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.     

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.     

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.     

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.     

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.     

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.     

Xer-mediated site-specific recombination in vitro.

The Xer site-specific recombination system acts at ColE1 cer and pSC101 psi sites to ensure that these plasmids are in a monomeric state prior to cell division. We show that four proteins, ArgR, PepA, XerC and XerD are necessary and sufficient for recombination between directly repeated cer sites on a supercoiled plasmid in vitro. Only PepA, XerC and XerD are required for recombination at psi in vitro. Recombination at cer and psi in vitro requires negative supercoiling and is exclusively intramolecular. Strand exchange at cer produces Holliday junction-containing products in which only the top strands have been exchanged. This reaction requires the catalytic tyrosine residue of Xer C but not that of XerD. Recombination at psi gives catenated circular resolution products. Strand exchange at psi is sequential. XerC catalyses the first (top) strand exchange to make a Holiday junction intermediate and XerD catalyses the second (bottom) strand exchange.     

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.     

Transposon-mediated site-specific recombination: A defined in vitro system

Transposition of the insertion element γδ is thought to involve formation of intermediates in which the element is present at each junction between donor and target replicons. In vivo these cointegrate structures are rapidly converted to the end products of transposition by site-specific recombination at a defined sequence, res, that is present in each directly repeated γδ element. Resolvase, an element encoded protein of molecular weight 21,000 is required for cointegrate resolution. I have demonstrated site-specific recombination in vitro using purified resolvase and a cointegrate analog substrate. The required components of the system described here are resolvase, negatively supercoiled substrate DNA, buffer and Mg²⁺. Neither host-encoded products nor high energy cofactors appear to be required for resolution in vitro. Catenated, resolved molecules are the major products of the reaction. Elimination of Mg²⁺ from the reaction yields different product molecules. The in vitro system described here provides an opportunity for detailed study of the resolution reaction.     

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.     

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.     

Cre-mediated somatic site-specific recombination in mice.

Conditional mutant mice equipped with heterologous recombination systems (Cre/lox or Flp/frt) are promising for studying tissue-specific gene function and for designing better models of human diseases. The utility of these mice depends on the cell target specificity, on the efficiency and on the control over timing of gene (in)activation. We have explored the utility of adenoviral vectors and transgenic mice expressing Cre under the control of tissue-specific promoters to achieve Cre/lox-mediated somatic recombination of the LacZ reporter gene, using a newly generated flox LacZ mouse strain. When adeno Cre viruses were administered via different routes, recombination and expression of LacZ was detected in a wide range of tissues. Whereas in liverbeta-galactosidase activity was quickly lost by turnover of expressing cells, even though the recombined allele was retained,beta-galactosidase in other tissues persisted for many months. Our data indicate that the flox LacZ transgenic line can be utilized effectively to monitor the level and functionality of Cre protein produced upon infection with adeno Cre virus or upon crossbreeding with different Cre transgenic lines.     

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.     

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.     

Highly efficient generation of recombinant baculoviruses by enzymatically medicated site-specific in vitro recombination.

We have used the Cre-lox system of bacteriophage P1 to develop a highly efficient in vitrosystem for construction of recombinant baculoviruses. A positive visual selection has been included to make identification of recombinant viral progeny rapid and straightforward. We report recombination frequencies as high as 5 x 10(7) recombinants/micrograms starting plasmid DNA and under certain conditions, up to 50% of the viral progeny are recombinants. Genes inserted into the baculovirus genome can be readily recovered in a simple one step process and re-inserted after manipulation if required. We have confirmed the structure of recovered plasmids by diagnostic restriction endonuclease digestion and the structure of recombinant viral genomes by Southern analysis. Possible uses and the significance of the system are discussed and experiments currently being done to improve it are described.     

Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site

Resolvase, the product of the tnpR gene of the transposable element gamma delta, mediates a site-specific recombination between two copies of the element directly repeated on the same replicon. The resolution site, res, at which resolvase acts lies in the intercistronic region between the tnpA and tnpR genes. We have studied this site-specific recombination in vitro. In the absence of Mg2+, a resolvase-res complex is formed, which contains DNA molecules that have been cleaved at res. Our data suggest that in this complex resolvase is covalently attached to the 5' ends of the cleaved DNA, leaving free 3' hydroxyl groups. DNA cleavage is stimulated by the interaction of two res sites on the same substrate molecule and appears to be an intermediate step in normal res site recombination. We show that the DNA is cut within a region previously identified as containing the crossover point at the palindromic sequence 5'- (see formula in text) to generate 3' extensions of two bases.     

The Xer activation factor of TLCΦ expands the possibilities for Xer recombination

The chromosome dimer resolution machinery of bacteria is generally composed of two tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific site, dif. The reaction depends on a cell division protein, FtsK, which activates XerD by protein-protein interactions. The toxin-linked cryptic satellite phage (TLCΦ) of Vibrio cholerae, which participates in the emergence of cholera epidemic strains, carries a dif-like attachment site (attP). TLCΦ exploits the Xer machinery to integrate into the dif site of its host chromosomes. The TLCΦ integration reaction escapes the control of FtsK because TLCΦ encodes for its own XerD-activation factor, XafT. Additionally, TLCΦ attP is a poor substrate for XerD binding, in apparent contradiction with the high integration efficiency of the phage. Here, we present a sequencing-based methodology to analyse the integration and excision efficiency of thousands of synthetic mini-TLCΦ plasmids with differing attP sites in vivo. This methodology is applicable to the fine-grained analyses of DNA transactions on a wider scale. In addition, we compared the efficiency with which XafT and the XerD-activation domain of FtsK drive recombination reactions in vitro. Our results suggest that XafT not only activates XerD-catalysis but also helps form and/or stabilize synaptic complexes between imperfect Xer recombination sites.