and excision reactions of CTnDOT. Unlike the well-characterized lambda Int, IntDOT tolerates heterology in the overlap region between the sites of cleavage and strand exchange. IntDOT also appears to have a different arrangement of active site catalytic residues. It is missing one of the arginine residues that is conserved in other tyrosine recombinases. The excision reaction of CTnDOT is complex, involving excision proteins Xis2c, Xis2d, and Exc, as well as IntDOT and a Bacteroides host factor. Xis2c and Xis2d are small, basic proteins like other recombination directionality factors (RDFs). Exc is a topoisomerase; however, the topoisomerase function is not required for the excision reaction. Exc has been shown to stimulate excision frequencies when there are mismatches in the overlap regions, suggesting that it may play a role in resolving Holliday junctions (HJs) containing heterology. Work is currently under way to elucidate the complex interactions involved with the formation of the CTnDOT excisive intasomes.

Interactions of NBU1 IntN1 and Orf2x proteins with attachment site DNA.

NBU1 is a mobilizable transposon found in Bacteroides spp. Mobilizable transposons require gene products from coresident conjugative transposons for excision and transfer to recipient cells. The integration of NBU1 requires IntN1, which has been identified as a tyrosine recombinase, as well as Bacteroides host factor BHFa. Excision of NBU1 is a more complicated process, involving five element-encoded proteins (IntN1, Orf2, Orf2x, Orf3, and PrmN1) as well as a Bacteroides host factor and a cis-acting DNA sequence. Little has been known about what role the proteins play in excision, although IntN1 and Orf2x have been shown to be the only proteins absolutely required for detectable excision. To determine where IntN1 and Orf2x bind during the excision of NBU1, both proteins were partially purified and tested in DNase I footprinting experiments with the excisive attachment sites attL and attR. The results demonstrate that IntN1 binds to four core-type sites that flank the region of cleavage and strand exchange, as well as six arm-type sites. A unique feature of the system is the location of DR2a and DR2b arm-type sites immediately downstream of the attL core. The DR1a, DR1b, DR3a, and DR3b arm-type sites were shown to be required for in vitro integration of NBU1. In addition, we have identified one Orf2x binding site (O1) on attL as well as a dA+dT-rich upstream element that is required for Orf2x interactions with O1.

Interactions of the excision proteins of CTnDOT in the attR intasome.

Excision of the conjugative transposon CTnDOT from the chromosome of Bacteroides spp. involves four CTnDOT-encoded proteins: IntDOT, Xis2c, Xis2d, and Exc along with a host factor. These proteins form excisive intasomes on the attR and attL sites which undergo synapsis and recombination to form the attDOT and attB sites. We recently developed an in vitro intramolecular excision reaction where the attL and attR sites are on the same plasmid. This reaction requires IntDOT, Xis2c, Xis2d, and is stimulated by Exc. We used this reaction to identify the binding sites of the IntDOT, Xis2c, and Xis2d. In this paper, we show that three of the six arm-type sites are absolutely required for excision. Furthermore, we also identified two binding sites for Xis2d and two possible binding sites for Xis2c on the attR site. We also showed that IntDOT interacts cooperatively with the Xis2c and Xis2d proteins on the attR site.

IntDOT interactions with core sites during integrative recombination.

Integrative and conjugative elements (ICEs), formerly called conjugative transposons, have been implicated in the proliferation of antibiotic resistance genes. CTnDOT is an extensively studied ICE found in Bacteroides spp. In addition to carrying resistance genes to both erythromycin and tetracycline, CTnDOT carries a gene that encodes a tyrosine recombinase called IntDOT that catalyzes integration into and excision out of the bacterial host chromosome. CTnDOT integrates into one of several known attB sites in the bacterial chromosome that consists of a pair of inverted repeat core sites called B and B' in attB. The attDOT site contains the core sites and D and D'. These sites flank the overlap regions where strand exchanges occur. A notable feature of all known attB sites is the conservation of the B core site sequence, which is also found in the D core site of attDOT. In this study, we used a mutational analysis to establish the importance of this conserved sequence for integration and characterize the interaction of IntDOT with individual base pairs. We identified important T-A base pairs at position -5 in the B and D core sites and position +5 in the poorly conserved B' core site that are important for integrative recombination. Base analog studies suggest that IntDOT may make specific contacts with the A residues in the major groove at positions -5 and +5. IntDOT interaction with the A at position -5 in the B core site is required for the first strand exchange.

The excision proteins of CTnDOT positively regulate the transfer operon.

The Bacteroides conjugative transposon, CTnDOT, is an integrated conjugative element (ICE), found in many human colonic Bacteroides spp. strains. It has a complex regulatory system for both excision from the chromosome and transfer and mobilization into a new host. It was previously shown that a cloned DNA segment encoding the xis2c, xis2d, orf3, and exc genes was required for tetracycline dependent activation of the P(tra) promoter. The Xis2c and Xis2d proteins are required for excision while the Exc protein stimulates excision. We report here that neither the Orf3 nor the Exc proteins are involved in activation of the P(tra) promoter. Deletion analysis and electromobility shift assays showed that the Xis2c and Xis2d proteins bind to the P(tra) promoter to activate the tra operon. Thus, the recombination directionality factors of CTnDOT excision also function as activator proteins of the P(tra) promoter.

Roles of Exc protein and DNA homology in the CTnDOT excision reaction.

Excision from the chromosome is the first step during the transfer of conjugative transposons (CTns) to a recipient. We previously showed that the excision of CTnDOT is more complex than the excision of lambdoid phages and CTns such as Tn916. The excision in vivo of CTnDOT utilizes four CTnDOT-encoded proteins, IntDOT, Xis2c, Xis2d, and Exc, and a host factor. We previously developed an in vitro excision reaction where the recombination sites attL and attR were located on different plasmids. The reaction was inefficient and did not require Exc, suggesting that the reaction conditions did not mimic in vivo conditions. Here, we report the development of an intramolecular excision reaction where the attL and attR sites are located on the same DNA molecule. We found that Exc stimulates the reaction 3- to 5-fold. The efficiency of the excision reaction was also dependent on the distance between the attL and attR sites and on the sequences of the overlap regions between the sites of the strand exchanges. Substrates with identical overlap sequences recombined more efficiently than ones with heterologous overlap sequences. This was surprising, because the integration reaction is not sensitive to heterology in the overlap regions of the attDOT and attB sites.

Resolution of Holliday junction recombination intermediates by wild-type and mutant IntDOT proteins.

CTnDOT encodes an integrase that is a member of the tyrosine recombinase family. The recombination reaction proceeds by sequential sets of genetic exchanges between the attDOT site in CTnDOT and an attB site in the chromosome. The exchanges are separated by 7 base pairs in each site. Unlike most tyrosine recombinases, IntDOT exchanges sites that contain different DNA sequences between the exchange sites to generate Holliday junctions (HJs) that contain mismatched bases. We demonstrate that IntDOT resolves synthetic HJs in vitro. Holliday junctions that contain identical sequences between the exchange sites are resolved into both substrates and products, while HJs that contain mismatches are resolved only to substrates. This result implies that resolution of HJs to products requires the formation of a higher-order nucleoprotein complex with natural sites containing IntDOT. We also found that proteins with substitutions of residues (V95, K94, and K96) in a putative alpha helix at the junction of the N and CB domains (coupler region) were defective in resolving HJs. Mutational analysis of charged residues in the coupler and the N terminus of the protein did not provide evidence for a charge interaction between the regions of the protein. V95 may participate in a hydrophobic interaction with another region of IntDOT.

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.

Homology-dependent interactions determine the order of strand exchange by IntDOT recombinase.

The Bacteroides conjugative transposon CTnDOT encodes an integrase, IntDOT, which is a member of the tyrosine recombinase family. Other members of this group share a strict requirement for sequence identity within the region of strand exchange, called the overlap region. Tyrosine recombinases catalyze recombination by making an initial cleavage, strand exchange and ligation, followed by strand swapping isomerization requiring sequence identity in the overlap region, followed by the second cleavage, strand exchange and ligation. IntDOT is of particular interest because it has been shown to utilize a three-step mechanism: a sequence identity-dependent initial strand exchange that requires two base pairs of complementary DNA at the site of cleavage; a sequence identity-independent strand swapping isomerization, followed by a sequence identity-independent cleavage, strand exchange and ligation. In addition to the sequence identity requirement in the overlap region, Lambda Int interactions with arm-type sites dictate the order of strand exchange regardless of the orientation of the overlap region. Although IntDOT has an arm-binding domain, we show here that the location of sequence identity within the overlap region dictates where the initial cleavage takes place and that IntDOT can recombine substrates containing mismatches in the overlap region so long as a single base of sequence identity exists at the site of initial cleavage.

Structure-function analysis of IntDOT.

CTnDOT integrase (IntDOT) is a member of the tyrosine family of site-specific DNA recombinases. IntDOT is unusual in that it catalyzes recombination between nonidentical sequences. Previous mutational analyses centered on mutants with substitutions of conserved residues in the catalytic (CAT) domain or residues predicted by homology modeling to be close to DNA in the core-binding (CB) domain. That work suggested that a conserved active-site residue (Arg I) of the CAT domain is missing and that some residues in the CB domain are involved in catalysis. Here we used a genetic approach and constructed an Escherichia coli indicator strain to screen for random mutations in IntDOT that disrupt integrative recombination in vivo. Twenty-five IntDOT mutants were isolated and characterized for DNA binding, DNA cleavage, and DNA ligation activities. We found that mutants with substitutions in the amino-terminal (N) domain were catalytically active but defective in forming nucleoprotein complexes, suggesting that they have altered protein-protein interactions or altered interactions with DNA. Replacement of Ala-352 of the CAT domain disrupted DNA cleavage but not DNA ligation, suggesting that Ala-352 may be important for positioning the catalytic tyrosine (Tyr-381) during cleavage. Interestingly, our biochemical data and homology modeling of the CAT domain suggest that Arg-285 is the missing Arg I residue of IntDOT. The predicted position of Arg-285 shows it entering the active site from a position on the polypeptide backbone that is not utilized in other tyrosine recombinases. IntDOT may therefore employ a novel active-site architecture to catalyze recombination.

Challenging a paradigm: the role of DNA homology in tyrosine recombinase reactions.

A classical feature of the tyrosine recombinase family of proteins catalyzing site-specific recombination, as exemplified by the phage lambda integrase and the Cre and Flp recombinases, is the ability to recombine substrates sharing very limited DNA sequence identity. Decades of research have established the importance of this short stretch of identity within the core regions of the substrates. Since then, several new enzymes that challenge this paradigm have been discovered and require the role of sequence identity in site-specific recombination to be reconsidered. The integrases of the conjugative transposons such as Tn916, Tn1545, and CTnDOT recombine substrates with heterologous core sequences. The integrase of the mobilizable transposon NBU1 performs recombination more efficiently with certain core mismatches. The integration of CTX phage and capture of gene cassettes by integrons also occur by altered mechanisms. In these systems, recombination occurs between mismatched sequences by a single strand exchange. In this review, we discuss literature that led to the formulation of the current strand-swapping isomerization model for tyrosine recombinases. The review then focuses on recent developments on the recombinases that challenged the paradigm that was derived from the studies of early systems.

Mutational analysis and homology-based modeling of the IntDOT core-binding domain.

Tyrosine recombinases mediate a wide range of important genetic rearrangement reactions. Models for tyrosine recombinases have been based largely on work done on the integrase of phage lambda and recombinases like Cre, Flp, and XerC/D. All of these recombinases share a common amino acid signature that is important for catalysis. Several conjugative transposons (CTns) encode recombinases that are also members of the tyrosine recombinase family, but the reaction that they catalyze differs in that recombination does not require homology in the attachment sites. In this study, we examine the role of the core-binding (CB) domain of the CTnDOT integrase (IntDOT) that is located adjacent to the catalytic domain of the protein. Since there is no crystal structure for any of the CTn integrases, we began with a predicted three-dimensional structure produced by homology-based modeling. Amino acid substitutions were made at positions predicted by the model to be close to the DNA. Mutant proteins were tested for the ability to mediate integration in vivo and for in vitro DNA-binding, cleavage, and ligation activities. We identified for the first time nonconserved amino acid residues in the CB domain that are important for catalytic activity. Mutant proteins with substitutions at three positions in the CB domain are defective for DNA cleavage but still proficient in ligation. The positions of the residues in the complex suggest that the mutant residues affect the positioning of the cleaved phosphodiester bond in the active site without disruption of the ligation step.

Purification and characterization of bacteriophage P22 Xis protein.

The temperate bacteriophages lambda 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. lambda and P22 Xis proteins are both small proteins (lambda Xis, 72 amino acids; P22 Xis, 116 amino acids) and have basic isoelectric points (for P22 Xis, 9.42; for lambda Xis, 11.16). However, the P22 Xis and lambda 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 lambda, 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.

CTnDOT integrase performs ordered homology-dependent and homology-independent strand exchanges.

Although the integrase (IntDOT) of the Bacteroides conjugative transposon CTnDOT has been classified as a member of the tyrosine recombinase family, the reaction it catalyzes appears to differ in some features from reactions catalyzed by other tyrosine recombinases. We tested the ability of IntDOT to cleave and ligate activated attDOT substrates in the presence of mismatches. Unlike other tyrosine recombinases, the results revealed that IntDOT is able to perform ligation reactions even when all the bases within the crossover region are mispaired. We also show that there is a strong bias in the order of strand exchanges during integrative recombination. The top strands are exchanged first in reactions that appear to require 2 bp of homology between the partner sites adjacent to the sites of cleavage. The bottom strands are exchanged next in reactions that do not require homology between the partner sites. This mode of coordination of strand exchanges is unique among tyrosine recombinases.

Integration site selection by the Bacteroides conjugative transposon CTnBST.

A newly discovered Bacteroides conjugative transposon (CTn), CTnBST, integrates more site specifically than two other well-studied CTns, the Bacteroides CTn CTnDOT and the enterococcal CTn Tn916. Moreover, the integrase of CTnBST, IntBST, had the C-terminal 6-amino-acid signature that is associated with the catalytic regions of members of the tyrosine recombinase family, most of which integrate site specifically. Also, in most of these integrases, all of the conserved amino acids are required for integration. In the case of IntBST, however, we found that changing three of the six conserved amino acids in the signature, one of which was the presumed catalytic tyrosine, resulted in a 1,000-fold decrease in integration frequency. Changes in the other amino acids had little or no effect. Thus, although the CTnBST integrase still seems to be a member of the tyrosine recombinase family, it clearly differs to some extent from other members of the family in its catalytic site. We also determined the sequence requirements for CTnBST integration in the 18-bp region where the crossover occurs preferentially during integration. We found that CTnBST integrates in this preferred site about one-half of the time but can also use other sites. A consensus sequence was tentatively derived by comparison of a few secondary sites: AATCTGNNAAAT. We report here that within the consensus region, no single base change affected the frequency of integration. However, 3 bp at one end of the consensus sequence (CTG) proved to be essential for integration into the preferred site. This sequence appeared to be at one end of a 7-bp crossover region, CTGNNAA. The other bases could vary without affecting either integration frequency or specificity. Thus, in contrast to well-studied site-specific recombinases which require homology throughout the crossover region, integration of CTnBST requires homology at one end of the crossover region but not at the other end.

Interaction of the Gifsy-1 Xis protein with the Gifsy-1 attP sequence.

The Gifsy-1 phage integrates site specifically into the Salmonella chromosome via an integrase-mediated site-specific recombination mechanism. Initial genetic analysis suggests that Gifsy-1 integrase-mediated excision of the Gifsy-1 phage is influenced by proteins encoded by both the Gifsy-1 and the Gifsy-2 phages. Our studies show that the Gifsy-1 Xis protein regulates the directionality of integrase-mediated excision of the Gifsy-1 phage. Electrophoretic mobility shift assays, DNase I footprinting, dimethyl sulfate (DMS) interference assays, and DMS protection assays were used to identify a 31-base-pair sequence in the attP region to which the Gifsy-1 protein binds. The results suggest that this recombination directionality factor binds in vitro to three imperfect direct repeats, spaced 10 base pairs apart, in a sequential and cooperative manner in the absence of other phage-encoded proteins. Our studies suggest that, while the Gifsy-1 Xis does not require additional factors for specific and high-affinity binding, it may form a microfilament on DNA similar to that described for the phage lambda Xis protein.

IntDOT interactions with core- and arm-type sites of the conjugative transposon CTnDOT.

CTnDOT is a Bacteroides conjugative transposon (CTn) that has facilitated the spread of antibiotic resistances among bacteria in the human gut in recent years. Although the integrase encoded by CTnDOT (IntDOT) carries the C-terminal set of conserved amino acids that is characteristic of the tyrosine family of recombinases, the reaction it catalyzes involves a novel step that creates a short region of heterology at the joined ends of the element during recombination. Also, in contrast to tyrosine recombinases, IntDOT catalyzes a reaction that is not site specific. To determine what types of contacts IntDOT makes with the DNA during excision and integration, we first developed an agarose gel-based assay for CTnDOT recombination, which facilitated the purification of the native IntDOT protein. The partially purified IntDOT was then used for DNase I footprinting analysis of the integration site attDOT and the excision sites attL and attR. Our results indicate that CTnDOT has five or six arm sites that are likely to be involved in forming higher-order nucleoprotein complexes necessary for synapsis. In addition, there are four core sites that flank the sites of strand exchange during recombination. Thus, despite the fact that the reaction catalyzed by IntDOT appears to be different from that typically catalyzed by tyrosine recombinases, the protein-DNA interactions required for higher-order structures and recombination appear to be similar.

Characterization of the integrase of NBU1, a Bacteroides mobilizable transposon.

NBU1 is a Bacteroides mobilizable transposon (MTn) that is integrated within the host chromosome and requires CTnDOT functions for its excision and transfer into a new host. The NBU1 integrase IntN1 has been classified as a tyrosine recombinase based on the presence of conserved residues. We created alanine mutants of the residues R291, K314, H393, R396, H419 and the conserved substitution Y429F and tested them for integration efficiency. The results suggest that these residues in IntN1 are important for integration, and Y429 could be the catalytic nucleophile. We employed suicide substrates and partially purified IntN1 to determine the positions of IntN1 cleavage within the 14 bp common core region that is identical in both NBU1 att sites. We show that IntN1 makes 7 bp staggered cuts on the top and bottom strands. From previous mutational analysis of the att sites, we show that two specific mutations near the site of bottom strand cleavage within this 7 bp region increased integration, and mutations of the two bases near top strand cleavage site had no effect on integration. These results indicate that IntN1 lacks the strict requirement for homology between the recombining sites seen with other tyrosine recombinases. We also show that phosphorothioate substitutions at the cleavage site and 1 bp downstream inhibited cleavage by IntN1. This differs from other studied tyrosine recombinases where inhibition occurs by substitutions at the cleavage site only.