Site-specific Recombination Process Research Articles

Large T antigen mediated target gene replication improves site-specific recombination efficiency.

With advantages of high-fidelity, monoclonality and large cargo capacity, site-specific recombination (SSR) holds great promises for precise genomic modifications. However, broad applications of SSR have been hurdled by low integration efficiency, and the amount of donor DNA available in nucleus for SSR presents as a limiting factor. Inspired by the DNA replication mechanisms observed in double-stranded DNA virus SV40, we hypothesized that expression of SV40 large T antigen (TAg) can increase the copy number of the donor plasmid bearing an SV40 origin, and in consequence promote recombination events. This hypothesis was tested with dual recombinase-mediated cassette exchange (RMCE) in suspension 293F cells. Results showed that TAg co-transfection significantly enhanced SSR in polyclonal cells. In the monoclonal cell line carrying a single landing pad at an identified genomic locus, 12% RMCE efficiency was achieved, and such improvement was indeed correlated with donor plasmid amplification. The developed TAg facilitated RMCE (T-RMCE) was exploited for the construction of large libraries of >107 diversity, from which GFP variants with enhanced fluorescence were isolated. We expect the underlying principle of target gene amplification can be applicable to other SSR processes and gene editing approaches in general for directed evolution and large-scale genomic screening in mammalian cells.

Analysis of Sporulation in Bacillus cereus Biovar anthracis Which Contains an Insertion in the Gene for the Sporulation Factor σK.

Bacillus cereus biovar anthracis (Bcbva) is an untypical pathogen causing a fatal anthrax-like disease in a variety of wildlife species in African rainforest areas. In contrast to Bacillus anthracis and most species of the B. cereus group, all strains of the Bcbva cluster contain a 22 kb insertion in the sigK gene which encodes the essential late sporulation sigma factor σK. This insertion is excised during sporulation in a site-specific recombination process resulting in an intact sigK gene and a circular molecule. The sporulation kinetics of two strains each of Bcbva and B. anthracis were compared by the expression analysis of eight sporulation-associated genes, including sigK, using reverse transcriptase quantitative real-time PCR. In addition, morphological sporulation stages were analyzed and quantified by electron microscopy. Our results indicated that the necessary excision of the insertion in Bcbva neither delayed nor inhibited its sporulation. In two spontaneous mutants of Bcbva, the excision of the sigK insertion and sporulation were impeded due to mutations in the spo0A and spoVG regulator genes, respectively. The spo0A frameshift mutation was overcome by intragenic suppression in a revertant which was able to sporulate normally, despite an M171S amino acid exchange in the global regulator Spo0A. A screening of the NCBI database identified further strains of the B. cereus group which possess unrelated insertions in the sigK gene, and two strains containing almost identical insertions at the same gene position. Some of the sigK insertions encode putative prophages, whereas the Bcbva insertion encoded a type I restriction-modification system. The function of these insertions and if they are possibly essential for sporulation remains to be assessed.

Influence of class 2 integron integrase concentration on gene cassette insertion and excision in vivo.

Integron can capture and express antimicrobial resistance gene cassettes and plays important roles in horizontal gene transfer. The establishment of a complete in vitro reaction system will help to reveal integron integrase mediated site-specific recombination process and regulation mechanism. As an enzymatic reaction, the concentration of integrase is assumed to have a great influence on the reaction rate. To determine the influence of different concentrations of integrase on the reaction rate and to find the best range of enzyme concentration were essential to optimizing the in vitro reaction system. In this study, plasmids with gradient transcription levels of class 2 integron integrase gene intI2 under different promoters were constructed. Among plasmids pI2W16, pINTI2N, pI2W, and pI2NW, intI2 transcription levels ranged from about 0.61-fold to 49.65-fold of that in pINTI2N. And the frequencies of gene cassette sat2 integration and excision catalyzed by IntI2 were positively correlated with the transcription levels of intI2 within this range. Western blotting results indicated high expression of IntI2 partly existed in the form of an inclusion body. When compared with Pc of class 1 integron, the spacer sequence of PintI2 can increase the strength of PcW but decrease the strength of PcS. In conclusion, the frequencies of gene cassette integration and excision were positively correlated with the concentration of IntI2. intI2 driving by PcW with PintI2 spacer sequence can obtain the optimum IntI2 concentration required to achieve the maximum recombination efficiency in vivo in this study.

Germline DNA Retention in Murine and Human Rearranged T Cell Receptor Gene Coding Joints: Alternative Recombination Signal Sequences and V(D)J Recombinase Errors.

The genes coding for the antigenic T cell receptor (TR) subunits are assembled in thymocytes from discrete V, D, and J genes by a site-specific recombination process. A tight control of this activity is required to prevent potentially detrimental recombination events. V, D, and J genes are flanked by semi-conserved nucleotide motives called recombination signal sequences (RSSs). V(D)J recombination is initiated by the precise introduction of a DNA double-strand break exactly at the border of the genes and their RSSs by the RAG recombinase. RSSs are therefore physically separated from the coding region of the genes before assembly of a rearranged TR gene. During a high throughput profiling of TRB genes in mice, we identified rearranged TRB genes in which part or all of a flanking RSS was retained in V-D or D-J coding joints. In some instances, this retention of germline DNA resulted from the use of an upstream alternative RSS. However, we also identified TRB sequences where retention of germline DNA occurred in the absence of alternative RSS, suggesting that RAG activity was mis-targeted during recombination. Similar events were also identified in human rearranged TRB and TRG genes. The use of alternative RSSs during V(D)J recombination illustrates the complexity of RAG-RSSs interactions during V(D)J recombination. While the frequency of errors resulting from mis-targeted RAG activity is very low, we believe that these RAG errors may be at the origin of oncogenic translocations and are a threat for genetic stability in developing lymphocytes.

Transduction of the Streptococcus pyogenes bacteriophage Φm46.1, carrying resistance genes mef(A) and tet(O), to other Streptococcus species.

Φm46.1 – Streptococcus pyogenes bacteriophage carrying mef(A) and tet(O), respectively, encoding resistance to macrolides (M phenotype) and tetracycline – is widespread in S. pyogenes but has not been reported outside this species. Φm46.1 is transferable in vitro among S. pyogenes isolates, but no information is available about its transferability to other Streptococcus species. We thus investigated Φm46.1 for its ability to be transduced in vitro to recipients of different Streptococcus species. Transductants were obtained from recipients of Streptococcus agalactiae, Streptococcus gordonii, and Streptococcus suis. Retransfer was always achieved, and from S. suis to S. pyogenes occurred at a much greater frequency than in the opposite direction. In transductants Φm46.1 retained its functional properties, such as inducibility with mitomycin C, presence both as a prophage and as a free circular form, and transferability. The transductants shared the same Φm46.1 chromosomal integration site as the donor, at the 3′ end of a conserved RNA uracil methyltransferase (rum) gene, which is an integration hotspot for a variety of genetic elements. No transfer occurred to recipients of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus salivarius, even though rum-like genes were also detected in the sequenced genomes of these species. A largely overlapping 18-bp critical sequence, where the site-specific recombination process presumably takes place, was identified in the rum genes of all recipients, including those of the species yielding no transductants. Growth assays to evaluate the fitness cost of Φm46.1 acquisition disclosed a negligible impact on S. pyogenes, S. agalactiae, and S. gordonii transductants and a noticeable fitness advantage in S. suis. The S. suis transductant also displayed marked overexpression of the autolysin-encoding gene atl.

DNA Secondary Structure Formation in Bacterial Gene Capture Systems at Single-Molecule Resolution

Bacteria with multiple antibiotic resistances are a threat to human health. These resistances spread faster than could be expected from mutations alone. It was shown that bacteria exhibit a mechanism of exchanging and collecting genetic sections - coding for antibiotic resistances or other adaptive traits - between individuals or even across species boundaries.This mechanism relies on genetic elements termed integrons. They allow the incorporation and expression of exogenous gene cassettes through a site-specific recombination process. The process involves an enzyme, integrase, which mediates recombination between a double stranded integron recombination site (attI site) and a single-stranded cassette recombination site (attC site). The attC site is supposably recognized by integrase through specificity to the secondary structure of the DNA hairpin formed by the single-stranded attC site (1).This poses the question of how the DNA hairpin forms inside the living cell. It was shown in vivo that negative superhelicity promotes integron recombination, most likely through cruciform extrusion from double-stranded DNA (2).Here, we use single-molecule FRET and magnetic tweezers to study the formation of the postulated DNA hairpin in the presence of various proteins. We present data on the competition between SSB and integrase and on the extrusion of E. coli attC sites from dsDNA and their interaction with integrase hinting at possible molecular mechanisms underlying the function of this system in bacteria.1. Mazel, D. 2006. Integrons: agents of bacterial evolution. Nat Rev Micro. 4: 608-620.2. Loot, C., D. Bikard, A. Rachlin, and D. Mazel. 2010. Cellular pathways controlling integron cassette site folding. EMBO J. 29: 3745-3745.

Real-time single-molecule tethered particle motion experiments reveal the kinetics and mechanisms of Cre-mediated site-specific recombination

Tyrosine family recombinases (YRs) are widely utilized in genome engineering systems because they can easily direct DNA rearrangement. Cre recombinases, one of the most commonly used types of YRs, catalyze site-specific recombination between two loxP sites without the need for high-energy cofactors, other accessory proteins or a specific DNA target sequence between the loxP sites. Previous structural, analytical ultracentrifuge and electrophoretic analyses have provided details of the reaction kinetics and mechanisms of Cre recombinase activity; whether there are reaction intermediates or side pathways involved has been left unaddressed. Using tethered particle motion (TPM), the Cre-mediated site-specific recombination process has been delineated, from beginning to end, at the single-molecule level, including the formation of abortive complexes and wayward complexes blocking inactive nucleoprotein complexes from entering the recombination process. Reversibility in the strand-cleavage/-ligation process and the formation of a thermally stable Holliday junction intermediate were observed within the Cre-mediated site-specific recombination process. Rate constants for each elementary step, which explain the overall reaction outcomes under various conditions, were determined. Taking the findings of this study together, they demonstrate the potential of single-molecule methodology as an alternative approach for exploring reaction mechanisms in detail.

Analysis of the Site-Specific Integration System of the Streptomyces aureofaciens Phage μ1/6

The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3' end of a putative tRNA(Thr) gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.

Dual-In/Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure

BackgroundThe development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. The construction of plasmid-less marker-less strains has many advantages for the further practical exploitation of these bacteria in industry. Such producer strains are usually constructed by sequential chromosome modifications including deletions and integration of genetic material. For these purposes complex methods based on in vitro and in vivo recombination processes have been developed.ResultsHere, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant E. coli strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial φ80-attB sites into desired points of the chromosome followed by two site-specific recombination processes: first, the φ80 system is used for integration of the recombinant DNA based on selective marker-carrier conditionally-replicated plasmid with φ80-attP-site, and second, the λ system is used for excision of inserted vector part, including the plasmid ori-replication and the marker, flanked by λ-attL/R-sites.ConclusionThe developed Dual-In/Out strategy is a rather straightforward, but convenient combination of previously developed recombination methods: phages site-specific and general Red/ET-mediated. This new approach allows us to detail the design of future recombinant marker-less strains, carrying, in particular, rather large artificial insertions that could be difficult to introduce by usually used PCR-based Recombineering procedure. The developed strategy is simple and could be particularly useful for construction of strains for the biotechnological industry.

In vivo restriction endonuclease activity of the Anabaena PCC 7120 XisA protein in Escherichia coli

Anabaena PCC 7120 genome contains three elements, which get excised out during late stages of heterocyst differentiation by a site-specific recombination process. The XisA protein, which excises the nifD element, shows sequence homology with the integrase family of tyrosine recombinase. The 11 bp target site of XisA C GGAGTAA TCC contains a 3 bp inverted repeat. Here, we report restriction endonuclease activity of XisA by specific loss of plasmids containing single or double target sites. The pMX25 plasmid containing two target sites demonstrated endonuclease activity proportional to excision frequency. Different plasmid substrates containing one base pair mutation in the inverted repeat of the target site were monitored for endonuclease activity. Mutation of A4C retained endonuclease activity, while other modifications lost endonuclease activity. The presence of an additional copy of the target site enhanced endonuclease activity. These results suggest that the XisA protein could be an IIE type of restriction endonuclease in addition to being a recombinase.

Control and Regulation of KplE1 Prophage Site-specific Recombination

KplE1 is one of the 10 prophage regions of Escherichia coli K12, located at 2464 kb on the chromosome. KplE1 is defective for lysis, but it is fully competent for excisive recombination. In this study, we have mapped the binding sites of the recombination proteins, namely IntS, TorI, and IHF on attL and attR, and the organization of these sites suggests that the intasome is architecturally different from the lambda canonical form. We also measured the relative contribution of these proteins to both excisive and integrative recombination by using a quantitative in vitro assay. These experiments show a requirement of the TorI excisionase for excisive recombination and of the IntS integrase for both integration and excision. Moreover, we observed a strong influence of the supercoiled state of the substrates. The KplE1 recombination module, composed of the integrase and excisionase genes together with the attL and attR DNA regions, is highly similar to that of several phages infecting various E. coli strains as well as Shigella flexneri and Shigella sonnei. The in vitro recombination data reveal that HK620 and KplE1 att sequences are exchangeable. This study thus defines a new site-specific recombination module, and implications for the mechanism and regulation of recombination are discussed.

Construction of new stable strain over-expressing the glucose isomerase of the Streptomyces sp. SK strain

In order to over express the xylA gene of Streptomyces sp. SK strain, it was cloned under the control of the constitutive ermE-up promoter. This construct was integrated through site-specific recombination process into the chromosome of a Streptomyces violaceoniger glucose isomerase deficient strain using the non-replicative vector pTS55. The resulting CBS4 strain shows a perfect stability in the absence of selection pressure. Its glucose isomerase activity was about four and nine-fold greater, than that obtained from Streptomyces sp. SK, respectively fully induced or not by xylose.

The DNA-dependent protein kinase: the director at the end.

Efficient repair of DNA double-strand breaks is essential for the maintenance of chromosomal integrity. In higher eukaryotes, non-homologous end-joining (NHEJ) DNA is the primary pathway that repairs these breaks. NHEJ also functions in developing lymphocytes to repair strand breaks that occur during V(D)J recombination, the site-specific recombination process that provides for the assembly of functional antigen-receptor genes. If V(D)J recombination is impaired, B- and T-lymphocyte development is blocked resulting in severe combined immunodeficiency disease. In the last decade, an intensive research effort has focused on NHEJ resulting in a reasonable understanding of how double-strand breaks are resolved. Six distinct gene products have been identified that function in this pathway (Ku70, Ku86, XRCC4, DNA ligase IV, Artemis, and DNA-PKcs). Three of these comprise one complex, the DNA-dependent protein kinase (DNA-PK). This protein complex is central during NHEJ, because DNA-PK initially recognizes and binds to the damaged DNA and then targets the other repair activities to the site of DNA damage. In this review, we discuss recent developments that have provided insight into how DNA-PK functions, once bound to DNA ends.

Mechanisms of Site-Specific Recombination

Integration, excision, and inversion of defined DNA segments commonly occur through site-specific recombination, a process of DNA breakage and reunion that requires no DNA synthesis or high-energy cofactor. Virtually all identified site-specific recombinases fall into one of just two families, the tyrosine recombinases and the serine recombinases, named after the amino acid residue that forms a covalent protein-DNA linkage in the reaction intermediate. Their recombination mechanisms are distinctly different. Tyrosine recombinases break and rejoin single strands in pairs to form a Holliday junction intermediate. By contrast, serine recombinases cut all strands in advance of strand exchange and religation. Many natural systems of site-specific recombination impose sophisticated regulatory mechanisms on the basic recombinational process to favor one particular outcome of recombination over another (for example, excision over inversion or deletion). Details of the site-specific recombination processes have been revealed by recent structural and biochemical studies of members of both families.

Detecting, signalling and repairing DNA double-strand breaks.

DNA double-strand breaks (DSBs) can be generated by a variety of genotoxic agents, including ionizing radiation and radiomimetic chemicals. They can also occur when DNA replication complexes encounter other forms of DNA damage, and are produced as intermediates during certain site-specific recombination processes. It is crucial that cells recognize DSBs and bring about their efficient repair, because a single unrepaired cellular DSB can induce cell death, and defective DSB repair can lead to mutations or the loss of significant segments of chromosomal material. Eukaryotic cells have evolved a variety of systems to detect DNA DSBs, repair them, and signal their presence to the transcription, cell cycle and apoptotic machineries. In this review, I describe how work on mammalian cells and also on model organisms such as yeasts has revealed that such systems are highly conserved throughout evolution, and has provided insights into the molecular mechanisms by which DNA DSBs are recognized, signalled and repaired. I also explain how defects in the proteins that function in these pathways are associated with a variety of human pathological states.

Detecting, signalling and repairing DNA double-strand breaks

DNA double-strand breaks (DSBs) can be generated by a variety of genotoxic agents, including ionizing radiation and radiomimetic chemicals. They can also occur when DNA replication complexes encounter other forms of DNA damage, and are produced as intermediates during certain site-specific recombination processes. It is crucial that cells recognize DSBs and bring about their efficient repair, because a single unrepaired cellular DSB can induce cell death, and defective DSB repair can lead to mutations or the loss of significant segments of chromosomal material. Eukaryotic cells have evolved a variety of systems to detect DNA DSBs, repair them, and signal their presence to the transcription, cell cycle and apoptotic machineries. In this review, I describe how work on mammalian cells and also on model organisms such as yeasts has revelaed that such systems are highly conserved throughout evolution, and has provided insights into the molecular mechanisms by which DNA DSBs are recognized, signalled and repaired. I also explain how defects in the proteins that function in these pathways are associated with a variety of human pathological states.

Immunoglobulin Class Switch Recombination: Will Genetics Provide New Clues to Mechanism?

Because of space limitations, only a fraction of the most recent relevant references have been included. I thank the National Institutes of Health for its support of our research on class-switch recombination (grant R01 GM39799) and somatic hypermutation (grant R01 GM41712).

Accessibility control of variable region gene assembly during T-cell development.

T-cell development is a complex and ordered process that is regulated in part by the progressive assembly and expression of antigen receptor genes. T cells can be divided into two lineages based on expression of either an alpha beta or gamma delta T-cell antigen receptor (TCR). The genes that encode the TCR beta and gamma chains lie in distinct loci, whereas the genes that encode the TCR alpha and delta chains lie in a single locus (TCR alpha/delta locus). Assembly of TCR variable region genes is mediated by a site-specific recombination process that is common among all lymphocytes. Despite the common nature of this process, recombination of TCR genes is tightly regulated within the context of the developing T cell. TCR beta, gamma and delta variable region genes are assembled prior to TCR alpha variable region genes. Furthermore, assembly of TCR beta variable region genes is regulated within the context of allelic exclusion. The regulation of rearrangement and expression of genes within the TCR alpha/delta locus presents a complicated problem. TCR alpha and delta variable region genes are assembled at different stages of T-cell development, and fully assembled TCR alpha and delta variable region genes must be expressed in distinct lineages of T cells, alpha beta and gamma delta, respectively. We have developed several experimental approaches to assess the role of cis-acting elements in regulating recombination and expression of TCR genes. Here we describe these approaches and discuss our analyses of the regulation of accessibility of the TCR beta and TCR alpha/delta loci during T-cell development.

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction.

RAGged repair: what's new in V(D)J recombination.

Antigen-specific immunity is due to the generation of a multitude of both immunoglobulins and T-cell receptors through a process designated V(D)J recombination. In vitro reconstitution of this system has taught us a great deal about the molecular mechanism underlying this site-specific recombination process. Hence, it became obvious that the initial steps of the reaction are carried out by the lymphocyte-specific proteins RAG1 and RAG2 (recombination-activating genes), with the help of members of the high mobility group protein family of DNA-binding proteins, HMG1 or HMG2. Structural resemblance between RAG1 and a prokaryotic recombinase, the Salmonella Hin Recombinase, together with mechanistic similarities between V(D)J recombination and bacterial transposition reactions, make it likely that these different processes have evolved from a common ancestral recombination system. The second step in V(D)J recombination is catalysed by the ubiquitous DNA double-strand break repair machinery. The link between V(D)J recombination and double-strand break repair was established through some mutational complementation groups, including the murine SCID mutation (severe combined immunodeficiency), which were shown to be defective in both V(D)J recombination and double-strand break repair. The multisubunit DNA-dependent protein kinase appears to be a key player in these processes. Thus, from an evolutionary point of view, antigen-specific immunity in mammals, e.g., humans and mice, appears to be the result of an evolutionary combination of two unrelated systems involved in DNA metabolism.