LAGLIDADG Homing Endonucleases Research Articles

Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases

Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40–70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ∼70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.

LAHEDES: the LAGLIDADG homing endonuclease database and engineering server

LAGLIDADG homing endonucleases (LHEs) are DNA cleaving enzymes, also termed ‘meganucleases’ that are employed as gene-targeting reagents. This use of LHEs requires that their DNA specificity be altered to match sequences in genomic targets. The choice of the most appropriate LHE to target a particular gene is facilitated by the growing number of such enzymes with well-characterized activities and structures. ‘LAHEDES’ (The LAGLIDADG Homing Endonuclease Database and Engineering Server) provides both an online archive of LHEs with validated DNA cleavage specificities and DNA-binding interactions, as well as a tool for the identification of DNA sequences that might be targeted by various LHEs. Searches can be performed using four separate scoring algorithms and user-defined choices of LHE scaffolds. The webserver subsequently provides information regarding clusters of amino acids that should be interrogated during engineering and selection experiments. The webserver is fully open access and can be found at http://homingendonuclease.net.

Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space.

LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ∼20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering—many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.

Comprehensive homing endonuclease target site specificity profiling reveals evolutionary constraints and enables genome engineering applications

Homing endonucleases (HEs) promote the evolutionary persistence of selfish DNA elements by catalyzing element lateral transfer into new host organisms. The high site specificity of this lateral transfer reaction, termed homing, reflects both the length (14–40 bp) and the limited tolerance of target or homing sites for base pair changes. In order to better understand molecular determinants of homing, we systematically determined the binding and cleavage properties of all single base pair variant target sites of the canonical LAGLIDADG homing endonucleases I-CreI and I-MsoI. These Chlorophyta algal HEs have very similar three-dimensional folds and recognize nearly identical 22 bp target sites, but use substantially different sets of DNA-protein contacts to mediate site-specific recognition and cleavage. The site specificity differences between I-CreI and I-MsoI suggest different evolutionary strategies for HE persistence. These differences also provide practical guidance in target site finding, and in the generation of HE variants with high site specificity and cleavage activity, to enable genome engineering applications.

DNA recognition and transcriptional regulation by the WhiA sporulation factor

Sporulation in the filamentous bacteria Streptomyces coelicolor is a tightly regulated process involving aerial hyphae growth, chromosome segregation, septation and spore maturation. Genetic studies have identified numerous genes that regulate sporulation, including WhiA and the sigma factor WhiG. WhiA, which has been postulated to be a transcriptional regulator, contains two regions typically associated with DNA binding: an N-terminal domain similar to LAGLIDADG homing endonucleases, and a C-terminal helix-turn-helix domain. We characterized several in vitro activities displayed by WhiA. It binds at least two sporulation-specific promoters: its own and that of parABp2. DNA binding is primarily driven by its HTH domain, but requires full-length protein for maximum affinity. WhiA transcription is stimulated by WhiG, while the WhiA protein binds directly to WhiG (leading to inhibition of WhiG-dependent transcription). These separate activities, which resemble a possible feedback loop, may help coordinate the closely timed cessation of aerial growth and subsequent spore formation.

Tapping natural reservoirs of homing endonucleases for targeted gene modification

Homing endonucleases mobilize their own genes by generating double-strand breaks at individual target sites within potential host DNA. Because of their high specificity, these proteins are used for "genome editing" in higher eukaryotes. However, alteration of homing endonuclease specificity is quite challenging. Here we describe the identification and phylogenetic analysis of over 200 naturally occurring LAGLIDADG homing endonucleases (LHEs). Biochemical and structural characterization of endonucleases from one clade within the phylogenetic tree demonstrates strong conservation of protein structure contrasted against highly diverged DNA target sites and indicates that a significant fraction of these proteins are sufficiently stable and active to serve as engineering scaffolds. This information was exploited to create a targeting enzyme to disrupt the endogenous monoamine oxidase B gene in human cells. The ubiquitous presence and diversity of LHEs described in this study may facilitate the creation of many tailored nucleases for genome editing.

Comprehensive computational design of mCreI homing endonuclease cleavage specificity for genome engineering

Homing endonucleases (HEs) cleave long (∼20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements. In order to determine whether comprehensive computational design could be used as a general strategy to engineer new HE target site specificities, we used RosettaDesign (RD) to generate 3200 different variants of the mCreI LAGLIDADG HE towards 16 different base pair positions in the 22 bp mCreI target site. Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved. These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.

IDENTIFICATION OF CONSERVED FEATURES OF LAGLIDADG HOMING ENDONUCLEASES

LAGLIDADG family of homing endonucleases are rare-cutting enzymes which recognize long target sequences and are of great interest in genome engineering. Despite advances in homing endonuclease engineering, effective methods of broadening the range of cleaved sequences are still lacking. Here, we present a study of conserved structural features of LAGLIDADG homing endonucleases that might aid further development of such methods. The protein-DNA interface of LAGLIDADG homing endonucleases differs considerably with the particular nuclease, and the analysis of conserved protein-DNA interactions could not identify any residues crucial for DNA binding and common to most nucleases of the family. For the homing endonuclease PI-SceI, a comparison of structural and experimental data derived from literature helped to identify 23 residues that are likely to be important for DNA binding. Analysis of the LAGLIDADG domain dimerization interface allowed the choosing of six positions that contribute to dimerization specificity most, while comparison of 446 sequences of LAGLIDADG endonucleases revealed groups of residues in these positions that appear to be most favorable for dimerization.

Genes within Genes: Multiple LAGLIDADG Homing Endonucleases Target the Ribosomal Protein S3 Gene Encoded within an rnl Group I Intron of Ophiostoma and Related Taxa

In some ascomycete fungi, ribosomal protein S3 (Rps3) is encoded within a group I intron (mL2449) that is inserted in the U11 region of the mitochondrial large subunit rDNA (rnl) gene. Previous characterization of the mL2449 intron in strains of Ophiostoma novo-ulmi subspecies americana (Dutch Elm Disease) revealed a complex genes-within-genes arrangement whereby a LAGLIDADG homing endonuclease gene (HEG) is inserted into the RPS3 gene near the 3' terminus, creating a hybrid Rps3-LAGLIDADG fusion protein. Here, we examined 119 additional strains of Ophiostoma and related taxa representing 85 different species by a polymerase chain reaction- based survey and detected both short (approximately 1.6 kb) and long (>2.2 kb) versions of the mL2449 intron in 88 and 31 strains, respectively. Among the long versions encountered, 21 were sequenced, revealing the presence of either intact or degenerated HEG-coding regions inserted within the RPS3 gene. Surprisingly, we identified two new HEG insertion sites in RPS3; one near the original C-terminal insertion site and one near the N-terminus of RPS3. In all instances, the HEGs are fused in-frame with the RPS3-coding sequences to create fusion proteins. However, comparative sequence analysis showed that upon insertion, the HEGs displaced a portion of the RPS3-coding region. Remarkably, the displaced RPS3-coding segments are duplicated and fused in-frame to the 3' end of RPS3, restoring a full-length RPS3 gene. We cloned and expressed the LAGLIDADG portion of two Rps3-HEG fusions, and showed that I-OnuI and I-LtrI generate 4 nucleotide (nt), 3' overhangs, and cleave at or 1 nt upstream of the HEG insertion site, respectively. Collectively, our data indicate that RPS3 genes are a refuge for distinct types of LAGLIDADG HEGs that are defined by the presence of duplicated segments of the host gene that restore the RPS3 gene, thus minimizing the impact of the HEG insertion on Rps3 function.

Engineering XID(Btk) site specific Homing Endonucleases for gene repair in hematopoietic stem cells (42.19)

Abstract X-linked agammaglobulinemia (XLA), a human immunodeficiency caused by mutations in Btk, represents an ideal candidate target for HSC gene therapy. However, current gene replacement methods carry risks of insertional mutagenesis as well as gene silencing. In contrast, gene repair induced by site specific DNA double strand breaks (DSB) offers a novel means to treat genetic disease with reduced risk and the potential to utilize endogenous gene expression control elements. LAGLIDADG Homing Endonucleases (LHEs) have been shown to efficiently induce site specific DSB and markerless modification of genes without toxicity. Due to the limited number of LHEs currently available, engineering site specific LHEs represents a critical step for enabling technology for DSB-induced gene modification. In the current study we have begun to generate a site specific variant of the LHE I-AniI, targeted to recognize a unique mutation in the mouse BTK gene in the XID model of human XLA. Our approaches combine: protein computational design with random mutation and selection using yeast library surface display; or alternatively, iterative somatic hyper-mutation and selection within DT40 B cell library targeted to the light chain locus. Using these combinatorial methods we have generated a panel of I-AniI variants with novel DNA binding and cleavage specificity towards the XID site. Preliminary characterization of a subset of these I-AniI variants will be presented. Ultimately, new I-Anil variants and donor DNA repair template will be delivered into HSC derived from XID/Tec-/- mice; and evaluated for capacity to re-engraft and reconstitute B lineage development and function in vivo.

Generation of single-chain LAGLIDADG homing endonucleases from native homodimeric precursor proteins

Homing endonucleases (HEs) cut long DNA target sites with high specificity to initiate and target the lateral transfer of mobile introns or inteins. This high site specificity of HEs makes them attractive reagents for gene targeting to promote DNA modification or repair. We have generated several hundred catalytically active, monomerized versions of the well-characterized homodimeric I-CreI and I-MsoI LAGLIDADG family homing endonuclease (LHE) proteins. Representative monomerized I-CreI and I-MsoI proteins (collectively termed mCreIs or mMsoIs) were characterized in detail by using a combination of biochemical, biophysical and structural approaches. We also demonstrated that both mCreI and mMsoI proteins can promote cleavage-dependent recombination in human cells. The use of single chain LHEs should simplify gene modification and targeting by requiring the expression of a single small protein in cells, rather than the coordinate expression of two separate protein coding genes as is required when using engineered heterodimeric zinc finger or homing endonuclease proteins.

Recognition of a common rDNA target site in archaea and eukarya by analogous LAGLIDADG and His–Cys box homing endonucleases

The presence of a homing endonuclease gene (HEG) within a microbial intron or intein empowers the entire element with the ability to invade genomic targets. The persistence of a homing endonuclease lineage depends in part on conservation of its DNA target site. One such rDNA sequence has been invaded both in archaea and in eukarya, by LAGLIDADG and His–Cys box homing endonucleases, respectively. The bases encoded by this target include a universally conserved ribosomal structure, termed helix 69 (H69) in the large ribosomal subunit. This region forms the ‘B2a’ intersubunit bridge to the small ribosomal subunit, contacts bound tRNA in the A- and P-sites, and acts as a trigger for ribosome disassembly through its interactions with ribosome recycling factor. We have determined the DNA-bound structure and specificity profile of an archaeal LAGLIDADG homing endonuclease (I-Vdi141I) that recognizes this target site, and compared its specificity with the analogous eukaryal His–Cys box endonuclease I-PpoI. These homodimeric endonuclease scaffolds have arrived at similar specificity profiles across their common biological target and analogous solutions to the problem of accommodating conserved asymmetries within the DNA sequence, but with differences at individual base pairs that are fine-tuned to the sequence conservation of archaeal versus eukaryal ribosomes.

Thermodynamics of DNA target site recognition by homing endonucleases

The thermodynamic profiles of target site recognition have been surveyed for homing endonucleases from various structural families. Similar to DNA-binding proteins that recognize shorter target sites, homing endonucleases display a narrow range of binding free energies and affinities, mediated by structural interactions that balance the magnitude of enthalpic and entropic forces. While the balance of ΔH and TΔS are not strongly correlated with the overall extent of DNA bending, unfavorable ΔHbinding is associated with unstacking of individual base steps in the target site. The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured. The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific ΔSbinding.

Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases

LAGLIDADG homing endonucleases (LHEs) cleave 18–24 bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which facilitates analysis of their DNA binding and cleavage properties by flow cytometry. Cells expressing surface LHEs can be stained with fluorescently conjugated double-stranded oligonucleotides (dsOligos) containing their respective target sequences. The signal is absolutely sequence specific and undetectable with dsOligos carrying single base-pair substitutions. LHE–dsOligo interactions facilitate rapid enrichment and viable recovery of rare LHE expressing cells by both fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS). Additionally, dsOligos conjugated with unique fluorophores at opposite termini can be tethered to the cell surface and used to detect DNA cleavage. Recapitulation of DNA binding and cleavage by surface-displayed LHEs provides a high-throughput approach to library screening that should facilitate rapid identification and analysis of enzymes with novel sequence specificities.

Genome Comparison of Pseudomonas aeruginosa Large Phages

Pseudomonas aeruginosa phage EL is a dsDNA phage related to the giant phiKZ-like Myoviridae. The EL genome sequence comprises 211,215 bp and has 201 predicted open reading frames (ORFs). The EL genome does not share DNA sequence homology with other viruses and micro-organisms sequenced to date. However, one-third of the predicted EL gene products (gps) shares similarity (Blast alignments of 17-55% amino acid identity) with phiKZ proteins. Comparative EL and phiKZ genomics reveals that these giant phages are an example of substantially diverged genetic mosaics. Based on the position of similar EL and phiKZ predicted gene products, five genome regions can be delineated in EL, four of which are relatively conserved between EL and phiKZ. Region IV, a 17.7 kb genome region with 28 predicted ORFs, is unique to EL. Fourteen EL ORFs have been assigned a putative function based on protein similarity. Assigned proteins are involved in DNA replication and nucleotide metabolism (NAD+-dependent DNA ligase, ribonuclease HI, helicase, thymidylate kinase), host lysis and particle structure. EL-gp146 is the first chaperonin GroEL sequence identified in a viral genome. Besides a putative transposase, EL harbours predicted mobile endonucleases related to H-N-H and LAGLIDADG homing endonucleases associated with group I intron and intein intervening sequences.

Metal-Dependent DNA Cleavage Mechanism of the I-CreI LAGLIDADG Homing Endonuclease,

The LAGLIDADG homing endonucleases include free-standing homodimers, pseudosymmetric monomers, and related enzyme domains embedded within inteins. DNA-bound structures of homodimeric I-CreI and monomeric I-SceI indicate that three catalytic divalent metal ions are distributed across a pair of overlapping active sites, with one shared metal participating in both strand cleavage reactions. These structures differ in the precise position and binding interactions of the metals. We have studied the metal dependence for the I-CreI homodimer using site-directed mutagenesis of active site residues and assays of binding affinity and cleavage activity. We have also reassessed the binding of a nonactivating metal ion (calcium) in the wild-type enzyme-substrate complex, and determined the DNA-bound structure of two inactive enzyme mutants. The conclusion of these studies is that the catalytic mechanism of symmetric LAGLIDADG homing endonucleases, and probably many of their monomeric cousins, involves a canonical two-metal mechanism in each of two active sites, which are chemically and structurally tethered to one another by a shared metal ion. Failure to occupy the shared metal site, as observed in the presence of calcium or when the metal-binding side chain from the LAGLIDADG motif (Asp 20) is mutated to asparagine, prevents cleavage by the enzyme.

The Crystal Structure of the Gene Targeting Homing Endonuclease I- SceI Reveals the Origins of its Target Site Specificity

The I- SceI homing endonuclease enhances gene targeting by introducing double-strand breaks at specific chromosomal loci, thereby increasing the recombination frequency. Here, we report the crystal structure of the enzyme complexed to its DNA substrate and Ca 2+ determined at 2.25 Å resolution. The structure shows the prototypical β-saddle of LAGLIDADG homing endonucleases that is contributed by two pseudo-symmetric domains. The high specificity of I- SceI is explained by the large number of protein–DNA contacts, many that are made by a long β-hairpin loop that reaches into the major groove of the DNA. The DNA minor groove is compressed at the catalytic center, bringing the two scissile phosphodiester bonds into close proximity. The protein–Ca 2+–DNA structure shows the protein bound to its DNA substrate in a pre-reactive state that is defined by the presence of two asymmetric active sites, one of which appears poised to first cleave the DNA bottom strand.

Unusual evolutionary history of the tRNA splicing endonuclease EndA: relationship to the LAGLIDADG and PD-(D/E)XK deoxyribonucleases.

The tRNA splicing endoribonuclease EndA from Methanococcus jannaschii is a homotetramer formed via heterologous interaction between the two pairs of homodimers. Each monomer consists of two alpha/beta domains, the N-terminal domain (NTD) and the C-terminal domain (CTD) containing the RNase A-like active site. Comparison of the EndA coordinates with the publicly available protein structure database revealed the similarity of both domains to site-specific deoxyribonucleases: the NTD to the LAGLIDADG family and the CTD to the PD-(D/E)XK family. Superposition of the NTD on the catalytic domain of LAGLIDADG homing endonucleases allowed a suggestion to be made about which amino acid residues of the tRNA splicing nuclease might participate in formation of a presumptive cryptic deoxyribonuclease active site. On the other hand, the CTD and PD-(D/E)XK endonucleases, represented by restriction enzymes and a phage lambda exonuclease, were shown to share extensive similarities of the structural framework, to which entirely different active sites might be attached in two alternative locations. These findings suggest that EndA evolved from a fusion protein with at least two distinct endonuclease activities: the ribonuclease, which made it an essential "antitoxin" for the cells whose RNA genes were interrupted by introns, and the deoxyribonuclease, which provided the means for homing-like mobility. The residues of the noncatalytic CTDs from the positions corresponding to the catalytic side chains in PD-(D/E)XK deoxyribonucleases map to the surface at the opposite side to the tRNA binding site, for which no function has been implicated. Many restriction enzymes from the PD-(D/E)XK superfamily might have the potential to maintain an additional active or binding site at the face opposite the deoxyribonuclease active site, a property that can be utilized in protein engineering.

Rapid evolution of the DNA-binding site in LAGLIDADG homing endonucleases.

Sequence analysis of chloroplast and mitochondrial large subunit rRNA genes from over 75 green algae disclosed 28 new group I intron-encoded proteins carrying a single LAGLIDADG motif. These putative homing endonucleases form four subfamilies of homologous enzymes, with the members of each subfamily being encoded by introns sharing the same insertion site. We showed that four divergent endonucleases from the I-CreI subfamily cleave the same DNA substrates. Mapping of the 66 amino acids that are conserved among the members of this subfamily on the 3-dimensional structure of I-CreI bound to its recognition sequence revealed that these residues participate in protein folding, homodimerization, DNA recognition and catalysis. Surprisingly, only seven of the 21 I-CreI amino acids interacting with DNA are conserved, suggesting that I-CreI and its homologs use different subsets of residues to recognize the same DNA sequence. Our sequence comparison of all 45 single-LAGLIDADG proteins identified so far suggests that these proteins share related structures and that there is a weak pressure in each subfamily to maintain identical protein-DNA contacts. The high sequence variability we observed in the DNA-binding site of homologous LAGLIDADG endonucleases provides insight into how these proteins evolve new DNA specificity.

Identification of Lys-403 in the PI-SceI Homing Endonuclease as Part of a Symmetric Catalytic Center

Superposition of the PI-SceI and I-CreI homing endonuclease three-dimensional x-ray structures indicates general similarity between the I-CreI homodimer and the PI-SceI endonuclease domain. Saddle-shaped structures are present in each protein that are proposed to bind DNA. At the putative endonucleolytic active sites, the superposition reveals that two lysine (Lys-301 and Lys-403 in PI-SceI and Lys-98 and Lys-98' in I-CreI) and two aspartic acid residues (Asp-218 and Asp-326 in PI-SceI and Asp-20 and Asp-20' in I-CreI) are related by 2-fold symmetry. The critical role of Lys-301, Asp-218, and Asp-326 in the PI-SceI reaction pathway was reported previously. Here, we demonstrate the significance of the active-site symmetry by showing that alanine substitution at Lys-403 reduces cleavage activity by greater than 50-fold but has little effect on the DNA binding activity of the mutant enzyme. Substitution of Lys-403 with arginine, which maintains the positive charge, has only a modest effect on activity. Interestingly, even though the Lys-301 and Lys-403 residues display pseudosymmetry, PI-SceI mutant proteins with substitutions at these positions have different behaviors. The presence of similar basic and acidic residues in many LAGLIDADG homing endonucleases suggests that these enzymes use a common reaction mechanism to cleave double-stranded DNA.