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1.
Site-specific recombinases are the enzymes that catalyze site-specific recombination between two specific DNA sequences to
mediate DNA integration, excision, resolution, or inversion and that play a pivotal role in the life cycles of many microorganisms
including bacteria and bacteriophages. These enzymes are classified as tyrosine-type or serine-type recombinases based on
whether a tyrosine or serine residue mediates catalysis. All known tyrosine-type recombinases catalyze the formation of a
Holliday junction intermediate, whereas the catalytic mechanism of all known serine-type recombinases includes the 180° rotation
and rejoining of cleaved substrate DNAs. Both recombinase families are further subdivided into two families; the tyrosine-type
recombinases are subdivided by the recombination directionality, and the serine-type recombinases are subdivided by the protein
size. Over more than two decades, many different site-specific recombinases have been applied to in vivo genome engineering,
and some of them have been used successfully to mediate integration, deletion, or inversion in a wide variety of heterologous
genomes, including those from bacteria to higher eukaryotes. Here, we review the recombination mechanisms of the best characterized
recombinases in each site-specific recombinase family and recent advances in the application of these recombinases to genomic
manipulation, especially manipulations involving site-specific gene integration into heterologous genomes. 相似文献
2.
The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination. Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or ΦC31 integrase) are usually 30–50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes. 相似文献
3.
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. 相似文献
4.
Most site-specific recombinases can be grouped into two structurally and mechanistically different classes. Whereas recombination by tyrosine recombinases proceeds with little movements by the proteins, serine recombinases exchange DNA strands by a mechanism requiring large quaternary rearrangements. Here we use site-directed crosslinking to investigate the conformational changes that accompany the formation of the synaptic complex and the exchange of DNA strands by the Hin serine recombinase. Efficient crosslinking between residues corresponding to the ‘D-helix’ region provides the first experimental evidence for interactions between synapsed subunits within this region and distinguishes between different tetrameric conformers that have been observed in crystal structures of related serine recombinases. Crosslinking profiles between cysteines introduced over the 35 residue E-helix region that constitutes most of the proposed rotating interface both support the long helical structure of the region and provide strong experimental support for a subunit rotation mechanism that mediates DNA exchange. 相似文献
5.
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. 相似文献
6.
Construction of genetically isogenic strains of mycobacteria is complicated by poor recombination rates and the lack of generalized transducing phages for Mycobacterium tuberculosis . We report here a powerful method for introducing single point mutations into mycobacterial genomes using oligonucleotide-derived single-stranded DNA recombineering and mycobacteriophage-encoded proteins. Phage Che9c gp61-mediated recombination is sufficiently efficient that single base changes can be introduced without requirement for direct selection, with isogenic mutant strains identified simply by PCR. Efficient recombination requires only short (50 nucleotide) oligonucleotides, but there is an unusually strong strand bias and an oligonucleotide targeting lagging strand DNA synthesis can recombine more than 10 000-fold efficiently than its complementary oligonucleotide. This ssDNA recombineering provides a simple assay for comparing the activities of related phage recombinases, and we find that both Escherichia coli RecET and phage λ Red recombination proteins function inefficiently in mycobacteria, illustrating the utility of developing recombineering in new bacterial systems using host-specific bacteriophage recombinases. ssDNA mycobacterial recombineering provides a simple approach to characterizing antimycobacterial drug targets, and we have constructed and characterized single point mutations that confer resistance to isoniazid, rifampicin, ofloxacin and streptomycin. 相似文献
7.
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. 相似文献
8.
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. 相似文献
9.
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. 相似文献
10.
Summary: 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. 相似文献
11.
Toxigenic conversion of Vibrio cholerae bacteria results from the integration of a filamentous phage, CTXϕ. Integration is driven by the bacterial Xer recombinases, which catalyse the exchange of a single pair of strands between the phage single-stranded DNA and the host double-stranded DNA genomes; replication is thought to convert the resulting pseudo-Holliday junction (HJ) intermediate into the final recombination product. The natural tendency of the Xer recombinases to recycle HJ intermediates back into substrate should thwart this integration strategy, which prompted a search for additional co-factors aiding directionality of the process. Here, we show that Endo III, a ubiquitous base excision repair enzyme, facilitates CTXϕ-integration in vivo. In vitro, we show that it prevents futile Xer recombination cycles by impeding new rounds of strand exchanges once the pseudo-HJ is formed. We further demonstrate that this activity relies on the unexpected ability of Endo III to bind to HJs even in the absence of the recombinases. These results explain how tandem copies of the phage genome can be created, which is crucial for subsequent virion production. 相似文献
12.
Site-specific recombinases (SSRs) are valuable tools for genetic engineering due to their ability to manipulate DNA in a highly specific manner. Engineered zinc-finger and TAL effector recombinases, in particular, are two classes of SSRs composed of custom-designed DNA-binding domains fused to a catalytic domain derived from the resolvase/invertase family of serine recombinases. While TAL effector and zinc-finger proteins can be assembled to recognize a wide range of possible DNA sequences, recombinase catalytic specificity has been constrained by inherent base requirements present within each enzyme. In order to further expand the targeted recombinase repertoire, we used a genetic screen to isolate enhanced mutants of the Bin and Tn21 recombinases that recognize target sites outside the scope of other engineered recombinases. We determined the specific base requirements for recombination by these enzymes and demonstrate their potential for genome engineering by selecting for variants capable of specifically recombining target sites present in the human CCR5 gene and the AAVS1 safe harbor locus. Taken together, these findings demonstrate that complementing functional characterization with protein engineering is a potentially powerful approach for generating recombinases with expanded targeting capabilities. 相似文献
13.
The tyrosine family of recombinases produces two smaller DNA circles when acting on circular DNA harboring two recombination sites in head-to-tail orientation. If the substrate is supercoiled, these circles can be unlinked or form multiply linked catenanes. The topological complexity of the products varies strongly even for similar recombination systems. This dependence has been solved here. Our computer simulation of the synapsis showed that the bend angles, phi, created in isolated recombination sites by protein binding before assembly of the full complex, determine the product topology. To verify the validity of this theoretical finding we measured the values of phi for Cre/loxP and Flp/FRT systems. The measurement was based on cyclization of the protein-bound short DNA fragments in solution. Despite the striking similarity of the synapses for these recombinases, action of Cre on head-to-tail target sites produces mainly unlinked circles, while that of Flp yields multiply linked catenanes. In full agreement with theoretical expectations we found that the values of phi for these systems are very different, close to 35 degrees and 80 degrees, respectively. Our findings have general implications in how small protein machines acting locally on large DNA molecules exploit statistical properties of their substrates to bring about directed global changes in topology. 相似文献
14.
Nucleoprotein filaments made up of Rad51 or Dmc1 recombinases, the core structures of recombination, engage in ATP-dependent DNA-strand exchange. The ability of recombinases to form filaments is enhanced by recombination factors termed 'mediators'. Here, we show that the Schizosaccharomyces pombe Swi5-Sfr1 complex, a conserved eukaryotic protein complex, at substoichiometric concentrations stimulates strand exchange mediated by Rhp51 (the S. pombe Rad51 homolog) and Dmc1 on long DNA substrates. Reactions mediated by both recombinases are completely dependent on Swi5-Sfr1, replication protein A (RPA) and ATP, although RPA inhibits the reaction when it is incubated with single-stranded DNA (ssDNA) before the recombinase. The Swi5-Sfr1 complex overcomes, at least partly, the inhibitory effect of RPA, representing a novel class of mediator. Notably, the Swi5-Sfr1 complex preferentially stimulates the ssDNA-dependent ATPase activity of Rhp51, and it increases the amounts of Dmc1 bound to ssDNA. 相似文献
15.
The conjugative transposon Tn916 encodes a protein called INT(Tn916) which, based on DNA sequence comparisons, is a member of the integrase family of site-specific recombinases. Integrase proteins such as INT(lambda), FLP, and XERC/D that promote site-specific recombination use characteristic, conserved amino acid residues to catalyze the cleavage and ligation of DNA substrates during recombination. The reaction proceeds by a two-step transesterification reaction requiring the formation of a covalent protein-DNA intermediate. Different requirements for homology between recombining DNA sites during integrase-mediated site-specific recombination and Tn916 transposition suggest that INT(Tn916) may use a reaction mechanism different from that used by other integrase recombinases. We show that purified INT(Tn916) mediates specific cleavage of duplex DNA substrates containing the Tn916 transposon ends and adjacent bacterial sequences. Staggered cleavages occur at both ends of the transposon, resulting in 5' hydroxyl protruding ends containing coupling sequences. These are sequences that are transferred with the transposon from donor to recipient during conjugative transposition. The nature of the cleavage products suggests that a covalent protein-DNA linkage occurs via a residue of INT(Tn916) and the 3'-phosphate group of the DNA. INT(Tn916) alone is capable of executing the strand cleavage step required for recombination during Tn916 transposition, and this reaction probably occurs by a mechanism similar to that of other integrase family site-specific recombinases. 相似文献
16.
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. 相似文献
17.
In Escherichia coli, the ATP-dependent DNA translocase FtsK transports DNA across the site of cell division and activates recombination by the XerCD recombinases at a specific site on the chromosome, dif, to ensure the last stages of chromosome segregation. DNA transport by FtsK is oriented by 8-base-pair asymmetric sequences ('KOPS'). Here we provide evidence that KOPS promote FtsK loading on DNA and that translocation is oriented at this step. 相似文献
18.
Integrons are genetic elements capable of integrating genes by a site-specific recombination system catalyzed by an integrase. Integron integrases are members of the tyrosine recombinase family and possess the four invariant residues (RHRY) and conserved motifs (boxes I and II and patches I, II, and III). An alignment of integron integrases compared to other tyrosine recombinases shows an additional group of residues around the patch III motif. We have analyzed the DNA binding and recombination properties of class I integron integrase (IntI1) variants carrying mutations at residues that are well conserved among all tyrosine recombinases and at some residues from the additional motif that are conserved among the integron integrases. The well-conserved residues studied were H277 from the conserved tetrad RHRY (about 90% conserved), E121 found in the patch I motif (about 80% conserved in prokaryotic recombinases), K171 from the patch II motif (near 100% conserved), W229 and F233 from the patch III motif, and G302 of box II (about 80% conserved in prokaryotic recombinases). Additional IntI1 mutated residues were K219 and a deletion of the sequence ALER215. We observed that E121, K171, and G302 play a role in the recombination activity but can be mutated without disturbing binding to DNA. W229, F233, and the conserved histidine (H277) may be implicated in protein folding or DNA binding. Some of the extra residues of IntI1 seem to play a role in DNA binding (K219) while others are implicated in the recombination activity (ALER215 deletion). 相似文献
19.
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. 相似文献
20.
Efficient methods of stacking genes into plant genomes are needed to expedite transfer of multigenic traits to crop varieties of diverse ecosystems. Over two decades of research has identified several DNA recombinases that carryout efficient cis and trans recombination between the recombination sites artificially introduced into the plant chromosome. The specificity and efficiency of recombinases make them extremely attractive for genome engineering. In plant biotechnology, recombinases have mostly been used for removing selectable marker genes and have rarely been extended to more complex applications. The reversibility of recombination, a property of the tyrosine family of recombinases, does not lend itself to gene stacking approaches that involve rounds of transformation for integrating genes into the engineered sites. However, recent developments in the field of recombinases have overcome these challenges and paved the way for gene stacking. Some of the key advancements include the application of unidirectional recombination systems, modification of recombination sites and transgene site modifications to allow repeated site‐specific integrations into the selected site. Gene stacking is relevant to agriculturally important crops, many of which are difficult to transform; therefore, development of high‐efficiency gene stacking systems will be important for its application on agronomically important crops, and their elite varieties. Recombinases, by virtue of their specificity and efficiency in plant cells, emerge as powerful tools for a variety of applications including gene stacking. 相似文献
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