RESUMEN
A key step in cut-and-paste DNA transposition is the pairing of transposon ends before the element is excised and inserted at a new site in its host genome. Crystallographic analyses of the paired-end complex (PEC) formed from precleaved transposon ends and the transposase of the eukaryotic element Mos1 reveals two parallel ends bound to a dimeric enzyme. The complex has a trans arrangement, with each transposon end recognized by the DNA binding region of one transposase monomer and by the active site of the other monomer. Two additional DNA duplexes in the crystal indicate likely binding sites for flanking DNA. Biochemical data provide support for a model of the target capture complex and identify Arg186 to be critical for target binding. Mixing experiments indicate that a transposase dimer initiates first-strand cleavage and suggest a pathway for PEC formation.
Asunto(s)
Elementos Transponibles de ADN , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Transposasas/metabolismo , Animales , Dominio Catalítico , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Modelos Moleculares , Estructura Terciaria de Proteína , Transposasas/química , Difracción de Rayos XRESUMEN
The structure and diversity of all open microbial communities are shaped by individual births, deaths, speciation and immigration events; the precise timings of these events are unknowable and unpredictable. This randomness is manifest as ecological drift in the population dynamics, the importance of which has been a source of debate for decades. There are theoretical reasons to suppose that drift would be imperceptible in large microbial communities, but this is at odds with circumstantial evidence that effects can be seen even in huge, complex communities. To resolve this dichotomy we need to observe dynamics in simple systems where key parameters, like migration, birth and death rates can be directly measured. We monitored the dynamics in the abundance of two genetically modified strains of Escherichia coli, with tuneable growth characteristics, that were mixed and continually fed into 10 identical chemostats. We demonstrated that the effects of demographic (non-environmental) stochasticity are very apparent in the dynamics. However, they do not conform to the most parsimonious and commonly applied mathematical models, where each stochastic event is independent. For these simple models to reproduce the observed dynamics we need to invoke an 'effective community size', which is smaller than the census community size.
Asunto(s)
Microbiota , Escherichia coli/genética , Modelos Teóricos , Dinámica PoblacionalRESUMEN
A device that counts and records the number of events experienced by an individual cell could have many uses in experimental biology and biotechnology. Here, we report a DNA-based 'latch' that switches between two states upon each exposure to a repeated stimulus. The key component of the latch is a DNA segment whose orientation is inverted by the actions of ÏC31 integrase and its recombination directionality factor (RDF). Integrase expression is regulated by an external input, while RDF expression is controlled by the state of the latch, such that the orientation of the invertible segment switches efficiently each time the device receives an input pulse. Recombination occurs over a time scale of minutes after initiation of integrase expression. The latch requires a delay circuit, implemented with a transcriptional repressor expressed in only one state, to ensure that each input pulse results in only one inversion of the DNA segment. Development and optimization of the latch in living cells was driven by mathematical modelling of the recombination reactions and gene expression regulated by the switch. We discuss how N latches built with orthogonal site-specific recombination systems could be chained together to form a binary ripple counter that could count to 2N - 1.
Asunto(s)
ADN/genética , Integrasas/genética , Recombinación Genética , Proteínas Virales/química , Bacteriófagos/genética , ADN/química , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Integrasas/química , Serina/genética , Análisis de la Célula Individual , Proteínas Virales/genéticaRESUMEN
Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The 'reverse' reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL × attR recombination and inhibits attP × attB recombination. We show here that serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL × attR recombination in vivo and in vitro, whereas attP × attB recombination efficiency is reduced. We provide evidence that activation of attL × attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein. Minor changes in the length and sequence of the integrase-RDF linker peptide did not affect fusion protein recombination activity. The efficiency and single-protein convenience of integrase-RDF fusion proteins make them potentially very advantageous for biotechnology/synthetic biology applications. Here, we demonstrate efficient gene cassette replacement in a synthetic metabolic pathway gene array as a proof of principle.
Asunto(s)
Bacteriófagos/enzimología , Integrasas/metabolismo , Recombinación Genética , Serina/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Sitios de Ligazón Microbiológica/genética , Bacteriófagos/genética , Fusión Génica , Integrasas/genética , Modelos Genéticos , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina/genética , Proteínas Virales/genéticaRESUMEN
Serine integrases, DNA site-specific recombinases used by bacteriophages for integration and excision of their DNA to and from their host genomes, are increasingly being used as tools for programmed rearrangements of DNA molecules for biotechnology and synthetic biology. A useful feature of serine integrases is the simple regulation and unidirectionality of their reactions. Recombination between the phage attP and host attB sites is promoted by the serine integrase alone, giving recombinant attL and attR sites, whereas the 'reverse' reaction (between attL and attR) requires an additional protein, the recombination directionality factor (RDF). Here, we present new experimental data on the kinetics and regulation of recombination reactions mediated by ÏC31 integrase and its RDF, and use these data as the basis for a mathematical model of the reactions. The model accounts for the unidirectionality of the attP × attB and attL × attR reactions by hypothesizing the formation of structurally distinct, kinetically stable integrase-DNA product complexes, dependent on the presence or absence of RDF. The model accounts for all the available experimental data, and predicts how mutations of the proteins or alterations of reaction conditions might increase the conversion efficiency of recombination.
Asunto(s)
Sitios de Ligazón Microbiológica/genética , Simulación por Computador , ADN/genética , ADN/metabolismo , Integrasas/química , Integrasas/metabolismo , Recombinación Genética , Bioensayo , Factores Biológicos/metabolismo , Estabilidad de Enzimas , Cinética , Modelos Biológicos , Plásmidos/genética , Plásmidos/metabolismo , Termodinámica , Proteínas Virales/metabolismoRESUMEN
Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.
Asunto(s)
Integrasas/metabolismo , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Recombinación Genética , Bacteriófagos/enzimología , Vías Biosintéticas/genética , Clonación Molecular/métodos , Orden Génico , Ribosomas/metabolismo , Biología Sintética/métodosRESUMEN
Integrases, such as that of the Streptomyces temperate bacteriophage ÏC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ÏC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ÏC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a ÏC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive "360° rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.
Asunto(s)
Bacteriófagos/enzimología , Integrasas/metabolismo , Recombinación Genética , Bacteriófagos/genética , ADN Viral/genética , Integrasas/genéticaRESUMEN
Xer site-specific recombination at cer and psi converts bacterial plasmid multimers into monomers so that they can be efficiently segregated to both daughter cells at cell division. Recombination is catalysed by the XerC and XerD recombinases acting at ~30 bp core sites, and is regulated by the action of accessory proteins bound to accessory DNA sequences adjacent to the core sites. Recombination normally occurs only between sites in direct repeat in a negatively supercoiled circular DNA molecule, and yields two circular products linked together in a right-handed four-node catenane with antiparallel sites. These and other topological results are explained by a model in which the accessory DNA sequences are interwrapped around the accessory proteins, trapping three negative supercoils so that strand exchange by the XerC and XerD yields the observed four-node catenane.
Asunto(s)
ADN Nucleotidiltransferasas/metabolismo , Conformación de Ácido Nucleico , Plásmidos/metabolismo , Recombinación Genética , Modelos MolecularesRESUMEN
BACKGROUND: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro. METHODS AND MAJOR RESULTS: In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ÏR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the ß-carotenoid pathway in Escherichia coli, using six different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up "poly-part" gene assembly and editing. CONCLUSIONS AND IMPLICATIONS: We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications.
Asunto(s)
Bacteriófagos , Integrasas , Integrasas/genética , Serina/metabolismo , ADN/genética , Plásmidos/genética , Bacteriófagos/genética , Bacteriófagos/metabolismoRESUMEN
Transposons of the Tc1/mariner family have been used to integrate foreign DNA stably into the genome of a large variety of different cell types and organisms. Integration is at TA dinucleotides located essentially at random throughout the genome, potentially leading to insertional mutagenesis, inappropriate activation of nearby genes, or poor expression of the transgene. Here, we show that fusion of the zinc-finger DNA-binding domain of Zif268 to the C-terminus of ISY100 transposase leads to highly specific integration into TA dinucleotides positioned 6-17 bp to one side of a Zif268 binding site. We show that the specificity of targeting can be changed using Zif268 variants that bind to sequences from the HIV-1 promoter, and demonstrate a bacterial genetic screen that can be used to select for increased levels of targeted transposition. A TA dinucleotide flanked by two Zif268 binding sites was efficiently targeted by our transposase-Zif268 fusion, suggesting the possibility of designer 'Z-transposases' that could deliver transgenic cargoes to chosen genomic locations.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Marcación de Gen , Transposasas/metabolismo , Animales , Sitios de Unión , Proteína 1 de la Respuesta de Crecimiento Precoz/química , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Ratones , Plásmidos/genética , Proteínas Recombinantes de Fusión/metabolismo , Transposasas/genética , Dedos de ZincRESUMEN
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.
Asunto(s)
ADN Superhelicoidal/química , Plásmidos/química , Recombinación Genética , ADN Cruciforme/química , Dimerización , Presión Osmótica , Recombinasas/metabolismoRESUMEN
Decatenation is a crucial in vivo reaction of DNA topoisomerases in DNA replication and is frequently used in in vitro drug screening. Usually this reaction is monitored using kinetoplast DNA as a substrate, although this assay has several limitations. Here we have engineered a substrate for Tn3 resolvase that generates a singly-linked catenane that can readily be purified from the DNA substrate after restriction enzyme digestion and centrifugation. We show that this catenated substrate can be used with high sensitivity in topoisomerase assays and drug-inhibition assays.
Asunto(s)
ADN-Topoisomerasas/metabolismo , ADN Encadenado/metabolismo , Pruebas de Enzimas/métodos , Secuencia de Bases , Recombinación Genética/genética , Especificidad por Sustrato , Resolvasas de Transposones/metabolismoRESUMEN
Transposons are invaluable biological tools for the genetic manipulation of microorganisms. ISY100 from Synechocystis sp. PCC6803 is a member of the Tc1/mariner/IS630 superfamily, and is characterized by high transposition efficiency and a strong preference for TA target sequences. In this paper, we describe the design and application of a mini-ISY100 suicide vector for the in vivo creation of stable random transposon insertion libraries. The system was successfully applied in seven species belonging to four different orders of γ proteobacteria. In all cases, delivery using conjugation consistently showed the highest transposition efficiency compared to chemical transformation or electroporation. We determined the frequency of transposon insertions in all the species and proved the utility of the system by identifying genes involved in colony coloration in Shewanella oneidensis. The ease and the efficiency of the protocol developed here allow the creation of complete knock-out libraries in an extensive range of host microorganisms in less than a week with no requirement for preparatory modification.
RESUMEN
Dual-state genetic switches that can change their state in response to input signals can be used in synthetic biology to encode memory and control gene expression. A transcriptional toggle switch (TTS), with two mutually repressing transcription regulators, was previously used for switching between two expression states. In other studies, serine integrases have been used to control DNA inversion switches that can alternate between two different states. Both of these switches use two different inputs to switch ON or OFF. Here, we use mathematical modelling to design a robust one-input binary switch, which combines a TTS with a DNA inversion switch. This combined circuit switches between the two states every time it receives a pulse of a single-input signal. The robustness of the switch is based on the bistability of its TTS, while integrase recombination allows single-input control. Unidirectional integrase-RDF-mediated recombination is provided by a recently developed integrase-RDF fusion protein. We show that the switch is stable against parameter variations and molecular noise, making it a promising candidate for further use as a basic element of binary counting devices.
Asunto(s)
ADN/metabolismo , Integrasas/metabolismo , Modelos Genéticos , Recombinación Genética , Biología Sintética , Transcripción Genética , ADN/química , ADN/genética , Integrasas/química , Integrasas/genéticaRESUMEN
By placing loxP adjacent to the accessory sequences from the Xer/psi multimer resolution system, we have imposed topological selectivity and specificity on Cre/loxP recombination. In this hybrid recombination system, the Xer accessory protein PepA binds to psi accessory sequences, interwraps them, and brings the loxP sites together such that the product of recombination is a four-node catenane. Here, we investigate communication between PepA and Cre by varying the distance between loxP and the accessory sequences, and by altering the orientation of loxP. The yield of four-node catenane and the efficiency of recombination in the presence of PepA varied with the helical phase of loxP with respect to the accessory sequences. When the orientation of loxP was reversed, or when half a helical turn was added between the accessory sequences and loxP, PepA reversed the preferred order of strand exchange by Cre at loxP. The results imply that PepA and the accessory sequences define precisely the geometry of the synapse formed by the loxP sites, and that this overcomes the innate preference of Cre to initiate recombination on the bottom strand of loxP. Further analysis of our results demonstrates that PepA can stimulate strand exchange by Cre in two distinct synaptic complexes, with the C-terminal domains of Cre facing either towards or away from PepA. Thus, no specific PepA-recombinase interaction is required, and correct juxtaposition of the loxP sites is sufficient to activate Cre in this system.
Asunto(s)
Integrasas/metabolismo , Plásmidos , Recombinación Genética , Proteínas Virales/metabolismo , Animales , Secuencia de Bases , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Etidio/metabolismo , Indicadores y Reactivos/metabolismo , Integrasas/genética , Modelos Genéticos , Conformación de Ácido Nucleico , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Virales/genéticaRESUMEN
Serine integrases catalyse site-specific recombination to integrate and excise bacteriophage genomes into and out of their host's genome. These enzymes exhibit remarkable directionality; in the presence of the integrase alone, recombination between attP and attB DNA sites is efficient and irreversible, giving attL and attR products which do not recombine further. However, in the presence of the bacteriophage-encoded recombination directionality factor (RDF), integrase efficiently promotes recombination between attL and attR to re-form attP and attB The DNA substrates and products of both reactions are approximately isoenergetic, and no cofactors (such as adenosine triphosphate) are required for recombination. The thermodynamic driving force for directionality of these reactions is thus enigmatic. Here, we present a minimal mathematical model which can explain the directionality and regulation of both 'forward' and 'reverse' reactions. In this model, the substrates of the 'forbidden' reactions (between attL and attR in the absence of RDF, attP and attB in the presence of RDF) are trapped as inactive protein-DNA complexes, ensuring that these 'forbidden' reactions are extremely slow. The model is in good agreement with the observed in vitro kinetics of recombination by ÏC31 integrase, and defines core features of the system necessary and sufficient for directionality.
Asunto(s)
Sitios de Ligazón Microbiológica , ADN/química , Integrasas/química , Modelos Químicos , Modelos Genéticos , Recombinación Genética , ADN/metabolismo , Integrasas/metabolismoRESUMEN
Assembling multiple DNA fragments into functional plasmids is an important and often rate-limiting step in engineering new functions in living systems. Bacteriophage integrases are enzymes that carry out efficient recombination reactions between short, defined DNA sequences known as att sites. These DNA splicing reactions can be used to assemble large numbers of DNA fragments into a functional circular plasmid in a method termed serine integrase recombinational assembly (SIRA). The resulting DNA assemblies can easily be modified by further recombination reactions catalyzed by the same integrase in the presence of its recombination directionality factor (RDF). Here we present a set of protocols for the overexpression and purification of bacteriophage ÏC31 and Bxb1 integrase and RDF proteins, their use in DNA assembly reactions, and subsequent modification of the resulting DNA assemblies.
Asunto(s)
ADN Nucleotidiltransferasas/genética , Integrasas/genética , Ingeniería Metabólica/métodos , Plásmidos/metabolismo , Siphoviridae/genética , Proteínas Virales/genética , Sitios de Ligazón Microbiológica , ADN Nucleotidiltransferasas/aislamiento & purificación , ADN Nucleotidiltransferasas/metabolismo , ADN Circular/genética , ADN Circular/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Integrasas/aislamiento & purificación , Integrasas/metabolismo , Plásmidos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Recombinación Genética , Serina/metabolismo , Siphoviridae/metabolismo , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismoRESUMEN
The product of Xer recombination at directly repeated psi sites on a circular unknotted DNA molecule is a right-hand four-noded catenane. Here, we use tangle equations to analyze the topological changes associated with Xer recombination at psi. This mathematical method allows computation of all possible topological pathways consistent with the experimental data. We give a rigorous mathematical proof that, under reasonable biological assumptions, there are only three solutions to the tangle equations. One of the solutions corresponds to a synaptic complex with antiparallel alignment of recombination core sites, the other two correspond to parallel alignment of cores. We show that all three solutions can be unified into a single three-dimensional model for Xer recombination. Thus the three distinct mathematical solutions do not necessarily represent distinct three-dimensional pathways, and in this case the three distinct tangle solutions are different planar projections of the same three-dimensional configuration.
Asunto(s)
Proteínas de Escherichia coli/química , Integrasas/química , Conformación Proteica , Recombinación GenéticaRESUMEN
In the lysogenic state, bacteriophage P1 is maintained as a low copy-number circular plasmid. Site-specific recombination at loxP by the phage-encoded Cre protein keeps P1 monomeric, thus helping to ensure stable plasmid inheritance. Two Escherichia coli DNA-binding proteins, PepA and ArgR, were recently reported to be necessary for maintenance or establishment of P1 lysogeny. PepA and ArgR bind to regulatory DNA sequences upstream of the ColE1 cer recombination site to regulate site-specific recombination by the XerCD recombinases. This recombination keeps ColE1 in a monomeric state and helps to ensure stable plasmid maintenance. It has been suggested that ArgR and PepA play a similar role in P1 maintenance, regulating Cre recombination by binding to DNA sequences upstream of loxP. Here, we show that ArgR does not bind to its proposed binding site upstream of loxP, and that Cre recombination at loxP in its natural P1 context is not affected by PepA and ArgR in vitro. When sequences upstream of loxP were mutated to allow ArgR binding, PepA and ArgR still had no effect on Cre recombination. Our results demonstrate that PepA requires specific DNA sequences for binding, and that PepA and ArgR have no direct role in Cre recombination at P1 loxP.
Asunto(s)
Sitios de Ligazón Microbiológica/genética , Bacteriófago P1/genética , ADN Viral/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Integrasas/metabolismo , Recombinación Genética/genética , Proteínas Represoras/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Unión ProteicaRESUMEN
The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.