When reverse genetics meets physiology: the use of site-specific recombinases in mice

The use of site-specific recombinases enables the precise introduction of defined genetic mutations into the mouse genome. In theory, any deletion, point mutation, inversion or translocation can be modeled in mice. Because gene targeting is controlled both spatially and temporally, the function of a given gene can be studied in the desired cell types and at a specific time point. This 'genetic dissection' allows to define gene function in development, physiology or behavior. In this review, we focus on the technical possibilities of Cre and other site-specific recombinases but also discuss their limitations.     

Half-site recombinations mediated by yeast site-specific recombinases Flp and R.

The Flp recombinase of Saccharomyces cerevisae and the related R recombinase of Zygosaccharomyces rouxii can efficiently catalyze strand cleavage and strand exchange reactions in half recombination sites. A half-site consists of one recombinase binding element, a recombinase cleavage site on one strand and a 5' spacer hydroxyl group on the other that can initiate the strand exchange reaction. We have studied the various types of strand exchanges that half-sites can participate in. Reaction between a left half-site and a right half-site generates a full recombination site. Strand transfer between two left half-sites or between two right half-sites produces pseudo-full-sites. Strand transfer within a half-site results in a stem-loop or hairpin product. The half-site strand transfer reaction is fairly indifferent to the spacer sequence of the substrate per se and is less sensitive to variations in spacer lengths than a full-site recombination reaction. The optimal spacer length of eight to ten nucleotides observed for the Flp half-site reaction likely permits the most productive catalytic interactions between two Flp monomers bound to each of two partner half-sites. When reacted with a full-site, the half-site can give rise to a normal or reverse recombinant, corresponding to homologous or non-homologous alignments of the spacer sequences during substrate synapsis. The contrary recombination (resulting from non-homologous spacer alignment), whose level is low relative to normal recombination, is partly suppressed when the half-site spacer ends in a 5'-phosphate rather than a 5'-hydroxyl group. Thus, the early steps of recombination, namely synapsis and initial stand transfer, are not dependent on complete spacer homology between the two recombining substrates. The selection of properly aligned substrate partners must occur at the homology dependent branch migration step. In reactions containing a mixture of Flp and R half-sites, Flp and R catalyze strand transfer, almost exclusively, within or between their respective cognate substrates. However, under conditions where self-crosses are inhibited, strand exchange between a Flp half-site and an R half-site appears to be stimulated by a combination of R and Flp.     

Roles of two large serine recombinases in mobilizing the methicillin‐resistance cassette SCCmec

Methicillin‐resistant Staphylococcus aureus (MRSA) emerged via acquisition of a mobile element, staphylococcal cassette chromosome mec (SCCmec). Integration and excision of SCCmec is mediated by an unusual site‐specific recombination system. Most variants of SCCmec encode two recombinases, CcrA and CcrB, that belong to the large serine family. Since CcrA and CcrB are always found together, we sought to address their specific roles. We show here that CcrA and CcrB can carry out both excisive and integrative recombination in Escherichia coli in the absence of any host‐specific or SCCmec‐encoded cofactors. CcrA and CcrB are promiscuous in their substrate choice: they act on many non‐canonical pairs of recombination sites in addition to the canonical ones, which may explain tandem insertions into the SCCmec attachment site. Moreover, CcrB is always required, but CcrA is only required if one of the four half‐sites is present. Recombinational activity correlates with DNA binding: CcrA recognizes only that half‐site, which overlaps a conserved coding frame on the host chromosome. Therefore, we propose that CcrA serves as a specificity factor that emerged through modular evolution to enable recognition of a bacterial recombination site that is not an inverted repeat.     

High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography

A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.     

Dissecting the hydrogen exchange properties of insulin under amyloid fibril forming conditions: a site-specific investigation by mass spectrometry

We have examined the hydrogen exchange properties of bovine insulin under solution conditions that cause it to aggregate and eventually form amyloid fibrils. The results have been obtained at the residue-specific level using peptic digestion and mass spectrometry. A total of 19 peptides were assigned to regions of the protein and their exchange properties monitored for a period of 24 hours. The results of the peptic digestion show that residues A13 to A21 and B11 to B30 are more susceptible to proteolysis than the N-terminal regions of the protein. A total of 15 slowly exchanging amides were observed for insulin under these solution conditions. Location of the protected amides was carried out using a peptic-digestion protocol at low pH. Chromatographic separation was not required. This enabled a direct comparison of the peptides within the same mass spectrum. From kinetic analysis of the rates slow exchange has been located to 4(+/-1) backbone amides in the A13-A19 helix and 6(+/-1) in the B chain helix. The remaining 5(+/-1) are assigned to helix A2-A8. Taken together the results from digestion and hydrogen exchange show that at low pH and relatively high concentrations the C termini of both chains are susceptible to proteolysis but that the solution structure contains the native state helices. More generally the results demonstrate that mass spectrometry can be applied to study site-specific hydrogen exchange properties of proteins even under conditions where they are known to be partially folded and aggregate extensively in solution.     

In vivo site-specific integration of transgene in silkworm via PhiC31 integrase-mediated cassette exchange

Current techniques for genetic engineering of the silkworm Bombyx mori genome utilize transposable elements, which result in positional effects and insertional mutagenesis through random insertion of exogenous DNA. New methods for introducing transgenes at specific positions are therefore needed to overcome the limitations of transposon-based strategies. Although site-specific recombination systems have proven powerful tools for genome manipulation in many organisms, their use has not yet been well established for the integration of transgenes in the silkworm. We describe a method for integrating target genes at pre-defined chromosomal sites in the silkworm via phiC31/att site-specific recombination system-mediated cassette exchange. Successful recombinase-mediated cassette exchange (RMCE) was observed in the two transgenic target strains with an estimated transformation efficiency of 3.84–7.01%. Our results suggest that RMCE events between chromosomal attP/attP target sites and incoming attB/attB sites were more frequent than those in the reciprocal direction. This is the first report of in vivo RMCE via phiC31 integrase in the silkworm, and thus represents a key step toward establishing genome manipulation technologies in silkworms and other lepidopteran species.     

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

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

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

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

The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange

Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.     

Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments

Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.     

The integrase family of site-specific recombinases: regional similarities and global diversity.

A combination of two methods for detecting distant relationships in protein primary sequences was used to compare the site-specific recombination proteins encoded by bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1. This group of proteins exhibits an unexpectedly large diversity of sequences. Despite this diversity, all of the recombinases can be aligned in their C-terminal halves. A 40-residue region near the C terminus is particularly well conserved in all the proteins and is homologous to a region near the C terminus of the yeast 2 mu plasmid Flp protein. This family of recombinases does not appear to be related to any other site-specific recombinases. Three positions are perfectly conserved within this family: histidine, arginine and tyrosine are found at respective alignment positions 396, 399 and 433 within the well-conserved C-terminal region. We speculate that these residues contribute to the active site of this family of recombinases, and suggest that tyrosine-433 forms a transient covalent linkage to DNA during strand cleavage and rejoining.     

Carbon dioxide exchange and canopy conductance of two coniferous forests under various sky conditions

Sky conditions play an important role in the Earth’s climate system and CO₂ uptake by plants. We used eddy covariance and meteorological data, including global and diffuse photosynthetic photon flux density (PPFD), recorded over the 2008 and 2009 growing season at two Sitka spruce [Picea sitchensis (Bong.) Carr.] forest sites in northern Britain, in order to establish relationships between physiological properties under diverse sky conditions, i.e. (1) sunny, (2) cloudy, and (3) overcast, and several canopy activity-related properties. These properties are: (1) response to PPFD, (2) photosynthetic light use efficiency, and (3) canopy stomatal conductance. We found that Sitka spruce forests utilise PPFD in a more efficient way when solar radiation is dominated by diffuse radiation. Furthermore, our results show that diffuse radiation enhances canopy stomatal conductance, an effect which may be the result of both blue light enrichment within the canopy and the reduction in vapour pressure deficit during cloudy and overcast weather. Diffuse radiation does not only influence short-term (hourly, daily, monthly) canopy activity but also long-term forest growth.     

Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination.

Genomic manipulations using site-specific recombinases rely on their applied characteristics in living systems. To understand their applied properties so that they can be optimally deployed, we compared the recombinases FLP and Cre in two assays. In both Escherichia coli and in vitro, FLP shows a different temperature optimum than Cre. FLP is more thermolabile, having an optimum near 30 degrees C and little detectable activity above 39 degrees C. Cre is optimally efficient at 37 degrees C and above. Consistent with FLP thermolability, recombination in a mammalian cell line mediated by a ligand- regulated FLP-androgen receptor fusion protein is more efficient at 35 degrees C than at higher temperatures. We also document a mutation in a commercially available FLP plasmid (FLP-F70L) which renders this recombinase even more thermolabile. The different temperature optima of FLP, FLP-F70L and Cre influence their strategies of usage. Our results recommend the use of Cre for applications in mice that require efficient recombination. The thermolabilities of FLP and FLP-F70L can be usefully exploited for gain of function and cell culture applications.     

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

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

Similarities and differences among 105 members of the Int family of site-specific recombinases.

Alignments of 105 site-specific recombinases belonging to the Int family of proteins identified extended areas of similarity and three types of structural differences. In addition to the previously recognized conservation of the tetrad R-H-R-Y, located in boxes I and II, several newly identified sequence patches include charged amino acids that are highly conserved and a specific pattern of buried residues contributing to the overall protein fold. With some notable exceptions, unconserved regions correspond to loops in the crystal structures of the catalytic domains of lambda Int (Int c170) and HP1 Int (HPC) and of the recombinases XerD and Cre. Two structured regions also harbor some pronounced differences. The first comprises beta-sheets 4 and 5, alpha-helix D and the adjacent loop connecting it to alpha-helix E: two Ints of phages infecting thermophilic bacteria are missing this region altogether; the crystal structures of HPC, XerD and Cre reveal a lack of beta-sheets 4 and 5; Cre displays two additional beta-sheets following alpha-helix D; five recombinases carry large insertions. The second involves the catalytic tyrosine and is seen in a comparison of the four crystal structures. The yeast recombinases can theoretically be fitted to the Int fold, but the overall differences, involving changes in spacing as well as in motif structure, are more substantial than seen in most other proteins. The phenotypes of mutations compiled from several proteins are correlated with the available structural information and structure-function relationships are discussed. In addition, a few prokaryotic and eukaryotic enzymes with partial homology with the Int family of recombinases may be distantly related, either through divergent or convergent evolution. These include a restriction enzyme and a subgroup of eukaryotic RNA helicases (D-E-A-D proteins).     

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

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

Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

Background  

Oncolytic herpes simplex virus (HSV) vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC) technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology.