Functional analysis of the C-terminal domains of the site-specific recombinases XerC and XerD

The tyrosine family site-specific recombinases XerC and XerD convert dimers of the Escherichia coli chromosome and many natural plasmids to monomers. The heterotetrameric recombination complex contains two molecules of XerC and two of XerD, with each recombinase mediating one pair of DNA strand exchanges. The two pairs of strand exchanges are separated in time and space. This demands that the catalytic activity of the four recombinase molecules be controlled so that only XerC or XerD is active at any given time, there being a switch in the recombinase activity state at the Holliday junction intermediate stage. Here, we analyse chimeras and deletion variants within the recombinase C-terminal domains in order to probe determinants that may be specific to either XerC or XerD, and to further understand how XerC-XerD interactions control catalysis in a recombining heterotetramer. The data confirm that the C-terminal "end" region of each recombinase plays an important role in coordinating catalysis within the XerCD heterotetramer and suggest that the interactions between the end regions of XerC and XerD and their cognate receptors within the partner recombinase are structurally and functionally different. The results support the hypothesis that the "normal" state in the heterotetrameric complex, in which XerC is catalytically active and XerD is inactive, depends on the interactions between the C-terminal end region of XerC and its receptor region within the C-terminal domain of XerD; interference with these interactions leads to a switch in the catalytic state, so that XerD is now preferentially active.     

Action of site-specific recombinases XerC and XerD on tethered Holliday junctions.

In Xer site-specific recombination, two related recombinases, XerC and XerD, mediate the formation of recombinant products using Holliday junction-containing DNA molecules as reaction intermediates. Each recombinase catalyses the exchange of one pair of specific strands. By using synthetic Holliday junction-containing recombination substrates in which two of the four arms are tethered in an antiparallel configuration by a nine thymine oligonucleotide, we show that XerD catalyses efficient strand exchange only when its substrate strands are 'crossed'. XerC also catalyses very efficient strand exchange when its substrate strands are 'crossed', though it also appears to be able to mediate strand exchange when its substrate strands are 'continuous'. By using chemical probes of Holliday junction structure in the presence and absence of bound recombinases, we show that recombinase binding induces unstacking of the bases in the centre of the recombination site, indicating that the junction branch point is positioned there and is distorted as a consequence of recombinase binding.     

Interactions of the site-specific recombinases XerC and XerD with the recombination site dif.

The Xer site-specific recombination system of Escherichia coli is involved in the stable inheritance of circular replicons. Multimeric replicons, produced by homologous recombination, are converted to monomers by the action of two related recombinases XerC and XerD. Site-specific recombination at a locus, dif, within the chromosomal replication terminus region is thought to convert dimeric chromosomes to monomers, which can then be segregated prior to cell division. The recombinases XerC and XerD bind cooperatively to dif, where they catalyse recombination. Chemical modification of specific bases and the phosphate-sugar backbone within dif was used to investigate the requirements for binding of the recombinases. Site-directed mutagenesis was then used to alter bases implicated in recombinase binding. Characterization of these mutants by in vitro recombinase binding and in vivo recombination, has demonstrated that the cooperative interactions between XerC and XerD can partially overcome DNA alterations that should interfere with specific recombinase-dif interactions.     

Reciprocal control of catalysis by the tyrosine recombinases XerC and XerD: an enzymatic switch in site-specific recombination

In Xer site-specific recombination, sequential DNA strand exchange reactions are catalyzed by a heterotetrameric complex composed of two recombinases, XerC and XerD. It is demonstrated that XerC and XerD catalytic activity is controlled by an interaction involving the C-terminal end of each protein (the donor region) and an internal region close to the active site (the acceptor region). Mutations in these regions reciprocally alter the relative activity of XerC and XerD, with their combination producing synergistic effects on catalysis. The data support a model in which C-terminal intersubunit interactions contribute to coupled protein-DNA conformational changes that lead to sequential activation and reciprocal inhibition of pairs of active sites in the recombinase tetramer during recombination.     

Detection of XerC and XerD recombinases in gram-negative bacteria of the family Enterobacteriaceae.

XerC and XerD are site-specific recombinases of the lambda integrase family which resolve multimeric replicons to monomers by acting at specific sites such as cer, ckr, nmr, parB, and psi, which are found in plasmids, or at the dif site found in the Escherichia coli chromosome. By using Southern hybridizations to cloned E. coli xerC and xerD genes and a cer-nmr plasmid-based resolution assay, the presence of these genes in several species of Enterobacteriaceae is shown.     

Sequential strand exchange by XerC and XerD during site-specific recombination at dif

Successful segregation of circular chromosomes in Escherichia coli requires that dimeric replicons, produced by homologous recombination, are converted to monomers prior to cell division. The Xer site-specific recombination system uses two related tyrosine recombinases, XerC and XerD, to catalyze resolution of circular dimers at the chromosomal site, dif. A 33-base pair DNA fragment containing the 28-base pair minimal dif site is sufficient for the recombinases to mediate both inter- and intramolecular site-specific recombination in vivo. We show that Xer-mediated intermolecular recombination in vitro between nicked linear dif "suicide" substrates and supercoiled plasmid DNA containing dif is initiated by XerC. Furthermore, on the appropriate substrate, the nicked Holliday junction intermediate formed by XerC is converted to a linear product by a subsequent single XerD-mediated strand exchange. We also demonstrate that a XerC homologue from Pseudomonas aeruginosa stimulates strand cleavage by XerD on a nicked linear substrate and promotes initiation of strand exchange by XerD in an intermolecular reaction between linear and supercoiled DNA, thereby reversing the normal order of strand exchanges.     

A dicentric bacterial chromosome requires XerC/D site-specific recombinases for resolution

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FtsK Is a DNA motor protein that activates chromosome dimer resolution by switching the catalytic state of the XerC and XerD recombinases

FtsK acts at the bacterial division septum to couple chromosome segregation with cell division. We demonstrate that a truncated FtsK derivative, FtsK(50C), uses ATP hydrolysis to translocate along duplex DNA as a multimer in vitro, consistent with FtsK having an in vivo role in pumping DNA through the closing division septum. FtsK(50C) also promotes a complete Xer recombination reaction between dif sites by switching the state of activity of the XerCD recombinases so that XerD makes the first pair of strand exchanges to form Holliday junctions that are then resolved by XerC. The reaction between directly repeated dif sites in circular DNA leads to the formation of uncatenated circles and is equivalent to the formation of chromosome monomers from dimers.     

Coordinated control of XerC and XerD catalytic activities during Holliday junction resolution

Site-specific recombinases XerC and XerD function in the segregation of circular bacterial replicons. In a recombining nucleoprotein complex containing two molecules each of XerC and XerD, coordinated reciprocal switches in recombinase activity ensure that only XerC or XerD is active at any one time. Mutated recombinases that carry sub?stitutions of a catalytic arginine residue stimulate cleavage and strand exchange mediated by the partner recombinase on DNA substrates that are normally recombined poorly by the partner. This is associated with a reciprocal impairment of the recombinase's own ability to initiate catalysis. The extent of this switch in catalysis is modulated by changes in recombination site sequence and is not a direct consequence of any catalytic defect. We propose that altered interactions between the mutated proteins and their wild-type partners lead to an increased level of an alternative Holliday junction intermediate that has a conformation appropriate for resolution by the partner recombinase. The results indicate how subtle changes in protein-DNA architecture at a Holliday junction can redirect recombination outcome.     

Catalysis by site-specific recombinases

Site-specific recombination reactions bring about controlled rearrangements of DNA molecules by cutting the DNA at precise points and rejoining the ends to new partners. The recombinases that catalyse these reactions can be grouped into two families by amino acid sequence homology. We describe our current understanding of how these proteins catalyse recombination, and show how the catalytic mechanisms of the two families differ.     

Genome engineering using site-specific recombinases

The targeted modification of the mammalian genome has a variety of applications in research, medicine, and biotechnology. Site-specific recombinases have become significant tools in all of these areas. Conditional gene targeting using site-specific recombinases has enabled the functional analysis of genes, which cannot be inactivated in the germline. The site-specific integration of adeno-associated virus, a major gene therapy vehicle, relies on the recombinase activity of the viral rep proteins. Site-specific recombinases also allow the precise integration of open reading frames encoding pharmaceutically relevant proteins into highly active gene loci in cell lines and transgenic animals. These goals have been accomplished by using a variety of genetic strategies but only a few recombinase proteins. However, the vast repertoire of recombinases, which has recently become available as a result of large-scale sequencing projects, may provide a rich source for the development of novel strategies to precisely alter mammalian genomes.     

Crystal structure of the site-specific recombinase, XerD.

The structure of the site-specific recombinase, XerD, that functions in circular chromosome separation, has been solved at 2.5 A resolution and reveals that the protein comprises two domains. The C-terminal domain contains two conserved sequence motifs that are located in similar positions in the structures of XerD, lambda and HP1 integrases. However, the extreme C-terminal regions of the three proteins, containing the active site tyrosine, are very different. In XerD, the arrangement of active site residues supports a cis cleavage mechanism. Biochemical evidence for DNA bending is encompassed in a model that accommodates extensive biochemical and genetic data, and in which the DNA is wrapped around an alpha-helix in a manner similar to that observed for CAP complexed with DNA.     

Genome engineering in actinomycetes using site-specific recombinases

The rational modification of the actinomycetes genomes has a variety of applications in research, medicine, and biotechnology. The use of site-specific recombinases allows generation of multiple mutations, large DNA deletions, integrations, and inversions and may lead to significant progress in all of these fields. Despite their huge potential, site-specific recombinase-based technologies have primarily been used for simple marker removal from a chromosome. In this review, we summarise the site-specific recombination approaches for genome engineering in various actinomycetes.     

The XerC recombinase of Proteus mirabilis: characterization and interaction with other tyrosine recombinases

XerC and XerD are two site-specific recombinases, which act on different sites to maintain replicons in a monomeric state. This system, which was first discovered and studied in Escherichia coli, is present in several species including Proteus mirabilis, where the XerD recombinase was previously characterized by our laboratory. In this paper, we report the presence of the xerC gene in P. mirabilis. Using in vitro reactions, we show that the two P. mirabilis recombinases display binding and cleavage activity on the E. coli dif site and the ColE1 cer site, together or in collaboration with E. coli recombinases. In vivo, P. mirabilis XerC and XerD are able to resolve and monomerize a plasmid containing two cer sites, increasing its stability. However, P. mirabilis XerC, in combination with E. coli XerD, is unable to perform these functions.     

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.     

Topoisomerases and site-specific recombinases: similarities in structure and mechanism

The processes of DNA topoisomerization and site-specific recombination are fundamentally similar: DNA cleavage by forming a phospho-protein covalent linkage, DNA topological rearrangement, and DNA ligation coupled with protein regeneration. Type IB DNA topoisomerases are structurally and mechanistically homologous to tyrosine recombinases. Both enzymes nick DNA double helices independent of metal ions, form 3′-phosphotyrosine intermediates, and rearrange the free 5′ ends relative to the uncut strands by swiveling. In contrast, serine recombinases generate 5′-phospho-serine intermediates. A 180° relative rotation of the two halves of a 100?kDa terameric serine recombinase and DNA complex has been proposed as the mechanism of strand exchange. Here I propose an alternative mechanism. Interestingly, the catalytic domain of serine recombinases has structural similarity to the TOPRIM domain, conserved among all Type IA and Type II topoisomerases and responsible for metal binding and DNA cleavage. TOPRIM topoisomerases also cleave DNA to generate 5′-phosphate and 3′-OH groups. Based on the existing biochemical data and crystal structures of topoisomerase II and serine recombinases bound to pre- and post-cleavage DNA, I suggest a strand passage mechanism for DNA recombination by serine recombinases. This mechanism is reminiscent of DNA topoisomerization and does not require subunit rotation.     

Topoisomerases and site-specific recombinases: similarities in structure and mechanism

The processes of DNA topoisomerization and site-specific recombination are fundamentally similar: DNA cleavage by forming a phospho-protein covalent linkage, DNA topological rearrangement, and DNA ligation coupled with protein regeneration. Type IB DNA topoisomerases are structurally and mechanistically homologous to tyrosine recombinases. Both enzymes nick DNA double helices independent of metal ions, form 3'-phosphotyrosine intermediates, and rearrange the free 5' ends relative to the uncut strands by swiveling. In contrast, serine recombinases generate 5'-phospho-serine intermediates. A 180° relative rotation of the two halves of a 100 kDa terameric serine recombinase and DNA complex has been proposed as the mechanism of strand exchange. Here I propose an alternative mechanism. Interestingly, the catalytic domain of serine recombinases has structural similarity to the TOPRIM domain, conserved among all Type IA and Type II topoisomerases and responsible for metal binding and DNA cleavage. TOPRIM topoisomerases also cleave DNA to generate 5'-phosphate and 3'-OH groups. Based on the existing biochemical data and crystal structures of topoisomerase II and serine recombinases bound to pre- and post-cleavage DNA, I suggest a strand passage mechanism for DNA recombination by serine recombinases. This mechanism is reminiscent of DNA topoisomerization and does not require subunit rotation.     

Pairing of single mutations yields obligate Cre-type site-specific recombinases

Tyrosine site-specific recombinases (SSRs) represent a versatile genome editing tool with considerable therapeutic potential. Recent developments to engineer and evolve SSRs into heterotetramers to improve target site flexibility signified a critical step towards their broad utility in genome editing. However, SSR monomers can form combinations of different homo- and heterotetramers in cells, increasing their off-target potential. Here, we discover that two paired mutations targeting residues implicated in catalysis lead to simple obligate tyrosine SSR systems, where the presence of all distinct subunits to bind as a heterotetramer is obligatory for catalysis. Therefore, only when the paired mutations are applied as single mutations on each recombinase subunit, the engineered SSRs can efficiently recombine the intended target sequence, while the subunits carrying the point mutations expressed in isolation are inactive. We demonstrate the utility of the obligate SSR system to improve recombination specificity of a designer-recombinase for a therapeutic target in human cells. Furthermore, we show that the mutations render the naturally occurring SSRs, Cre and Vika, obligately heteromeric for catalytic proficiency, providing a straight-forward approach to improve their applied properties. These results facilitate the development of safe and effective therapeutic designer-recombinases and advance our mechanistic understanding of SSR catalysis.     

Genetic characterization of gram-positive homologs of the XerCD site-specific recombinases

Homologs of the XerCD enzymes, which in Escherichia coli have been shown to be responsible for resolving chromosomal multimers prior to chromosome segregation, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Phylogenetic and conservation pattern analysis suggests that the S. aureus gene products are orthologs of XerC and D. A S. aureus xerC null mutant displayed in vitro characteristics consistent with the segregation defect reported for E. coli xer mutants, and was found to be attenuated in a murine infection model. Strikingly, the S. aureus xerD gene appears to be absolutely required for viability, and may therefore be the first example of an essential gene of the lambda integrase family. In contrast, phylogenetic and conservation pattern analysis show that the S. pneumoniae gene products are more closely related to phage integrases than to XerCD. S. pneumoniae xer1, 2 and 3 null mutants were each found to be attenuated in a murine infection model, suggesting that they may control processes which affect virulence.