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David J. Sherratt - One of the best experts on this subject based on the ideXlab platform.
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Coupled catalysis in a recombination machine: Mutated XerC and XerD Recombinases that stimulate strand exchange by their partner Recombinase
Journal of molecular biology, 2000Co-Authors: Lidia K. Arciszewska, Rachel Baker, Bernard Hallet, David J. SherrattAbstract:Abstract 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.
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Xer site-specific recombination. DNA strand rejoining by Recombinase XerC.
The Journal of biological chemistry, 1999Co-Authors: Ian Grainge, David J. SherrattAbstract: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.
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Action of site-specific Recombinases XerC and XerD on tethered Holliday junctions.
The EMBO journal, 1997Co-Authors: Lidia K. Arciszewska, Ian Grainge, David J. SherrattAbstract: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.
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Interactions of the site-specific Recombinases XerC and XerD with the recombination site dif
Nucleic acids research, 1994Co-Authors: G W Blakely, David J. SherrattAbstract: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.
Carlos F. Barbas - One of the best experts on this subject based on the ideXlab platform.
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Redesigning Recombinase Specificity for Safe Harbor Sites in the Human Genome
PloS one, 2015Co-Authors: Mark C. Wallen, Thomas Gaj, Carlos F. BarbasAbstract: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.
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Expanding the zinc-finger Recombinase repertoire: directed evolution and mutational analysis of serine Recombinase specificity determinants
Nucleic acids research, 2014Co-Authors: Shannon J. Sirk, Thomas Gaj, Andreas Jonsson, Andrew C. Mercer, Carlos F. BarbasAbstract:The serine Recombinases are a diverse family of modular enzymes that promote high-fidelity DNA rearrangements between specific target sites. Replacement of their native DNA-binding domains with custom-designed Cys2–His2 zinc-finger proteins results in the creation of engineered zinc-finger Recombinases (ZFRs) capable of achieving targeted genetic modifications. The flexibility afforded by zinc-finger domains enables the design of hybrid Recombinases that recognize a wide variety of potential target sites; however, this technology remains constrained by the strict recognition specificities imposed by the ZFR catalytic domains. In particular, the ability to fully reprogram serine Recombinase catalytic specificity has been impeded by conserved base requirements within each Recombinase target site and an incomplete understanding of the factors governing DNA recognition. Here we describe an approach to complement the targeting capacity of ZFRs. Using directed evolution, we isolated mutants of the β and Sin Recombinases that specifically recognize target sites previously outside the scope of ZFRs. Additionally, we developed a genetic screen to determine the specific base requirements for site-specific recombination and showed that specificity profiling enables the discovery of unique genomic ZFR substrates. Finally, we conducted an extensive and family-wide mutational analysis of the serine Recombinase DNA-binding arm region and uncovered a diverse network of residues that confer target specificity. These results demonstrate that the ZFR repertoire is extensible and highlights the potential of ZFRs as a class of flexible tools for targeted genome engineering.
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Targeted Plasmid Integration into the Human Genome by Engineered Recombinases
Site-directed insertion of transgenes, 2012Co-Authors: Charles A. Gersbach, Carlos F. BarbasAbstract:The targeted integration of transgenes into cellular genomes is central to numerous applications in biotechnology, basic science, and medicine. In recent years, a variety of advances have improved upon conventional methods for site-specific transgene integration. Most of these methods involve nucleases that cleave DNA to activate DNA repair pathways including homologous recombination or integrases that fully catalyze the integration reaction but are limited in their capacity to target new sites in the genome. Recently, zinc-finger Recombinases have emerged as a class of engineered enzymes that combines the strengths of both of these previous methods. Zinc-finger Recombinases can fully and autonomously catalyze plasmid integration into the genome of mammalian cells without creating free DNA breaks. In addition, they can be engineered to target new genomic recognition sites by exchanging the modular and programmable DNA-binding domain and through directed evolution of the serine Recombinase catalytic domain. This chapter reviews the development of the zinc-finger Recombinase technology, including discussions of its strengths and weaknesses and the future directions necessary to translate this technology into routine use for transgene integration into cellular genomes.
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Directed evolution of Recombinase specificity by split gene reassembly
Nucleic acids research, 2010Co-Authors: Charles A. Gersbach, Thomas Gaj, Russell M. Gordley, Carlos F. BarbasAbstract:The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific Recombinases with novel substrate specificities. In this system, Recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding β-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-Recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered Recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of Recombinases. This enhanced evolution system will translate Recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications.
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Evolution of programmable zinc finger-Recombinases with activity in human cells.
Journal of molecular biology, 2007Co-Authors: Russell M. Gordley, Justin D. Smith, Torbjörn Gräslund, Carlos F. BarbasAbstract:Site-specific Recombinases are important tools for genomic engineering in many living systems. Applications of Recombinases are, however, constrained by the DNA targeting endemic of the Recombinase used. A tremendous range of Recombinase applications can be envisioned if the targeting of Recombinase specificity can be made readily programmable. To address this problem we sought to generate zinc finger-Recombinase fusion proteins (RecZFs) capable of site-specific function in a diversity of genetic contexts. Our first RecZF, Tn3Ch15X2, recombined substrates derived from the native Tn3 resolvase recombination site. Substrate Linked Protein Evolution (SLiPE) was used to optimize the catalytic domains of the enzymes Hin, Gin, and Tn3 for resolution between non-homologous sites. One of the evolved clones, GinL7C7, catalyzed efficient, site-specific recombination in a variety of sequence contexts. When introduced into human cells by retroviral transduction, GinL7C7 excised a 1.4 kb EGFP cassette out of the genome, diminishing fluorescence in ∼17% of transduced cells. Following this template of rational design and directed evolution, RecZFs may eventually mediate gene therapies, facilitate the genetic manipulation of model organisms and cells, and mature into powerful new tools for molecular biology and medicine.
G. D. Van Duyne - One of the best experts on this subject based on the ideXlab platform.
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Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination.
The EMBO journal, 1998Co-Authors: D. N. Gopaul, Feng Guo, G. D. Van DuyneAbstract:We have determined the X‐ray crystal structures of two DNA Holliday junctions (HJs) bound by Cre Recombinase. The HJ is a four‐way branched structure that occurs as an intermediate in genetic recombination pathways, including site‐specific recombination by the λ‐integrase family. Cre Recombinase is an integrase family member that recombines 34 bp loxP sites in the absence of accessory proteins or auxiliary DNA sequences. The 2.7 A structure of Cre Recombinase bound to an immobile HJ and the 2.5 A structure of Cre Recombinase bound to a symmetric, nicked HJ reveal a nearly planar, twofold‐symmetric DNA intermediate that shares features with both the stacked‐X and the square conformations of the HJ that exist in the unbound state. The structures support a protein‐mediated crossover isomerization of the junction that acts as the switch responsible for activation and deactivation of Recombinase active sites. In this model, a subtle isomerization of the Cre Recombinase–HJ quaternary structure dictates which strands are cleaved during resolution of the junction via a mechanism that involves neither branch migration nor helical restacking.
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structure of cre Recombinase complexed with dna in a site specific recombination synapse
Nature, 1997Co-Authors: Feng Guo, D. N. Gopaul, G. D. Van DuyneAbstract:During site-specific DNA recombination, which brings about genetic rearrangement in processes such as viral integration and excision and chromosomal segregation, Recombinase enzymes recognize specific DNA sequences and catalyse the reciprocal exchange of DNA strands between these sites. The bacteriophage Recombinase Cre catalyses site-specific recombination between two 34-base-pair loxP sites. The crystal structure at 2.4 A resolution of Cre bound to a loxP substrate reveals an intermediate in the recombination reaction, in which a Cre molecule has cleaved the substrate to form a covalent 3'-phosphotyrosine linkage with the DNA. Four Recombinases and two loxP sites form a synapsed structure in which the DNA resembles models of four-way Holliday-Junction intermediates. The Cre-loxP complex challenges models of site-specific recombination that require large changes in quaternary structure. Subtle allosteric changes at the carboxy termini of the Cre subunits may instead coordinate the cleavage and strand-exchange reactions.
Jerome Bonnet - One of the best experts on this subject based on the ideXlab platform.
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Computational Methods for the Design of Recombinase Logic Circuits.
Methods in molecular biology (Clifton N.J.), 2020Co-Authors: Sarah Guiziou, Jerome BonnetAbstract:Synthetic biology aims at engineering new biological systems and functions that can be used to provide new technological solutions to worldwide challenges. Detection and processing of multiple signals are crucial for many synthetic biology applications. A variety of logic circuits operating in living cells have been implemented. One particular class of logic circuits uses site-specific Recombinases mediating specific DNA inversion or excision. Recombinase logic offers many interesting features, including single-layer architectures, memory, low metabolic footprint, and portability in many species. Here, we present two automated design strategies for Recombinase-based logic circuits, one based on the distribution of computation within a multicellular consortia and the other one being a single-cell design. The two design strategies are complementary and are both adapted for none expert as a design web-interface exits for each strategy, the CALIN and RECOMBINATOR web-interface for respectively the multicellular and single-cell design strategy. In this book chapter, we are guiding the reader step by step through Recombinase-logic circuit design from selecting the design strategy fitting to his/her final system of interest to obtaining the final design using one of our design web-interface.
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Hierarchical composition of reliable Recombinase logic devices
Nature communications, 2019Co-Authors: Sarah Guiziou, Pauline Mayonove, Jerome BonnetAbstract:A major goal of synthetic biology is to reprogram living organisms to solve pressing challenges in manufacturing, environmental remediation, and healthcare. Recombinase devices can efficiently encode complex logic in many species, yet current designs are performed on a case-by-case basis, limiting their scalability and requiring time-consuming optimization. Here we provide a systematic framework for engineering reliable Recombinase logic devices by hierarchical composition of well-characterized, optimized Recombinase switches. We apply this framework to build a Recombinase logic device family supporting up to 4-input Boolean logic within a multicellular system. This work enables straightforward implementation of multicellular Recombinase logic and will support the predictable engineering of several classes of Recombinase devices to reliably control cellular behavior.
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Hierarchical composition of reliable Recombinase logic devices.
2018Co-Authors: Sarah Guiziou, Pauline Mayonove, Jerome BonnetAbstract:We provide a systematic framework for engineering reliable Recombinase logic devices by hierarchical composition of well-characterized, optimized Recombinase switches. We apply this framework to build a Recombinase logic device family supporting up to 4-input Boolean logic. This work will support the predictable engineering of several classes of Recombinase devices to reliably control cellular behavior.
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An Automated Design Framework for Multicellular Recombinase Logic
ACS Synthetic Biology, 2018Co-Authors: Sarah Guiziou, Federico Ulliana, Violaine Moreau, Michel Leclère, Jerome BonnetAbstract:Tools to systematically reprogram cellular behavior are crucial to address pressing challenges in manufacturing, environment, or healthcare. Recombinases can very efficiently encode Boolean and history-dependent logic in many species, yet current designs are performed on a case-by-case basis, limiting their scalability and requiring time-consuming optimization. Here we present an automated workflow for designing Recombinase logic devices executing Boolean functions. Our theoretical framework uses a reduced library of computational devices distributed into different cellular subpopulations, which are then composed in various manners to implement all desired logic functions at the multicellular level. Our design platform called CALIN (Composable Asynchronous Logic using Integrase Networks) is broadly accessible via a web server, taking truth tables as inputs and providing corresponding DNA designs and sequences as outputs (available at http://synbio.cbs.cnrs.fr/calin). We anticipate that this automated design workflow will streamline the implementation of Boolean functions in many organisms and for various applications.
Yuri Voziyanov - One of the best experts on this subject based on the ideXlab platform.
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Genome targeting by hybrid Flp-TAL Recombinases.
Scientific reports, 2020Co-Authors: Eugenia Voziyanova, Riddhi Shah, Yuri VoziyanovAbstract:Genome engineering is a rapidly evolving field that benefits from the availability of different tools that can be used to perform genome manipulation tasks. We describe here the development of the Flp-TAL Recombinases that can target genomic FRT-like sequences in their native chromosomal locations. Flp-TAL Recombinases are hybrid enzymes that are composed of two functional modules: a variant of site-specific tyrosine Recombinase Flp, which can have either narrow or broad target specificity, and the DNA-binding domain of the transcription activator-like effector, TAL. In Flp-TAL, the TAL module is responsible for delivering and stabilizing the Flp module onto the desired genomic FRT-like sequence where the Flp module mediates recombination. We demonstrate the functionality of the Flp-TAL Recombinases by performing integration and deletion experiments in human HEK-293 cells. In the integration experiments we targeted a vector to three genomic FRT-like sequences located in the β-globin locus. In the deletion experiments we excised ~ 15 kilobases of DNA that contained a fragment of the integrated vector sequence and the neighboring genome sequence. On average, the efficiency of the integration and deletion reactions was about 0.1% and 20%, respectively.
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Dual Recombinase-Mediated Cassette Exchange by Tyrosine Site-Specific Recombinases.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Eugenia Voziyanova, Rachelle P. Anderson, Yuri VoziyanovAbstract:Recombinase-mediated cassette exchange, or RMCE, is a genome engineering tool that can be used to swap DNA fragments of interest between two DNA molecules. In a variation of RMCE, called dual RMCE, the exchange of DNA fragments is mediated by two Recombinases in contrast to one Recombinase in the classic RMCE reaction. Under optimal conditions, the efficiency of dual RMCE can be quite high: up to ~45% of the transfected cells depending on the Recombinase pair used to mediate the replacement reaction. Here we describe protocols for preparing for, performing, and optimizing the parameters of dual RMCE.
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Efficient Genome Manipulation by Variants of Site-Specific Recombinases R and TD.
Journal of Molecular Biology, 2016Co-Authors: Eugenia Voziyanova, Rachelle P. Anderson, Riddhi Shah, Yuri VoziyanovAbstract:Abstract Genome engineering benefits from the availability of DNA modifying enzymes that have different target specificities and have optimized performance in different cell types. This variety of site-specific enzymes can be used to develop complex genome engineering applications at multiple loci. Although eight yeast site-specific tyrosine Recombinases are known, only Flp is actively used in genome engineering. To expand the pool of the yeast site-specific tyrosine Recombinases capable of mediating genome manipulations in mammalian cells, we engineered and analyzed variants of two tyrosine Recombinases: R and TD. The activity of the evolved variants, unlike the activity of the native R and TD Recombinases, is suitable for genome engineering in Escherichia coli and mammalian cells. Unexpectedly, we found that R Recombinase benefits from the shortening of its C-terminus. We also found that the activity of wild-type R can be modulated by its non-consensus “head” sequence but this modulation became not apparent in the evolved R variants. The engineered Recombinase variants were found to be active in all recombination reactions tested: excision, integration, and dual Recombinase-mediated cassette exchange. The analysis of the latter reaction catalyzed by the R/TD Recombinase pair shows that the condition supporting the most efficient replacement reaction favors efficient TD-mediated integration reaction while favoring efficient R-mediated integration and deletion reactions.
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High efficiency of a sequential Recombinase-mediated cassette exchange reaction in Escherichia coli.
Journal of molecular microbiology and biotechnology, 2010Co-Authors: Natalia Malchin, Yuri Voziyanov, Tatiana Molotsky, Ilya Borovok, Alexander Kotlyar, Ezra Yagil, Mikhail KolotAbstract:A comparison between the efficiency of Recombinase-mediated cassette exchange (RMCE) reactions catalyzed in Escherichia coli by the site-specific Recombinases Flp of yeast and Int of coliphage HK022 has revealed that an Flp-catalyzed RMCE reaction is more efficient than an Int-HK022 catalyzed reaction. In contrast, an RMCE reaction with 1 pair of frt sites and 1 pair of att sites catalyzed in the presence of both Recombinases is very inefficient. However, the same reaction catalyzed by each Recombinase individually supplied in a sequential order is very efficient, regardless of the order. Atomic force microscopy images of Flp with its DNA substrates show that only 1 pair of recombination sites forms a synaptic complex with the Recombinase. The results suggest that the RMCE reaction is sequential.