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.     

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.     

mwr Xer site-specific recombination is hypersensitive to DNA supercoiling

The multiresistance plasmid pJHCMW1, first identified in a Klebsiella pneumoniae strain isolated from a neonate with meningitis, includes a Xer recombination site, mwr, with unique characteristics. Efficiency of resolution of mwr-containing plasmid dimers is strongly dependent on the osmotic pressure of the growth medium. An increase in supercoiling density of plasmid DNA was observed as the osmotic pressure of the growth culture decreased. Reporter plasmids containing directly repeated mwr, or the related cer sites were used to test if DNA topological changes were correlated with significant changes in efficiency of Xer recombination. Quantification of Holliday junctions showed that while recombination at cer was efficient at all levels of negative supercoiling, recombination at mwr became markedly less efficient as the level of supercoiling was reduced. These results support a model in which modifications at the level of supercoiling density caused by changes in the osmotic pressure of the culture medium affects resolution of mwr-containing plasmid dimers, a property that separates mwr from other Xer recombination target sites.     

Asymmetric activation of Xer site-specific recombination by FtsK

Chromosome dimers, which frequently form in Escherichia coli, are resolved by the combined action of two tyrosine recombinases, XerC and XerD, acting at a specific site on the chromosome, dif, together with the cell division protein FtsK. The C-terminal domain of FtsK (FtsK(C)) is a DNA translocase implicated in helping synapsis of the dif sites and in locally promoting XerD strand exchanges after synapse formation. Here we show that FtsK(C) ATPase activity is directly involved in the local activation of Xer recombination at dif, by using an intermolecular recombination assay that prevents significant DNA translocation, and we confirm that FtsK acts before Holliday junction formation. We show that activation only occurs with a DNA segment adjacent to the XerD-binding site of dif. Only one such DNA extension is required. Taken together, our data suggest that FtsK needs to contact the XerD recombinase to switch its activity on using ATP hydrolysis.     

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.     

Direct interaction of aminopeptidase A with recombination site DNA in Xer site-specific recombination.

Xer site-specific recombination at ColE1 cer converts plasmid multimers into monomers, thus ensuring the heritable stability of ColE1. Two related recombinase proteins, XerC and XerD, catalyse the strand exchange reaction at a 30 bp recombination core site. In addition, two accessory proteins, PepA and ArgR, are required for recombination at cer. These two accessory proteins are thought to act at 180 bp of accessory sequences adjacent to the cer recombination core to ensure that recombination only occurs between directly repeated sites on the same molecule. Here, we demonstrate that PepA and ArgR interact directly with cer, forming a complex in which the accessory sequences of two cer sites are interwrapped approximately three times in a right-handed fashion. We present a model for this synaptic complex, and propose that strand exchange can only occur after the formation of this complex.     

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.     

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.     

Decatenation of kinetoplast DNA by topoisomerases

Kinetoplast DNA is the mitochondrial DNA of trypanosomatids such as Crithidia fasciculata. This DNA is in the form of networks containing thousands of DNA circles which are apparently catenated (interlocked). Some topoisomerases, such as T4 phage topoisomerase and DNA gyrase, catalyze a decatenation of the networks to form individual covalently closed circles.     

Control of Cre recombination by regulatory elements from Xer recombination systems

Site-specific recombination by the Cre recombinase takes place at a simple DNA site (loxP), requires no additional proteins and gives topologically simple recombination products. In contrast, cer and psi sites for Xer recombination contain approximately 150 bp of accessory sequences, require accessory proteins PepA, ArgR and ArcA, and the products are specifically linked to form a four-noded catenane. Here, we use hybrid sites consisting of accessory sequences of cer or psi fused to loxP to probe the function of accessory proteins in site-specific recombination. We show that PepA instructs Cre to produce four-noded catenane, but is not required for recombination at these hybrid sites. Mutants of Cre that require PepA and accessory sequences for efficient recombination were selected. PepA-dependent Cre gave products with a specific topology and displayed resolution selectivity. Our results reveal that PepA acts autonomously in the synapsis of psi and cer accessory sequences and is the main architectural element responsible for intertwining accessory site DNA. We suggest that accessory proteins can activate recombinases simply by synapsing the regulatory DNA sequences, thus bringing the recombination sites together with a specific geometry. This may occur without the need for protein-protein interactions between accessory proteins and the recombinases.     

Peptidase activity of Escherichia coli aminopeptidase A is not required for its role in Xer site-specific recombination

Xer site-specific recombination is required for the stable inheritance of multicopy plasmids and the normal segregation of the bacterial chromosome in Escherichia coli.Two related recombinases and two accessory proteins are essential for Xer-mediated recombination at cer, a recombination site in the plasmid ColE1 The accessory proteins, ArgR and PepA, function in ensuring that the Xer recombination reaction acts exclusively intramolecularly, converting plasmid dimers into monomers and not vice versa. PepA is an amino-exopeptidase, but its molecular role in the Xer recombination mechanism is unclear. Here we show that a mutation directed at the presumptive active site of PepA creates a protein with no detectable peptidase activity in vitro or in vivo, but which still functions normality in Xer site-specific recombination at cer.     

The cyclization of linear DNA in Escherichia coli by site-specific recombination

The efficiency with which linearized plasmid DNA can transform competent Escherichia coli can be significantly increased by use of the Cre-lox site-specific recombination system of phage P1. Linear plasmid molecules containing directly repeated loxP sites (lox² plasmids) are cyclized in Cre⁺ E. coli strains after introduction either by transformation or by mini-Mu transduction, Exonuclease V activity of the RecBC enzyme inhibits efficient cyclization of linearized lox² plasmids after transformation. By use of E. coli mutants which lack exonuclease V activity, Cre-mediated cyclization results in transformation efficiencies for linearized lox² plasmids identical to those obtained with covalently closed circular plasmid DNA. Moreover, Cre⁺ E. coli recBC strains allow the efficient recovery of lox² plasmids integrated within large linear DNA molecules such as the 150-kb genome of pseudorabies virus.     

Integrons: Novel DNA elements which capture genes by site-specific recombination

Integrons are unusual DNA elements which include a gene encoding a site-specific DNA recombinase, a DNA integrase, and an adjacent site at which a wide variety of antibiotic resistance and other genes are found as inserts. One or more genes can be found in the insert region, but each gene is part of an independent gene cassette. The inserted genes are expressed from a promoter in the conserved sequences located 5 to the genes, and integrons are thus natural expression vectors. A model for gene insertion in which circular gene cassettes are inserted individually via a single site-specific recombination event has been proposed and verified experimentally. The gene cassettes include a gene coding region and, at the 3 end of the gene an imperfect inverted repeat, a 59-base element. The 59-base elements are a diverse family of elements which function as sites recognized by the DNA integrase. Site-specific insertion of individual genes thus represents a further mechanism which contributes to the evolution of the genomes of Gram-negative bacteria and their plasmids and transposons.Members of the most studied class of integrons, which include thesulI gene in the conserved sequences, are believed to be mobile DNA elements on the basis that they are found in many independent locations, and a discrete boundary is found at the outer end of the 5-conserved segment. However, the length of the 3-conserved segment is variable in the integrons examined to date, and it is likely that this variability has arisen as the result of insertion and deletion events. Though the true extent of the 3-conserved segment remains to be determined, it seems likely that these integrons are mobile DNA elements. The second known class of integrons comprises members of the Tn7 transposon family.     

Self-control in DNA site-specific recombination mediated by the tyrosine recombinase TnpI

Tn4430 is a distinctive transposon of the Tn3 family that encodes a tyrosine recombinase (TnpI) to resolve replicative transposition intermediates. The internal resolution site of Tn4430 (IRS, 116 bp) contains two inverted repeats (IR1 and IR2) at the crossover core site, and two additional TnpI binding motifs (DR1 and DR2) adjacent to the core. Deletion analysis demonstrated that DR1 and DR2 are not required for recombination in vivo and in vitro. Their function is to provide resolution selectivity to the reaction by stimulating recombination between directly oriented sites on a same DNA molecule. In the absence of DR1 and/or DR2, TnpI-mediated recombination of supercoiled DNA substrates gives a mixture of topologically variable products, while deletion between two wild-type IRSs exclusively produces two-noded catenanes. This demonstrates that TnpI binding to the accessory motifs DR1 and DR2 contributes to the formation of a specific synaptic complex in which catalytically inert recombinase subunits act as architectural elements to control recombination sites pairing and strand exchange. A model for the organization of TnpI/IRS recombination complex is presented.     

Topology of Xer recombination on catenanes produced by lambda integrase.

Xer site-specific recombination at the psi site from plasmid pSC101 displays topological selectivity, such that recombination normally occurs only between directly repeated sites on the same circular DNA molecule. This intramolecular selectivity is important for the biological role of psi, and is imposed by accessory proteins PepA and ArcA acting at accessory DNA sequences adjacent to the core recombination site. Here we show that the selectivity for intramolecular recombination at psi can be bypassed in multiply interlinked catenanes. Xer site-specific recombination occurred relatively efficiently between antiparallel psi sites located on separate rings of right-handed torus catenanes containing six or more nodes. This recombination introduced one additional node into the catenanes. Antiparallel sites on four-noded right-handed catenanes, the normal product of Xer recombination at psi, were not recombined efficiently. Furthermore, parallel psi sites on right-handed torus catenanes were not substrates for Xer recombination. These findings support a model in which psi sites are plectonemically interwrapped, trapping a precise number of supercoils that are converted to four catenation nodes by Xer strand exchange.     

The ArcA/ArcB two-component regulatory system of Escherichia coli is essential for Xer site-specific recombination at psi

Two recombinases, XerC and XerD, act at the recombination sites psi and cer in plasmids pSC101 and ColE1 respectively. Recombination at these sites maintains the plasmids in a monomeric state and helps to promote stable plasmid inheritance. The accessory protein PepA acts at both psi and cer to ensure that only intramolecular recombination takes place. An additional accessory protein, ArgR, is required for recombination at cer but not at psi . Here, we demonstrate that the ArcA/ArcB two-component regulatory system of Escherichia coli , which mediates adaptation to anaerobic growth conditions, is required for efficient recombination in vivo at psi . Phosphorylated ArcA binds to psi in vitro and increases the efficiency of recombination at this site. Binding of ArcA to psi may contribute to the formation of a higher order synaptic complex between a pair of psi sites, thus helping to ensure that recombination is intramolecular.