Effects of DNA binding of the zinc finger and linkers for domain fusion on the catalytic activity of sequence-specific chimeric recombinases determined by a facile fluorescent system

Artificial zinc finger proteins (ZFPs) consist of Cys(2)-His(2)-type modules composed of ～30 amino acids with a ββα structure that coordinates a zinc ion. ZFPs that recognize specific DNA target sequences can substitute for the binding domains of enzymes that act on DNA to create designer enzymes with programmable sequence specificity. The most studied of these engineered enzymes are zinc finger nucleases (ZFNs). ZFNs have been widely used to model organisms and are currently in human clinical trials with an aim of therapeutic gene editing. Difficulties with ZFNs arise from unpredictable mutations caused by nonhomologous end joining and off-target DNA cleavage and mutagenesis. A more recent strategy that aims to address the shortcomings of ZFNs involves zinc finger recombinases (ZFRs). A thorough understanding of ZFRs and methods for their modification promises powerful new tools for gene manipulation in model organisms as well as in gene therapy. In an effort to design efficient and specific ZFRs, the effects of the DNA binding affinity of the zinc finger domains and the linker sequence between ZFPs and recombinase catalytic domains have been assessed. A plasmid system containing ZFR target sites was constructed for evaluation of catalytic activities of ZFRs with variable linker lengths and numbers of zinc finger modules. Recombination efficiencies were evaluated by restriction enzyme analysis of isolated plasmids after reaction in Escherichia coli and changes in EGFP fluorescence in mammalian cells. The results provide information relevant to the design of ZFRs that will be useful for sequence-specific genome modification.     

Zinc-finger recombinase activities in vitro

Zinc-finger recombinases (ZFRs) are chimaeric proteins comprising a serine recombinase catalytic domain linked to a zinc-finger DNA binding domain. ZFRs can be tailored to promote site-specific recombination at diverse 'Z-sites', which each comprise a central core sequence flanked by zinc-finger domain-binding motifs. Here, we show that purified ZFRs catalyse efficient high-specificity reciprocal recombination between pairs of Z-sites in vitro. No off-site activity was detected. Under different reaction conditions, ZFRs can catalyse Z-site-specific double-strand DNA cleavage. ZFR recombination activity in Escherichia coli and in vitro is highly dependent on the length of the Z-site core sequence. We show that this length effect is manifested at reaction steps prior to formation of recombinants (binding, synapsis and DNA cleavage). The design of the ZFR protein itself is also a crucial variable affecting activity. A ZFR with a very short (2 amino acids) peptide linkage between the catalytic and zinc-finger domains has high activity in vitro, whereas a ZFR with a very long linker was less recombination-proficient and less sensitive to variations in Z-site length. We discuss the causes of these phenomena, and their implications for practical applications of ZFRs.     

Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants

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.     

A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells

Zinc-finger recombinases (ZFRs) represent a potentially powerful class of tools for targeted genetic engineering. These chimeric enzymes are composed of an activated catalytic domain derived from the resolvase/invertase family of serine recombinases and a custom-designed zinc-finger DNA-binding domain. The use of ZFRs, however, has been restricted by sequence requirements imposed by the recombinase catalytic domain. Here, we combine substrate specificity analysis and directed evolution to develop a diverse collection of Gin recombinase catalytic domains capable of recognizing an estimated 3.77 × 10⁷ unique DNA sequences. We show that ZFRs assembled from these engineered catalytic domains recombine user-defined DNA targets with high specificity, and that designed ZFRs integrate DNA into targeted endogenous loci in human cells. This study demonstrates the feasibility of generating customized ZFRs and the potential of ZFR technology for a diverse range of applications, including genome engineering, synthetic biology and gene therapy.     

Evolution of programmable zinc finger-recombinases with activity in human cells

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 (Rec(ZF)s) capable of site-specific function in a diversity of genetic contexts. Our first Rec(ZF), Tn3Ch15(X2), 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 approximately 17% of transduced cells. Following this template of rational design and directed evolution, Rec(ZF)s 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.     

Site-specific recombinases as tools for heterologous gene integration

Site-specific recombinases are the enzymes that catalyze site-specific recombination between two specific DNA sequences to mediate DNA integration, excision, resolution, or inversion and that play a pivotal role in the life cycles of many microorganisms including bacteria and bacteriophages. These enzymes are classified as tyrosine-type or serine-type recombinases based on whether a tyrosine or serine residue mediates catalysis. All known tyrosine-type recombinases catalyze the formation of a Holliday junction intermediate, whereas the catalytic mechanism of all known serine-type recombinases includes the 180° rotation and rejoining of cleaved substrate DNAs. Both recombinase families are further subdivided into two families; the tyrosine-type recombinases are subdivided by the recombination directionality, and the serine-type recombinases are subdivided by the protein size. Over more than two decades, many different site-specific recombinases have been applied to in vivo genome engineering, and some of them have been used successfully to mediate integration, deletion, or inversion in a wide variety of heterologous genomes, including those from bacteria to higher eukaryotes. Here, we review the recombination mechanisms of the best characterized recombinases in each site-specific recombinase family and recent advances in the application of these recombinases to genomic manipulation, especially manipulations involving site-specific gene integration into heterologous genomes.     

Stimulation of homologous recombination through targeted cleavage by chimeric nucleases

Chimeric nucleases that are hybrids between a nonspecific DNA cleavage domain and a zinc finger DNA recognition domain were tested for their ability to find and cleave their target sites in living cells. Both engineered DNA substrates and the nucleases were injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and subsequent homologous recombination were observed. Specific cleavage required two inverted copies of the zinc finger recognition site in close proximity, reflecting the need for dimerization of the cleavage domain. Cleaved DNA molecules were activated for homologous recombination; in optimum conditions, essentially 100% of the substrate recombined, even though the DNA was assembled into chromatin. The original nuclease has an 18-amino-acid linker between the zinc finger and cleavage domains, and this enzyme cleaved in oocytes at paired sites separated by spacers in the range of 6 to 18 bp, with a rather sharp optimum at 8 bp. By shortening the linker, we found that the range of effective site separations could be narrowed significantly. With no intentional linker between the binding and cleavage domains, only binding sites exactly 6 bp apart supported efficient cleavage in oocytes. We also showed that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. Because the recognition specificity of zinc fingers can be altered experimentally, this approach holds great promise for inducing targeted recombination in a variety of organisms.     

Gene deletion from Plasmodium falciparum using FLP and Cre recombinases: Implications for applied site-specific recombination

The ability to manipulate the genome and induce site-specific recombination using either Flippase (FLP) or Cre recombinase has been useful in many systems including Plasmodium berghei for specific deletion events or to obtain conditional gene expression. To test whether these recombinases are active in Plasmodium falciparum we constructed gene knockouts that contain sequences recognised as templates for site-specific recombination. We tested the ability of FLP and Cre recombinases, expressed conditionally in P. falciparum, to mediate deletion of the human dihydrofolate reductase (hdhfr) drug resistance gene. We show that Cre recombinase is capable of efficient removal of hdhfr by site-specific recombination. In contrast, FLP recombinase is very inefficient, even at the optimum temperature of 30 °C for this enzyme. These results demonstrate that Cre recombinase can be utilised in P. falciparum for deletion of specific sequences such as drug resistance genes. This can be exploited for recycling of drug resistance cassettes and for the design of specific recombination events in P. falciparum.     

Directed evolution of recombinase specificity by split gene reassembly

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.     

Control of Cre recombination by regulatory elements from Xer recombination systems

Site-specific recombination by the Cre recombinase takes place at a simple DNA site (loxP), requires no additional proteins and gives topologically simple recombination products. In contrast, cer and psi sites for Xer recombination contain approximately 150 bp of accessory sequences, require accessory proteins PepA, ArgR and ArcA, and the products are specifically linked to form a four-noded catenane. Here, we use hybrid sites consisting of accessory sequences of cer or psi fused to loxP to probe the function of accessory proteins in site-specific recombination. We show that PepA instructs Cre to produce four-noded catenane, but is not required for recombination at these hybrid sites. Mutants of Cre that require PepA and accessory sequences for efficient recombination were selected. PepA-dependent Cre gave products with a specific topology and displayed resolution selectivity. Our results reveal that PepA acts autonomously in the synapsis of psi and cer accessory sequences and is the main architectural element responsible for intertwining accessory site DNA. We suggest that accessory proteins can activate recombinases simply by synapsing the regulatory DNA sequences, thus bringing the recombination sites together with a specific geometry. This may occur without the need for protein-protein interactions between accessory proteins and the recombinases.     

Switching catalytic activity in the XerCD site-specific recombination machine.

The tyrosine family site-specific recombinases, XerCD, function in the conversion of circular dimer replicons to monomers. In the recombining complex that contains two synapsed recombination sites and two molecules each of XerC and XerD, the DNA strand-exchange reactions are separated in time and space. XerC initiates recombination to form a Holliday junction intermediate, which undergoes a conformational change to provide a substrate for strand exchange by XerD. XerCD are two-domain proteins, whose C-terminal domains contain all of the catalytic residues. We show that XerC or XerD variants lacking their N-terminal domains are active in recombination when combined with their wild-type partner. Nevertheless, the normal pattern of catalysis is dramatically altered; strand exchange by the recombinase variant is stimulated, while that by the wild-type partner recombinase is impaired. The primary determinants for the mutant phenotype reside in the region of alpha-helix B of XerD. We propose that altered interactions within the recombining heterotetramer lead to changes in the relative concentrations of the two alternative Holliday junction substrates that are recombined by XerC or XerD, respectively.     

Challenging a Paradigm: the Role of DNA Homology in Tyrosine Recombinase Reactions

Summary: A classical feature of the tyrosine recombinase family of proteins catalyzing site-specific recombination, as exemplified by the phage lambda integrase and the Cre and Flp recombinases, is the ability to recombine substrates sharing very limited DNA sequence identity. Decades of research have established the importance of this short stretch of identity within the core regions of the substrates. Since then, several new enzymes that challenge this paradigm have been discovered and require the role of sequence identity in site-specific recombination to be reconsidered. The integrases of the conjugative transposons such as Tn916, Tn1545, and CTnDOT recombine substrates with heterologous core sequences. The integrase of the mobilizable transposon NBU1 performs recombination more efficiently with certain core mismatches. The integration of CTX phage and capture of gene cassettes by integrons also occur by altered mechanisms. In these systems, recombination occurs between mismatched sequences by a single strand exchange. In this review, we discuss literature that led to the formulation of the current strand-swapping isomerization model for tyrosine recombinases. The review then focuses on recent developments on the recombinases that challenged the paradigm that was derived from the studies of early systems.     

Mammalian genomes contain active recombinase recognition sites

Recombinases derived from microorganisms mediate efficient site-specific recombination. For example, the Cre recombinase from bacteriophage P1 efficiently carries out recombination at its loxP target sites. While this enzyme can function in mammalian cells, the 34bp loxP site is expected to be absent from mammalian genomes. We have discovered that sequences from the human and mouse genomes surprisingly divergent from loxP can support Cre-mediated recombination at up to 100% of the efficiency of the native loxP site in bacterial assays. Transient assays in human cells demonstrate that such pseudo-lox sites also support Cre-mediated integration and excision in the human cell environment. Pseudo sites for Cre and other recombinases may be useful for site-specific insertion of exogenous genes into mammalian genomes during gene therapy and other genetic engineering processes.     

Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre.

Site-specific DNA recombinases from bacteriophage and yeasts have been developed as novel tools for genome engineering both in prokaryotes and eukaryotes. The 38kDa Cre protein efficiently produces both inter- and intramolecular recombination between specific 34bp sites called loxP. We report here the in vivo use of Cre recombinase to manipulate the genome of the protozoan parasite Toxoplasma gondii. Cre catalyzes the precise removal of transgenes from T. gondii genome when flanked by two directly repeated loxP sites. The efficiency of excision has been determined using LacZ as reporter and indicates that it can easily be applied to the removal of undesired sequences such as selectable marker genes and to the determination of gene essentiality. We have also shown that the reversibility of the recombination reaction catalyzed by Cre offers the possibility to target site-specific integration of a loxP-containing vector in a chromosomally placed loxP target in the parasite. In mammalian systems, the Cre recombinase can be regulated by hormone and is used for inducible gene targeting. In T. gondii, fusions between Cre recombinase and the hormone-binding domain of steroids are constitutively active, hampering the utilization of this mode of post-translational regulation as inducible gene expression system.     

Site-specific photo-cross-linking between lambda integrase and its DNA recombination target

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to "arm-type" sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the "core-type" sequences where DNA cleavage and ligation are executed. We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position. When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically. The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of approximately 20%. The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly. Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the lambda Int family.     

Targeted modification of mammalian genomes

The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination.

Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or ΦC31 integrase) are usually 30–50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes.     

DNA topology and geometry in Flp and Cre recombination

The Flp recombinase of yeast and the Cre recombinase of bacteriophage P1 both belong to the lambda-integrase (Int) family of site-specific recombinases. These recombination systems recognize recombination-target sequences that consist of two 13bp inverted repeats flanking a 6 or 8bp spacer sequence. Recombination reactions involve particular geometric and topological relationships between DNA target sites at synapsis, which we investigate using nicked-circular DNA molecules. Examination of the tertiary structure of synaptic complexes formed on nicked plasmid DNAs by atomic-force microscopy, in conjunction with detailed topological analysis using the mathematics of tangles, shows that only a limited number of recombination-site topologies are consistent with the global structures of plasmids bearing directly and inversely repeated sites. The tangle solutions imply that there is significant distortion of the Holliday-junction intermediate relative to the planar structure of the four-way DNA junction present in the Flp and Cre co-crystal structures. Based on simulations of nucleoprotein structures that connect the two-dimensional tangle solutions with three-dimensional models of the complexes, we propose a recombination mechanism in which the synaptic intermediate is characterized by a non-planar, possibly near-tetrahedral, Holliday-junction intermediate. Only modest conformational changes within this structure are needed to form the symmetric, planar DNA junction, which may be characteristic of shorter-lived intermediates along the recombination pathway.     

Polyphosphate kinase is a component of the Escherichia coli RNA degradosome

Xer site-specific recombination functions in the stable inheritance of circular plasmids and bacterial chromosomes. Two related recombinases, XerC and XerD, mediate this recombination, which 'undoes' the potential damage of homologous recombination. Xer recombination on natural plasmid sites is preferentially intramolecular, converting plasmid multimers to monomers. In contrast, recombination at the Escherichia coli recombination site, dif , occurs both intermolecularly and intramolecularly, at least when dif is inserted into a multicopy plasmid. Here the DNA sequence features of a family of core recombination sites in which the XerC- and XerD-binding sites, which are separated by 6 bp, were analysed in order to ascertain what determines whether recombination will be preferentially intramolecular, or will occur both within and between molecules. Sequence changes in either the XerC- or XerD-binding site can alter the recombination outcome. Preferential intramolecular recombination between a pair of recombination sites requires additional accessory DNA sequences and accessory recombination proteins and is correlated with reduced affinities of recombinase binding to recombination core sites, reduced XerC-mediated cleavage in vitro , and an apparent increased overall bending in recombinase–core-site complexes.     

The effects of ethidium bromide and Mg++ ion on strand exchange in the Hin-mediated inversion reaction

The biochemical reaction of a site-specific recombinase such as Hin invertase or gammadelta resolvase starts with binding of the recombinase to its recombination site and cleavage of the DNA in the center of the site. This is followed by strand exchange and finally ligation of the ends of the recombined strands. Previous biochemical studies have shown that Hin invertase and gammadelta resolvase cannot proceed beyond DNA cleavage in the absence of Mg++ ion, indicating that these recombinases require Mg++ ion in the strand exchange process. We have observed that the intercalating agent, ethidium bromide (2 microM), does not interfere with DNA cleavage, but slows strand exchange in a concentration-dependent manner. Levels of Mg++ ion below 5 mM also slow strand exchange substantially. We infer that random intercalation of ethidium bromide inhibits unwinding of the double helix at the recombination site in the negatively supercoiled DNA and propose that Mg+ may be required for Hin to deform the secondary structure of B-DNA prior to strand exchange.