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.     

Site-specific recombination in human cells catalyzed by phage lambda integrase mutants

Phage lambda Integrase (Int) is the prototype of the so-called integrase family of conservative site-specific recombinases, which includes Cre and FLP. The natural function of Int is to execute integration and excision of the phage into and out of the Escherichia coli genome, respectively. In contrast to Cre and FLP, however, wild-type Int requires accessory proteins and DNA supercoiling of target sites to catalyze recombination. Here, we show that two mutant Int proteins, Int-h (E174 K) and its derivative Int-h/218 (E174 K/E218 K), which do not require accessory factors, are proficient to perform intramolecular integrative and excisive recombination in co-transfection assays inside human cells. Intramolecular integrative recombination is also detectable by Southern analysis in human reporter cell lines harboring target sites attB and attP as stable genomic sequences. Recombination by wild-type Int, however, is not detectable by this method. The latter result implies that eukaryotic co-factors, which could functionally replace the prokaryotic ones normally required for wild-type Int, are most likely not present in human cells.     

Xer site-specific recombination in vitro.

Two related recombinases, XerC and XerD, belonging to the lambda integrase family of enzymes, are required for Xer site-specific recombination in vivo. In order to understand the roles of these proteins in the overall reaction mechanism, an in vitro recombination system using a synthetic Holliday junction-containing substrate has been developed. Recombination of this substrate is efficient and requires both XerC and XerD. However, only exchange of one pair of strands, the one corresponding to the conversion of the Holliday junction intermediate back to the substrate, has been observed. Recombination reactions using XerC and XerD derivatives that are mutant in their presumptive catalytic residues, or are maltose-binding fusion recombinase derivatives, have demonstrated that this pair of strand exchanges is catalysed by XerC. The site of XerC-mediated cleavage has been located to between the last nucleotide of the XerC binding site and the first nucleotide of the central region. Cleavage at this site generates a free 5'-OH and a covalent complex between XerC and the 3' end of the DNA.     

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.     

Xer site-specific recombination. DNA strand rejoining by recombinase XerC

Xer site-specific recombination functions in the stable maintenance of circular replicons in Escherichia coli. Each of two related recombinase proteins, XerC and XerD, cleaves a specific pair of DNA strands, exchanges them, and rejoins them to the partner DNA molecule during a complete recombination reaction. The rejoining activity of recombinase XerC has been analyzed using isolated covalent XerC-DNA complexes resulting from DNA cleavage reactions upon Holliday junction substrates. These covalent protein-DNA complexes are competent in the rejoining reaction, demonstrating that covalently bound XerC can catalyze strand rejoining in the absence of other proteins. This contrasts with a recombinase-mediated cleavage reaction, which requires the presence of both recombinases, the recombinase mediating catalysis at any given time requiring activation by the partner recombinase. In a recombining nucleoprotein complex, both cleavage and rejoining can occur prior to dissociation of the complex.     

Decatenation of DNA circles by FtsK-dependent Xer site-specific recombination

DNA replication results in interlinked (catenated) sister duplex molecules as a consequence of the intertwined helices that comprise duplex DNA. DNA topoisomerases play key roles in decatenation. We demonstrate a novel, efficient and directional decatenation process in vitro, which uses the combination of the Escherichia coli XerCD site-specific recombination system and a protein, FtsK, which facilitates simple synapsis of dif recombination sites during its translocation along DNA. We propose that the FtsK-XerCD recombination machinery, which converts chromosomal dimers to monomers, may also function in vivo in removing the final catenation links remaining upon completion of DNA replication.     

Asymmetric DNA requirements in Xer recombination activation by FtsK

In bacteria with circular chromosomes, homologous recombination events can lead to the formation of chromosome dimers. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover by two tyrosine recombinases, XerC and XerD, at a specific site on the chromosome, dif. Recombination depends on a direct contact between XerD and a cell division protein, FtsK, which functions as a hexameric double stranded DNA translocase. Here, we have investigated how the structure and composition of DNA interferes with Xer recombination activation by FtsK. XerC and XerD each cleave a specific strand on dif, the top and bottom strand, respectively. We found that the integrity and nature of eight bottom-strand nucleotides and three top-strand nucleotides immediately adjacent to the XerD-binding site of dif are crucial for recombination. These nucleotides are probably not implicated in FtsK translocation since FtsK could translocate on single stranded DNA in both the 5′–3′ and 3′–5′ orientation along a few nucleotides. We propose that they are required to stabilize FtsK in the vicinity of dif for recombination to occur because the FtsK–XerD interaction is too transient or too weak in itself to allow for XerD catalysis.     

Unlinking chromosome catenanes in vivo by site-specific recombination

A challenge for chromosome segregation in all domains of life is the formation of catenated progeny chromosomes, which arise during replication as a consequence of the interwound strands of the DNA double helix. Topoisomerases play a key role in DNA unlinking both during and at the completion of replication. Here we report that chromosome unlinking can instead be accomplished by multiple rounds of site-specific recombination. We show that step-wise, site-specific recombination by XerCD-dif or Cre-loxP can unlink bacterial chromosomes in vivo, in reactions that require KOPS-guided DNA translocation by FtsK. Furthermore, we show that overexpression of a cytoplasmic FtsK derivative is sufficient to allow chromosome unlinking by XerCD-dif recombination when either subunit of TopoIV is inactivated. We conclude that FtsK acts in vivo to simplify chromosomal topology as Xer recombination interconverts monomeric and dimeric chromosomes.     

FtsK-dependent and -independent pathways of Xer site-specific recombination.

Homologous recombination between circular chromosomes generates dimers that cannot be segregated at cell division. Escherichia coli Xer site-specific recombination converts chromosomal and plasmid dimers to monomers. Two recombinases, XerC and XerD, act at the E. coli chromosomal recombination site, dif, and at related sites in plasmids. We demonstrate that Xer recombination at plasmid dif sites occurs efficiently only when FtsK is present and under conditions that allow chromosomal dimer formation, whereas recombination at the plasmid sites cer and psi is independent of these factors. We propose that the chromosome dimer- and FtsK-dependent process that activates Xer recombination at plasmid dif also activates Xer recombination at chromosomal dif. The defects in chromosome segregation that result from mutation of the FtsK C-terminus are attributable to the failure of Xer recombination to resolve chromosome dimers to monomers. Conditions that lead to FtsK-independent Xer recombination support the hypothesis that FtsK acts on Holliday junction Xer recombination intermediates.     

Synaptic intermediates in bacteriophage lambda site-specific recombination: integrase can align pairs of attachment sites.

Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome. The recombination event is mediated by the recombinase integrase (Int) together with several accessory proteins through short specific DNA sequences known as attachment sites. A gel mobility shift assay has been used to show that, in the absence of accessory proteins, Int can align and hold together two DNA molecules, each with an attachment site, to form stable non-covalent 'bimolecular complexes'. Each attachment site must have both core and arm binding sites for Int to participate in a bimolecular complex. These stable structures can be formed between pairs of attL and attP attachment sites, but cannot include attB or attR sites; they are inhibited by integration host factor (IHF) protein. The bimolecular complexes are shown to represent a synaptic intermediate in the reaction in which Int protein promotes the IHF-independent recombination of two attL sites. These complexes should enable a detailed analysis of synapsis for this pathway.     

The topological mechanism of phage lambda integrase.

Bacteriophage lambda integrase (Int) is a versatile site-specific recombinase. In concert with other proteins, it mediates phage integration into and excision out of the bacterial chromosome. Int recombines intramolecular sites in inverse or direct orientation or sites on separate DNA molecules. This wide spectrum of Int-mediated reactions has, however, hindered our understanding of the topology of Int recombination. By systematically analyzing the topology of Int reaction products and using a mathematical method called tangles, we deduce a unified model for Int recombination. We find that, even in the absence of (-) supercoiling, all Int reactions are chiral, producing one of two possible enantiomers of each product. We propose that this chirality reflects a right-handed DNA crossing within or between recombination sites in the synaptic complex that favors formation of right-handed Holliday junction intermediates. We demonstrate that the change in linking number associated with excisive inversion with relaxed DNA is equally +2 and -2, reflecting two different substrates with different topology but the same chirality. Additionally, we deduce that integrative Int recombination differs from excisive recombination only by additional plectonemic (-) DNA crossings in the synaptic complex: two with supercoiled substrates and one with relaxed substrates. The generality of our results is indicated by our finding that two other members of the integrase superfamily of recombinases, Flp of yeast and Cre of phage P1, show the same intrinsic chirality as lambda Int.     

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.     

Modulation of lambda integrase synthesis by rare arginine tRNA

Lambda's int gene contains an anomalously high frequency of the rare arginine codons AGA and AGG when compared to genes of Escherichia coli or to the rest of phage lambda. These are the least frequent codons in genes of E. coli and are recognized by the rarest tRNAs. The presence of these codons reduces the translation rate and, depending on the context, this can strongly modulate translational efficiency by a variety of mechanisms. In this study, we show that expression of the natural int gene may also be modulated by rare arginine codon usage, and we explore this mechanism.     

The unconventional Xer recombination machinery of Streptococci/Lactococci

Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving two paralogous tyrosine recombinases, XerC and XerD, and a 28-bp recombination site (dif) located at the junction of the two replication arms. Xer recombination is tightly controlled by the septal protein FtsK. XerCD recombinases and FtsK are found on most sequenced eubacterial genomes, suggesting that the Xer recombination system as described in E. coli is highly conserved among prokaryotes. We show here that Streptococci and Lactococci carry an alternative Xer recombination machinery, organized in a single recombination module. This corresponds to an atypical 31-bp recombination site (dif_SL) associated with a dedicated tyrosine recombinase (XerS). In contrast to the E. coli Xer system, only a single recombinase is required to recombine dif_SL, suggesting a different mechanism in the recombination process. Despite this important difference, XerS can only perform efficient recombination when dif_SL sites are located on chromosome dimers. Moreover, the XerS/dif_SL recombination requires the streptococcal protein FtsK_SL, probably without the need for direct protein-protein interaction, which we demonstrated to be located at the division septum of Lactococcus lactis. Acquisition of the XerS recombination module can be considered as a landmark of the separation of Streptococci/Lactococci from other firmicutes and support the view that Xer recombination is a conserved cellular function in bacteria, but that can be achieved by functional analogs.     

The isomeric preference of Holliday junctions influences resolution bias by lambda integrase.

Lambda site-specific recombination proceeds by a pair of sequential strand exchanges that first generate and then resolve a Holliday junction intermediate. A family of synthetic Holliday junctions with the branch point constrained to the center of the 7 bp overlap region was used to show that resolution of the top strands and resolution of the bottom strands are symmetrical but stereochemically distinct processes. Lambda integrase is sensitive to isomeric structure, preferentially resolving the pair of strands that are crossed in the protein-free Holliday junction. At the branch point of stacked immobile Holliday junctions, the number of purines is preferentially maximized in the crossed (versus continuous) strands if there is an inequality of purines between strands of opposite polarity. This stacking preference was used to anticipate the resolution bias of freely mobile junctions and thereby to reinforce the conclusions with monomobile junctions. The results provide a strong indication that in the complete recombination reaction a restacking of helices occurs between the top and bottom strand exchanges.     

The Xer/dif site-specific recombination system of Campylobacter jejuni

Chromosome dimers, which form during the bacterial life cycle, represent a problem that must be solved by the bacterial cell machinery so that chromosome segregation can occur effectively. The Xer/dif site-specific recombination system, utilized by most bacteria, resolves chromosome dimers into monomers using two tyrosine recombinases, XerC and XerD, to perform the recombination reaction at the dif site which consists of 28–30 bp. However, single Xer recombinase systems have been recently discovered in several bacterial species. In Streptococci and Lactococci a single recombinase, XerS, is capable of completing the monomerisation reaction by acting at an atypical dif site called dif _SL (31 bp). It was recently shown that a subgroup of ε-proteobacteria including Campylobacter spp. and Helicobacter spp. had a phylogenetically distinct Xer/dif recombination system with only one recombinase (XerH) and an atypical dif motif (difH). In order to biochemically characterize this system in greater detail, Campylobacter jejuni XerH was purified and its DNA-binding activity was characterized. The protein showed specific binding to the complete difH site and to both halves separately. It was also shown to form covalent complexes with difH suicide substrates. In addition, XerH was able to catalyse recombination between two difH sites located on a plasmid in Escherichia coli in vivo. This indicates that this XerH protein performs a similar function as the related XerS protein, but shows significantly different binding characteristics.     

A general model for site-specific recombination by the integrase family recombinases.

We present here a general model for integrase family site-specific recombination using the geometric relationships of the cleavable phosphodiester bonds and the disposition of the recombinase monomers (defined by their binding planes) with respect to them. The 'oscillation model' is based largely on the conformations of the recombinase-bound DNA duplexes and their dynamics within Holliday junctions. The duplex substrate or the Holliday junction intermediate is capable of 'oscillating' between two cleavage-competent asymmetric states with respect to corres-ponding chemically inert 'equilibrium positions'. The model accommodates several features of the Flp system and predicts two modes of DNA cleavage during a normal recombination event. It is equally applicable to other systems that mediate recombination across 6, 7 or 8 bp long strand exchange regions (or spacers). The model is consistent with approximately 0-1, 1-2 and 2-3 bp of branch migration during recombination reactions involving 6, 7 and 8 bp spacers, respectively.     

Amino-terminal domain interactions of lambda integrase on arm-type DNA

In contrast to the other tyrosine recombinase family members, integrase protein (Int) of bacteriophage λ has an additional amino-terminal domain that binds to “arm-type” DNA sequences distant from those involved in strand exchange. The homomeric interaction between neighboring amino-terminal domains of Int is contributed by R30-D71 salt-bridge in a non-equivalent manner on Holliday-junction intermediates. In this report, R30 and D71 residues were investigated in regard to Int’s cooperative binding to “arm-type” DNA and the attenuating function of “arm-type” DNA. The results suggest the electrostatic interaction between residues 30 and 71 is dependent on “arm-type” DNA and contributes the “selective” inhibition of catalytic activity of λ Int by “arm-type” DNA.     

Genetic manipulation of mouse embryonic stem cells by mutant lambda integrase

Mutant lambda integrases catalyze site-specific recombination reactions inside mammalian cells. Here we demonstrate that the integrase system can be used to eliminate resistance marker genes from the genome of mouse embryonic stem cells. So-called integrative and excisive recombination pathways led to the precise deletion of the neomycin gene, which was inserted together with a flanking pair of directly repeated recombination sites into the ROSA26 locus by standard targeting techniques. The excision of the resistance gene led to the expression of enhanced green fluorescence protein, which served as a means to sort out cells that had undergone site-specific recombination. Southern analysis and DNA sequencing confirmed that strand exchange reactions had occurred in the genome as expected. Hence, the integrase system may be used in conjunction with other site-specific recombinases as a tool in genome manipulation protocols.     

Genetic analysis of second-site revertants of bacteriophage lambda integrase mutants.

Bacteriophage lambda site-specific recombination is catalyzed by the phage-encoded integrase (Int) protein. Using a collection of 21 recombination-defective Int mutants, we performed a second-site reversion analysis. One of the primary mutants contained a valine-to-glutamic acid change at position 175 (V175E), and a pseudorevertant with a lysine change at this site (V175K) was also isolated. Relative to the wild-type protein, the V175E protein was defective in its ability to form the attL complex and to catalyze excision in vivo and in vitro. A mutant containing an alanine substitution (V175A) was made by site-directed mutagenesis, and it was more efficient than the V175K protein in forming the attL complex and promoting excision. These results indicate that a nonpolar side chain at residue 175 is required for function. The second primary mutant contained a proline-to-leucine change at position 243 (P243L). A true second-site revertant was isolated that contained a glutamic acid-to-lysine change (E218K). The P243L-E218K protein promoted recombination and bound arm-type sites more efficiently than the original P243L protein but not as efficiently as the protein containing the E218K substitution alone. The E218K substitution also restored activity to a mutant with a threonine-to-isoleucine substitution at position 270 (T270I). This result showed that suppression by the E218K change is not allele specific and suggests that the substitution improves an inherent activity of Int rather than directly compensating for the defect caused by the primary substitutions. Results with challenge phages carrying attL sites with altered core sites indicate that the E218K change may improve binding to the core site.