Homing Endonucleases: from Microbial Genetic Invaders to Reagents for Targeted DNA Modiﬁcation

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector Structure Review

Homing Endonucleases: From Microbial Genetic Invaders to Reagents for Targeted DNA Modiﬁcation

Barry L. Stoddard1,* 1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., A3-025, Seattle, WA 98109, USA *Correspondence: [email protected] DOI 10.1016/j.str.2010.12.003

Homing endonucleases are microbial DNA-cleaving enzymes that mobilize their own reading frames by generating double strand breaks at specific genomic invasion sites. These proteins display an economy of size, and yet recognize long DNA sequences (typically 20 to 30 base pairs). They exhibit a wide range of fidelity at individual nucleotide positions in a manner that is strongly influenced by host constraints on the coding sequence of the targeted gene. The activity of these proteins leads to site-specific recombination events that can result in the insertion, deletion, mutation, or correction of DNA sequences. Over the past fifteen years, the crystal structures of representatives from several homing endonuclease families have been solved, and methods have been described to create variants of these enzymes that cleave novel DNA targets. Engineered homing endonucleases proteins are now being used to generate targeted genomic modifications for a variety of biotech and medical applications.

Endonuclease enzymes are ubiquitous catalysts that are A series of studies conducted in several laboratories over the involved in genomic modification, rearrangement, protection, ensuing years led to the discovery of homing endonucleases and and repair. Their specificity spans at least nine orders of magni- mobile introns and inteins within a wide variety of additional tude, ranging from nonspecific degradative enzymes up to microbial genomes (reviewed in Belfort and Perlman, 1995). a variety of gene-specific endonucleases. The most specific of The transfer, duplication, and transmission of these sequences these latter enzymes, termed homing endonucleases, generate was shown to be extremely efficient, leading to unidirectional double-strand breaks at individual loci in their host genomes, gene conversion events in diploid genomes (Jacquier and Dujon, and thereby drive site-specific gene conversion events. 1985), or genetic competition in mixed phage infections (Good- Homing is a process in which microbial self-splicing inter- rich-Blair and Shub, 1996). Homing was found to occur even vening sequences—group I or group II introns or inteins—are between different subcellular compartments in unrelated organ- specifically duplicated into recipient alleles of their host gene isms (Turmel et al., 1995) and to be capable of driving the rapid that lack such a sequence (Belfort and Perlman, 1995; Belfort spread of mobile introns into related target sites throughout and Roberts, 1997; Chevalier and Stoddard, 2001; Dujon, a broad range of biological hosts (Cho et al., 1998). 1989; Lambowitz and Belfort, 1993). The first observation of Although homing endonucleases can also be encoded by free- homing dates to experiments conducted at the Pasteur Institute standing reading frames, their association with self-splicing in the early 1970s. In these studies, investigators noted the domi- sequences allows them to invade highly conserved sequences nant inheritance of a genetic marker, termed ‘‘omega,’’ during in protein- and RNA-encoding host genes without disrupting yeast mating experiments (mitochondrial genes are passed on their function and then to persist in microbial genomes that are biparentally in such studies and are thus subject to Mendelian otherwise subject to strong selective pressure to eliminate laws of inheritance). ‘‘Omega’’ was determined to be located extraneous genetic elements (Edgell et al., 2000). The ability of within the mitochondrial gene that encodes the large ribosomal homing endonuclease genes to accumulate within microbial RNA subunit (LSrRNA) (Bolotin et al., 1971) and was inherited genomes is extraordinary. For example, fifteen separate homing- at near 100% frequency in experiments involving homozygous endonuclease genes correspond to 11% of the total coding ‘‘omega-plus’’ and ‘‘omega-minus’’ yeast strains (Netter et al., sequence of the T4 phage genome (Edgell et al., 2010). 1974). In subsequent experiments, omega was found to correspond to an intervening sequence (later recognized as a self- Intron-Encoded Proteins: Families and Structures splicing group I intron) (Bos et al., 1978; Faye et al., 1979). Homing endonucleases are extremely widespread and are found The dominant inheritance of this intron was eventually deter- in microbes from all biological kingdoms, as well as in those mined to be induced by a site-specific endonuclease (now organisms’ corresponding phage and viruses. Despite the close termed I-SceI) that is encoded by an open reading frame proximity and the frequent symbiotic relationship between multi- harbored within the intron sequence (Jacquier and Dujon, cellular eukaryotes and various microbial species, no examples 1985). This enzyme was shown to generate a DNA double-strand have been reported of homing endonuclease genes within break within a long DNA target sequence in the LSrRNA gene genomes of those more complex organisms. that contains the eventual intron insertion site. Repair via homol- At least five different families of homing endonucleases have ogous recombination, while using the intron-containing allele as been identified and characterized; each is primarily associated a corrective template, leads to duplication of the intron and its with a particular biological host range (Stoddard, 2005). Bioinfor- endonuclease gene into the target site (Figure 1). matic and crystallographic studies of representatives from each

upon antiparallel b sheets that dock into the major grooves of their DNA target sites (Flick et al., 1998; Jurica et al., 1998). There they establish a collection of sequence-specific and nonspecific contacts that are distributed nonuniformly across multiple consecutive base pairs (Chevalier et al., 2003; Scalley-Kim et al., 2007)(Figure 3). In contrast, the less specific homing endonucleases found primarily in phage and bacteria form a more heterogeneous collection of DNA contacts within both the major and minor groove of their target sites. The phage-derived GIY- YIG and HNH enzymes (typified by I-TevI and I-HmuI, respectively) (see Figure 2A) display extended, multidomain protein structures in which disparate structural elements (including individual a helices, zinc fingers, and/or helix-turn-helix domains) contact a series of DNA regions that can span almost 30 base pairs in length (Shen et al., 2004; Van Roey et al., 2001). Finally, examinations of microbial and metagenomic sequence databases indicate that additional homing endonuclease fami- Figure 1. Homing Endonucleases and Genetic Homing (A) Mobile element consisting of a homing endonuclease gene (red bar) that is lies remain to be discovered and characterized. A recent analysis embedded within a self-splicing intron or intein (blue bars) resides within a host of the Global Ocean Sampling (GOS) metagenomic sequence gene (gray bars). The homing endonuclease (red star) is expressed and database (initially with the goal of finding novel split genes) cleaves a target site (green bar) that is found in a homologous allele of the host gene lacking the entire element. The resulting double-strand break is resulted in the discovery of a previously undescribed homing repaired by cellular machinery, generally leading either to repair via nonhomol- endonuclease family (Dassa et al., 2009). These proteins display ogous end-joining (not shown) or via homologous recombination (HR). If HR a unique domain organization, with a putative N-terminal DNA successfully uses the intron-containing host allele (I+) as a corrective template, binding region fused to a C-terminal catalytic domain that then the original uninterrupted allele (intron-minus [I-]) is converted to an allele that now contains the intron and homing endonuclease gene (intron-plus [I+]). resembles the very short patch repair (Vsr) endonuclease. The (B) A variety of biotechnology and gene therapy applications can potentially Vsr endonuclease is a bacterial mismatch repair enzyme that make use of the properties of a homing endonuclease, if such an enzyme is contains a variation of the canonical PD-(D/E)xK active site. introduced into or expressed in a living cell, to drive a gene conversion process. Depending on the presence or absence (as well as the sequence) Therefore, two different forms of the PD-(D/E)xK catalytic motif of a corrective DNA template for break repair, and on the catalytic properties appear to be found in two very different homing endonuclease of the endonuclease, such applications can lead to mutation, knockout, modi- lineages (similar to the HNH nuclease motif, as described above). fication, or insertion of exogenous coding DNA into the gene target. Subsequent to its identification in the metagenomic sequence database, a representative of this new family was found to of these families (Figure 2) have indicated that they contain already have been sequenced within the Bacillus thuriengensis unique catalytic cores and are presumably descended from phage 0305f genome. However, this protein was not annotated different ancestral nucleases. Three of these families (corre- until its relatives were identified based on their position within sponding to the GIY-YIG, PD-(D/E)xK, and LAGLIDADG homing metagenomic intervening sequence elements. This protein has endonucleases) are largely constrained to phage, bacterial, and now been renamed I-Bth0305I (B.L. Stoddard, unpublished archael/eukaryotic hosts, respectively. Two additional structural data), following the nomenclature convention established for families of homing endonucleases that each contain a permuta- homing endonucleases (Roberts et al., 2003). tion of an ‘‘HNH’’ nuclease active site have also evolved. Enzymes from these latter families display completely different Adaptation of Homing Endonucleases for Host-Specific protein scaffolds that are optimized for DNA homing in phage Functions and in protists, respectively. In general, homing endonucleases have evolved to act as oppor- Crystallographic structures of DNA-bound representatives tunistic selfish DNA that provide little benefit (but also no signif- from each of the known homing endonuclease families (Flick icant cost) to their hosts (Stoddard and Belfort, 2010). However, et al., 1998; Jurica et al., 1998; Moure et al., 2002, 2003; Shen a fraction of homing endonucleases have acquired a more bene- et al., 2004; Van Roey et al., 2001; Zhao et al., 2007) have ficial role for their hosts. In bacteriophage, some homing endo- provided a clear answer to the question of how these small nucleases confer an advantage during mixed infections by proteins recognize their long target sites. Two strategies are specifically cleaving the DNA of competing phage (Goodrich- observed: they either form highly elongated protein folds with Blair and Shub, 1996). At least one homing endonuclease minimal hydrophobic cores (Shen et al., 2004; Van Roey et al., (I-TevI) acts as a transcriptional regulator by autorepressing its 2001), or they multimerize and thereby double their DNA-contact own transcription (Edgell et al., 2004a). Many homing endonu- surface (Flick et al., 1998; Jurica et al., 1998; Zhao et al., 2008). cleases can also participate in the posttranscriptional splicing The latter strategy is employed at the cost of being constrained of their host intron by assisting the folding of their cognate to the recognition of DNA sequences with significant palindromic RNA intron, a function termed ‘‘maturase’’ activity (Delahodde symmetry. et al., 1989; Geese et al., 2003; Goguel et al., 1992; Henke DNA recognition mechanisms vary widely across homing et al., 1995; Ho et al., 1997; Longo et al., 2005; Szczepanek endonucleases. The LAGLIDADG and His-Cys box enzymes and Lazowska, 1996; Wenzlau et al., 1989). Finally, some homing (which are the most sequence-specific of these enzymes) rely endonucleases have been adopted by the host to act directly as

Figure 2. Homing Endonuclease Structural Families In all panels, divalent cations associated with the enzyme active sites are indicated by light green spheres. Bound zinc ions (D) are indicated by smaller dark green spheres. (A) The phage-encoded HNH endonuclease I-HmuI (Shen et al., 2004) and GIY-YIG endonuclease I-TevI (Van Roey et al., 2001, 2002). These proteins display an extended monomeric structure consisting of an N-terminal nuclease catalytic domain and a C-terminal DNA binding region that incorporates a C-terminal helix-turn-helix domain. The diagram of I-TevI is a composite of two separate crystal structures of the C-terminal region bound to DNA, and the unbound catalytic domain. (B) The cyanobacterial PD-(D/E)xK homing endonuclease I-Ssp6803I (a tetrameric intron-associated endonuclease that recognizes a target site in a tRNA host gene) (Zhao et al., 2007). (C) The algal LAGLIDADG homing endonuclease I-CreI, which is a homodimer that recognizes a target site in the 23S rRNA-encoding gene in the chloroplast genome of Chlamydomonas rein- hardtii (Jurica et al., 1998). Similar endonucleases are encoded in fungal mitochondrial genomes and in archaea. These enzymes are found both as homodimers and as tandem repeats of two LAGLIDADG domains that form single chain monomeric proteins. (D) The His-Cys box homing endonuclease I-PpoI, which is encoded in the nuclear genome in the slime mold Physarum polycephalum (Flick et al., 1998). This enzyme contains a variant of the HNH nuclease active site, and is therefore distantly related to the phage endonuclease I-HmuI (A), but the catalytic cores of these two enzymes have been incorporated into two very different structural scaffolds. freestanding endonucleases that drive biologically important resembling the I-PpoI endonuclease (Grishin, 2001). Similarly, gene conversion events. For example, the HO endonuclease in the DNA binding domain of the AP2/ERF family of plant tran- yeast, which is responsible for the mating-type genetic switch scription regulators contain a recognizable HNH endonuclease in that organism, is a LAGLIDADG protein that appears to be domain (Magnani et al., 2004). derived from an intein-associated homing endonuclease (Jin Finally, two separate bioinformatics reports (Knizewski and et al., 1997). Ginalski, 2007; Xie et al., 2007) and a recent crystallographic In addition to the direct employment of homing endonucleases analysis (Kaiser et al., 2009) have demonstrated that proteins for host-specific activities, more distant relatives of these from the bacterial DUF199 gene family contain highly diverged proteins are used for a wide variety of biological functions LAGLIDADG domains (which no longer contain recognizable (Figure 4). The HNH catalytic motif is found in a wide variety of nuclease active site residues) fused to helix-turn-helix domains. nonspecific bacterial and fungal nucleases, (Friedhoff et al., The best studied of these factors (known as ‘‘WhiA’’ from 1999; Kuhlmann et al., 1999), structure-specific endonucleases Streptomyces coelicolor) appear to act as an autoregulatory (Biertumpfel et al., 2007), restriction endonucleases such as transcription factor for an operon-like cluster of genes that R.Hpy99I and R.PacI (Shen et al., 2010; Sokolowska et al., includes its own reading frame (Ainsa et al., 2000). Found in virtu- 2009), and many transposases, polymerase editing domains, ally all gram-positive bacteria, the DUF199/WhiA proteins are and DNA packaging factors (Dalgaard et al., 1997; Mehta hypothesized to be involved in the regulation of networks of et al., 2004). Similarly, the GIY-YIG catalytic motif is also found genes and proteins that are involved in growth transitions during in bacterial restriction enzymes such as R.Eco29kI and various bacterial life cycles. R.Hpy88I (Mak et al., 2010; Sokolowska et al., 2010), and in a wide variety of enzymes involved in DNA repair, recombination, Engineering Gene Targeting Reagents and fidelity (reviewed in Dunin-Horkawicz et al., 2006). with LAGLIDADG Proteins In the cases outlined above, the host-specific activities still The LAGLIDADG homing endonuclease (LHE) family (Figure 2C), involve the ability of these enzymes to catalyze phosphotransfer which is the primary source of the enzymes that are used for reactions or to promote the rearrangement of nucleic acid gene targeting applications, is primarily encoded within archaea substrates. However, at least three instances have been docu- and in the chloroplast and mitochondrial genomes of algae and mented where the biological function of a homing endonuclease fungi (Chevalier et al., 2005; Dalgaard et al., 1997; Sethuraman scaffold has been more dramatically altered—with the protein et al., 2009). These enzymes are often referred to as ‘‘Meganu- in each case finding new employment as a transcription factor. cleases,’’ a term that reflects the name ‘‘Omega’’ that was In the first example, the DNA-binding domains found in ‘‘Smad’’ coined for the first known homing system as well as the length proteins (eukaryotic transcription factors involved in TGF-b of their DNA targets and the exceptional DNA cleavage speci- signaling) were found to be comprised of an endonuclease fold ficity displayed by most of these enzymes (Paques and

Figure 4. Evolution of Homing Endonucleases and Disparate Host Gene Products and Functions from Common Nuclease Ancestors (A) The GIY-YIG nuclease motif (center) has given rise both to phage-specific,- monomeric homing endonucleases such as I-TevI (left) (Van Roey et al., 2001, 2002), and to multimeric bacterial restriction endonucleases such as R.Eco29k (right) (Mak et al., 2010). The GIY-YIG catalytic motif in the core nuclease fold is shown in red in the upper panel. (B) The LAGLIDADG fold (with its namesake catalytic motif shown in red in the center panel) has given rise to monomeric and homodimeric homing endonucleases (such as the I-AniI endonuclease from Aspergillus nidulans, left) (Bol- duc et al., 2003), and to the broadly distributed DUF199 gene family, found in gram positive bacteria (right) (Kaiser et al., 2009; Knizewski and Ginalski, Figure 3. The DNA-Binding Surface and Contacts Formed by One 2007). In this latter gene family, the LAGLIDADG scaffold is fused to a C- Subunit of the I-CreI Homing Endonuclease terminal helix-turn-helix domain; members of this bacterial protein family (A) An antiparallel b sheet structure presents a group of broadly distributed side (such as the WhiA protein from Streptomyces coelicolor) are thought to act chains to the major groove of the DNA target site, where they make a variety of as genetic regulators during processes such as sporulation (Ainsa et al., 2000). specific and nonspecific contacts. (B) A schematic of the contacts made by the structure shown in (A). Hydrogen- bond acceptor and donor positions on each base pair are shown with concave and convex features on each base. Direct contacts are shown with red arrows; the individual halves of these DNA targets are largely segregated water-mediated contacts are shown with blue arrows and blue spheres, which to the two corresponding protein domains or subunits. A number denote the water molecules in the crystal structure. Individual base pairs of studies have indicated that LAGLIDADG proteins display high display a wide variation in the total number of contacts to protein atoms; this fidelity at many of the base pair positions in their target sites variation correlates approximately with the fidelity of recognition that is displayed at each position. Additional specificity is derived by indirect exploitation (Gimble et al., 2003; Scalley-Kim et al., 2007; Thyme et al., of sequence-specific conformational preferences of the DNA target, which is 2009). Those positions that display reduced fidelity often corre- often bent as part of endonuclease binding and cleavage. spond to the base pairs in the reading frame of the host gene that display coding degeneracy or ‘‘wobble’’ (Edgell et al., 2004b; Duchateau, 2007). LAGLIDADG homing endonucleases are the Gimble et al., 2003; Scalley-Kim et al., 2007). Much of their scaffold of choice, in addition to zinc finger nucleases, for use cleavage specificity appears to be realized at the transition state in genetic and cell biology applications that require the genera- of the reaction (Jarjour et al., 2009; Thyme et al., 2009). In other tion of targeted DNA strand breaks at individual chromosomal words, these proteins may bind many related DNA sequences loci. with comparable affinities, but they actually cleave a much LAGLIDADG endonucleases exist both as homodimers (where smaller subset of sequences. the two identical protein subunits are each typically 160 to 200 The sum of fidelities that is exhibited at individual DNA base residues in size), and as monomeric proteins where a tandem pair positions by a typical LAGLIDADG enzyme results in a con- repeat of two LAGLIDADG domains is connected by a variable servative estimate for their overall specificity during in vitro peptide linker. Compared with their homodimeric cousins, cleavage experiments of at least 1 in 107 to 108. Additional inter- the monomeric proteins are rather small; their individual dependence between adjacent base pair substitutions reduces domains are often only 100 to 120 residues in size. Monomeric this value to at least 1 in 109 (Argast et al., 1998; Arnould et al., LAGLIDADG enzymes can recognize fully asymmetric DNA 2006; Ashworth et al., 2010; Scalley-Kim et al., 2007). This value target sites, and they often display disparate rates of cleavage (which represents an enzyme’s activity and specificity under of the two DNA strands in their target sequences, thereby gener- optimal in vitro digest conditions in the absence of competing ating transient nicked DNA intermediates en route to the final protein factors) is likely to be a conservative estimate relative double-strand break (Geese et al., 2003; Moure et al., 2008). to the in vivo specificity displayed by a LAGLIDADG enzyme. The DNA-binding surfaces of LAGLIDADG endonucleases can Well before their first crystal structures had been determined, accommodate DNA targets of up to 24 base pairs; contacts to the sequence specificity of LAGLIDADG homing endonucleases

had been exploited in a series of experiments using the I-SceI patible with substitutions at adjacent positions. A recent struc- enzyme, which demonstrated the ability of LHEs to induce a tar- ture-based redesign approach using a closely related enzyme geted genetic modification in a complex eukaryotic genome. scaffold has recapitulated this result and provided a high-resolu- In these studies, I-SceI was expressed in transformed murine tion explanation for the unpredictable behavior of single-point cells, where its site-specific cleavage activity at a previously redesigns relative to ‘‘clusters’’ of altered protein-DNA contacts integrated I-SceI target site resulted in the generation of (Ashworth et al., 2010). a double-strand break and the eventual markerless modification At the same time that the experiments described above were of the organism’s germ line (Choulika et al., 1995; Rouet et al., being performed on contact points between amino acid side 1994). chains and DNA bases, several related studies demonstrated Based on the results in these and subsequent studies, it that entire domains or subunits from unrelated LAGLIDADG became obvious that modification of a homing endonuclease’s enzymes could be mixed and fused to create novel chimeric cleavage specificity would be required in order to target and homing endonucleases that recognize corresponding chimeric modify endogenous target sites in various biological genomes. DNA target sites (Chevalier et al., 2002; Epinat et al., 2003; Consequently, from 2002 to 2005, several academic groups Steuer et al., 2004). These studies demonstrated that the indi- and one biotech company described methods to create mutated vidual domains and subunits of LAGLIDADG enzymes are largely variants of LAGLIDADG enzymes that displayed altered DNA responsible for the recognition and binding of individual DNA recognition specificities, for the purpose of extending DNA tar- half-sites. Subsequent experiments reinforced this conclusion geting applications to endogenous chromosomal recognition (Fitzsimons-Hall et al., 2002; Steuer et al., 2004; Silva and sites (reviewed in Stoddard et al., 2007). These studies were Belfort, 2004; Silva et al., 2006). Together, these studies demon- facilitated by previously published descriptions of how protein- strated that the task of altering a homing endonuclease’s DNA recognition is largely accomplished by contacts with cleavage specificity can be ‘‘broken down’’ into two separate chemically compatible amino acid side chains (for example, as projects to individually target the left and right half-sites of summarized in Pabo and Sauer, 1992). a DNA target, by systematically altering the DNA-contacting resi- The earliest experiments to alter LAGLIDADG endonuclease dues of the protein’s N- and C-terminal domains and then specificity relied upon assays to visually identify mutated endo- combining the final solutions for each domain into a single nuclease constructs that displayed altered recognition speci- gene-targeting protein. ficity (Figure 5). These protocols utilized reporters of high affinity DNA binding (for example, through the use of a bacterial two- The Present and Future: Homing Endonuclease Variants hybrid screening strategy) (Gimble et al., 2003), or methods Directed toward Chromosomal Targets that coupled endonuclease activity to the elimination of a With tools now in hand to redirect the DNA cleavage specificities reporter gene (Doyon et al., 2006; Gruen et al., 2002; Rosen of homing endonucleases, these enzymes are being used to et al., 2006; Seligman et al., 2002; Sussman et al., 2004). A study fundamental questions that surround the molecular more direct assay method was described in 2005, in which endo- biology of DNA strand break repair and gene conversion. nuclease cleavage activity was coupled to the recombination For example, several laboratories have demonstrated that and reconstitution of a reporter gene (Chames et al., 2005). LAGLIDADG homing endonucleases can be altered to deliver Over the same period of time, experiments that relied solely on either double-strand breaks or single-strand breaks (i.e., nicks) structure-based redesign of the protein-DNA interface, either by in a site-specific manner (McConnell Smith et al., 2009; Niu direct physical examination or by the use of computational algo- et al., 2008; Silva and Belfort, 2004). Subsequent studies using rithms that repack and optimize new protein-DNA contacts, one of these altered endonucleases has demonstrated that were also discussed and reported (Ashworth et al., 2006; Cheva- a homing ‘‘nickase’’ can stimulate homologous recombination lier et al., 2003). Such methods can be used either to directly at the enzyme’s target site (albeit at a reduced efficiency as redesign homing endonuclease-DNA contacts at individual resi- compared with a double-strand break) but with greatly reduced dues (thus bypassing selection approaches altogether), or to frequency of end-joining and mutagenesis at that target, and facilitate more efficient mutational screening of enzyme libraries with a corresponding reduction in toxicity (Metzger et al., 2010). (by reducing the number of protein residues to be randomized). Such use of altered homing endonuclease activities are now By 2005, the literature contained multiple examples demon- being combined with new generations of cell-based reporter strating the mutation and alteration of DNA specificity of systems that can provide information on relative levels of a LAGLIDADG homing endonuclease (specifically I-CreI) at indi- competing strand break repair pathways (particularly homolo- vidual base pairs (Chames et al., 2005; Gruen et al., 2002; Selig- gous recombination versus end-joining) on a per cell basis. In man et al., 2002; Sussman et al., 2004). Soon thereafter, two such experiments, the strand cleavage activity and kinetic separate studies described high-throughput experiments to behaviors of a homing endonuclease, along with the genomic redirect endonuclease specificity across contiguous ‘‘pockets’’ background of the targeted cell population, can be systemati- of sequential base pairs (Arnould et al., 2006; Smith et al., cally varied and used to conduct a wide range of studies of 2006). In those experiments, alterations of specificity were found DNA repair and correction (Arnould et al., 2011). to display considerable context dependence. Alteration of indi- Using the protein design and selection approaches summa- vidual protein-DNA contacts that caused reduced activity or rized earlier, derivatives of the I-CreI homing endonuclease specificity were sometimes well tolerated in more extensively have been generated that display sequence-specific cleavage altered pockets; conversely, some alterations of protein-DNA and recombination activity against the human RAG1 gene at contacts that behaved well on their own were found to be incom- the site of mutations that give rise to a rare subset of severe

Figure 5. Selections and Direct Redesign of Homing Endonuclease Specificity (A) Several methods have been developed to assay the ability of mutated variants of homing endonucleases to recognize and cleave specific DNA target sites. These assays included the elimination of an integrated b-galactosidase gene in E. coli (top) (Seligman et al., 2002), the elimination of a cell death protein in E. coli (middle) (Doyon et al., 2006; Gruen et al., 2002), or the conversion of two inactive copies of a reporter gene into a single active copy as a result of endonuclease-induced recombination in yeast (bottom) (Chames et al., 2005). As described in the literature, this latter assay has been incorporated in to a high-throughput screen where activity against a large number of potential target sites is measured in parallel for individual homing endonuclease variants (Arnould et al., 2006; Smith et al., 2006). A change in the position of a positive clone in the array of possible targets (shown by arrows pointing to two adjacent colonies with different target sequences) demonstrates a shift in target site specificity. The basis of this latter assay, which directly measures endonuclease-induced recombination in a living cell, has been modified and used in many different eukaryotic cell contexts with a variety of reporters, particularly green fluorescent protein (GFP). (B) Direct examination of a homing endonuclease-DNA bound co-crystal structure, often with the use of structure-based computational tools, can also be used to redirect protein-DNA contacts and corresponding specificity (Ashworth et al., 2006; Chevalier et al., 2003). Such methods can be used directly to redirect specificity at individual contact positions, or can be used to focus subsequent mutations to reduced numbers of positions and possible mutated amino acid identities. combined immunodeficiency disease (or SCID) phenotypes tion group C) (Arnould et al., 2007) and dystrophin (which is asso- (Grizot et al., 2009). The altered I-CreI enzymes used in this study ciated with Duchenne muscular dystrophy) (Chapdelaine et al., induced high levels of gene correction via homologous recombi- 2010). Recently, strategies similar to those summarized above nation (up to 6% of the transformed cells) while exhibiting have been employed by a separate research group to create minimal toxicity. This study represented the first time that an a variant of the same well-behaved I-CreI enzyme that could engineered homing endonuclease was used in mammalian cells drive a targeted gene disruption event in corn (Gao et al., to target and modify a chromosomal target locus. 2010). This latter study demonstrated that the information and A number of subsequent studies reported by the same group tools developed over the past fifteen years by homing endonu- have described the production of additional homing endonuclease investigators may now be employed by a broader clease variants (each using the same wild-type I-CreI protein community of molecular biologists to create an increasingly as the initial scaffold) that are successfully directed at the human wide range of genome engineering tools for biotechnology, agri- genes encoding XPC (Xeroderma Pigmentosum complementa- culture, and medicine.

In concert with zinc-finger (Le Provost et al., 2010) and TAL Chapdelaine, P., Pichavant, C., Rousseau, J., Paques, F., and Tremblay, J.P. (transcription activator-like) proteins (Christian et al., 2010; Li (2010). Meganucleases can restore the reading frame of a mutated dystrophin. Gene Therapy 17, 846–858. et al., 2010), the discipline of site-specific genome engineering now enjoys a wealth of structural scaffolds for the continued Chevalier, B.S., and Stoddard, B.L. (2001). Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility. Nucleic Acids development of gene targeting proteins. Each of these proteins Res. 29, 3757–3774. displays properties that are particularly well suited for this field. Zinc-finger and TAL proteins contain highly modular architec- Chevalier, B.S., Kortemme, T., Chadsey, M.S., Baker, D., Monnat, R.J., Jr., and Stoddard, B.L. (2002). Design, activity and structure of a highly specific tures that appear broadly engineerable for unique DNA specific- artificial endonuclease. Mol. Cell 10, 895–905. ities, while LAGLIDADG homing endonuclease tightly couple Chevalier, B., Turmel, M., Lemieux, C., Monnat, R.J., and Stoddard, B.L. cognate site recognition to DNA strand cleavage activity and (2003). Flexible DNA target site recognition by divergent homing endonuclease possess small structures that can be coded by short reading isoschizomers I-CreI and I-MsoI. J. Mol. Biol. 329, 253–269. frames. Given the wide variety of applications that are envisioned Chevalier, B., Monnat, R.J., Jr., and Stoddard, B.L. (2005). The LAGLIDADG within this field, it seems likely that each of these protein scaf- homing endonuclease family. In Homing endonucleases and inteins, M. Bel- folds will find significant future employment in biotechnology fort, D. Wood, V. Derbyshire, and B. Stoddard, eds. (Berlin: Springer Verlag), and medicine. pp. 34–47. Cho, Y., Qiu, Y.-L., Kuhlman, P., and Palmer, J.D. (1998). Explosive invasion REFERENCES of plant mitochondria by a group I intron. Proc. Natl. Acad. Sci. USA 95, 14244–14249. Ainsa, J.A., Ryding, N.J., Hartley, N., Findlay, K.C., Bruton, C.J., and Chater, K.F. (2000). WhiA, a protein of unknown function conserved among gram-posi- Choulika, A., Perrin, A., Dujon, B., and Nicolas, J.F. (1995). Induction of homol- tive bacteria, is essential for sporulation in Streptomyces coelicolor A3(2). ogous recombination in mammalian chromosomes by using the I-SceI system J. Bacteriol. 182, 5470–5478. of Saccharomyces cerevisiae. Mol. Cell Biol. 15, 1968–1973.

Argast, G.M., Stephens, K.M., Emond, M.J., and Monnat, R.J., Jr. (1998). Christian, M., Cermak, T., Doyle, E.L., Schmidt, C., Zhang, F., Hummel, A., I-PpoI and I-CreI homing site sequence degeneracy determined by random Bogdanove, A.J., and Voytas, D.F. (2010). Targeting DNA double-strand mutagenesis and sequential in vitro enrichment. J. Mol. Biol. 280, 345–353. breaks with TAL effector nucleases. Genetics 186, 757–761.

Arnould, S., Chames, P., Perez, C., Lacroix, E., Duclert, A., Epinat, J.C., Dalgaard, J.Z., Klar, A.J., Moser, M.J., Holley, W.R., Chatterjee, A., and Mian, Stricher, F., Petit, A.S., Patin, A., Guillier, S., et al. (2006). Engineering of large I.S. (1997). Statistical modeling and analysis of the LAGLIDADG family of site- numbers of highly specific homing endonucleases that induce recombination specific endonucleases and identification of an intein that encodes a site- on novel DNA targets. J. Mol. Biol. 355, 443–458. specific endonuclease of the HNH family. Nucleic Acids Res. 25, 4626–4638.

Arnould, S., Perez, C., Cabaniols, J.-P., Smith, J., Gouble, A., Grizot, S., Dassa, B., London, N., Stoddard, B.L., Schueler-Furman, O., and Pietrokovski, Epinat, J.-C., Duclert, A., Duchateau, P., and Paques, F. (2007). Engineered S. (2009). Fractured genes: a novel genomic arrangement involving new split I-CreI derivatives cleaving sequences from the human XPC gene can induce inteins and a new homing endonuclease family. Nucleic Acids Res. 37, highly efﬁcient gene correction in mammalian cells. J. Mol. Biol. 371, 49–65. 2560–2573.

Arnould, S., Delenda, C., Grizot, S., Desseaux, C., Paques, F., Silva, G.H., and Delahodde, A., Goguel, V., Becam, A.M., Creusot, F., Perea, J., Banroques, J., Smith, J. (2011). The I-CreI meganuclease and its engineered derivatives: and Jacq, C. (1989). Site-speciﬁc DNA endonuclease and RNA maturase applications from cell modiﬁcation to gene therapy. Protein Eng. Des. Sel., activities of two homologous intron-encoded proteins from yeast mitochon- in press. Published online November 3, 2010. 10.1093/protein/gzq083. dria. Cell 56, 431–441.

Ashworth, J., Havranek, J.J., Duarte, C.M., Sussman, D., Monnat, R.J., Jr., Doyon, J.B., Pattanayak, V., Meyer, C.B., and Liu, D.R. (2006). Directed evolu- Stoddard, B.L., and Baker, D. (2006). Computational redesign of endonu- tion and substrate specificity profile of homing endonuclease I-SceI. J. Am. clease DNA binding and cleavage specificity. Nature 441, 656–659. Chem. Soc. 128, 2477–2484.

Ashworth, J., Taylor, G.K., Havranek, J.J., Quadri, S.A., Stoddard, B.L., and Dujon, B. (1989). Group I introns as mobile genetic elements: facts and mech- Baker, D. (2010). Computational reprogramming of homing endonuclease anistic speculations–a review. Gene 82, 91–114. speciﬁcity at multiple adjacent base pairs. Nucleic Acids Res. 38, 5601–5608. Dunin-Horkawicz, S., Feder, M., and Bujnicki, J.M. (2006). Phylogenomic Belfort, M., and Perlman, P.S. (1995). Mechanisms of intron mobility. J. Biol. analysis of the GIY-YIG nuclease superfamily. BMC Genomics 7,98. Chem. 270, 30237–30240. Edgell, D.R., Belfort, M., and Shub, D.A. (2000). Barriers to intron promiscuity Belfort, M., and Roberts, R.J. (1997). Homing endonucleases - keeping the in bacteria. J. Bacteriol. 182, 5281–5289. house in order. Nucleic Acids Res. 25, 3379–3388.

Biertumpfel, C., Yang, W., and Suck, D. (2007). Crystal structure of T4 endonu- Edgell, D.R., Derbyshire, V., Van Roey, P., LaBonne, S., Stanger, M.J., Li, Z., clease VII resolving a Holliday junction. Nature 449, 616–620. Boyd, T.M., Shub, D.A., and Belfort, M. (2004a). Intron-encoded homing endonuclease I-TevI also functions as a transcriptional autorepressor. Nat. Struct. Bolduc, J.M., Spiegel, P.C., Chatterjee, P., Brady, K.L., Downing, M.E., Mol. Biol. 11, 936–944. Caprara, M.G., Waring, R.B., and Stoddard, B.L. (2003). Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endo- Edgell, D.R., Stanger, M.J., and Belfort, M. (2004b). Coincidence of cleavage nuclease and group I intron splicing factor. Genes Dev. 17, 2875–2888. sites of intron endonuclease I-TevI and critical sequences of the host thymidy- late synthase gene. J. Mol. Biol. 343, 1231–1241. Bolotin, M., Coen, D., Deutsch, J., Dujon, B., Netter, P., Petrochilo, E., and Slonimski, P.P. (1971). La recombinaison des mitochondries chez Saccharo- Edgell, D.R., Gibb, E.A., and Belfort, M. (2010). Mobile DNA elements in T4 and myces cerevisiae. Bull. Inst. Pasteur 69, 215–239. related phages. Virol. J. 7, 290.

Bos, J.L., Heyting, C., Borst, P., Arnberg, A.C., and Van Bruggen, E.F. (1978). Epinat, J.C., Arnould, S., Chames, P., Rochaix, P., Desfontaines, D., Puzin, C., An insert in the single gene for the large ribosomal RNA in yeast mitochondrial Patin, A., Zanghellini, A., Paques, F., and Lacroix, E. (2003). A novel engineered DNA. Nature 275, 336–338. meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res. 31, 2952–2962. Chames, P., Epinat, J.C., Guillier, S., Patin, A., Lacroix, E., and Paques, F. (2005). In vivo selection of engineered homing endonucleases using double- Faye, G., Dennebouy, N., Kujawa, C., and Jacq, C. (1979). Inserted sequence strand break induced homologous recombination. Nucleic Acids Res. 33, in the mitochondrial 23S ribosomal RNA gene of the yeast Saccharomyces e178. cerevisiae. Mol. Gen. Genet. 168, 101–109.

Fitzsimons-Hall, M., Noren, C.J., Perler, F.B., and Schildkraut, I. (2002). Crea- Lambowitz, A.M., and Belfort, M. (1993). Introns as mobile genetic elements. tion of an artificial bifunctional intein by grafting a homing endonuclease into Ann. Rev. Biochem. 62, 587–622. a mini-intein. J. Mol. Biol. 323, 173–179. Le Provost, F., Lillico, S., Passet, B., Young, R., Whitelaw, B., and Vilotte, J.L. Flick, K.E., Jurica, M.S., Monnat, R.J., Jr., and Stoddard, B.L. (1998). DNA (2010). Zinc finger nuclease technology heralds a new era in mammalian binding and cleavage by the nuclear intron-encoded homing endonuclease transgenesis. Trends Biotechnol. 28, 134–141. I-PpoI. Nature 394, 96–101. Li, T., Huang, S., Jiang, W.Z., Wright, D., Spalding, M.H., Weeks, D.P., and Friedhoff, P., Franke, I., Meiss, G., Wende, W., Krause, K.L., and Pingoud, A. Yang, B. (2010). TAL nucleases (TALNs): hybrid proteins composed of TAL (1999). A similar active site for non-specific and specific endonucleases. effectors and FokI DNA-cleavage domain. Nucleic Acids Res., in press. Nat. Struct. Biol. 6, 112–113. Published online August 10, 2010. 10.1093/nar/gkq704.

Gao, H., Smith, J., Yang, M., Jones, S., Djukanovic, V., Nicholson, M.G., West, Longo, A., Leonard, C.W., Bassi, G.S., Berndt, D., Krahn, J.M., Tanaka-Hall, A., Bidney, D., Falco, S.C., Jantz, D., and Lyznik, L.A. (2010). Heritable T.M., and Weeks, K.M. (2005). Evolution from DNA to RNA recognition by targeted mutagenesis in maize using a designed endonuclease. Plant J 61, the bI3 LAGLIDADG maturase. Nat. Struct. Mol. Biol. 12, 779–787. 176–187. Magnani, E., Sjolander, K., and Hake, S. (2004). From endonucleases to tran- Geese, W.J., Kwon, Y.K., Wen, X., and Waring, R.B. (2003). In vitro analysis of scription factors: evolution of the AP2 DNA binding domain in plants. Plant Cell the relationship between endonuclease and maturase activities in the bi-func- 16, 2265–2277. tional group I intron-encoded protein, I-AniI. Eur. J. Biochem. 270, 1543–1554. Mak, A.N.-S., Lambert, A.R., and Stoddard, B.L. (2010). Folding, DNA recog- Gimble, F.S., Moure, C.M., and Posey, K.L. (2003). Assessing the plasticity of nition and function of GIY-YIG endonucleases: crystal structures of R.Eco29kI. DNA target site recognition of the PI-SceI homing endonuclease using a bacte- Structure 18, 1321–1331. rial two-hybrid selection system. J. Mol. Biol. 334, 993–1008. McConnell Smith, A., Takeuchi, R., Pellenz, S., Davis, L., Maizels, N., Monnat, Goguel, V., Delahodde, A., and Jacq, C. (1992). Connections between RNA R.J., Jr., and Stoddard, B.L. (2009). Generation of a nicking enzyme that splicing and DNA intron mobility in yeast mitochondria: RNA maturase and stimulates site-speciﬁc gene conversion from the I-AniI LAGLIDADG homing DNA endonuclease switching experiments. Mol. Cell. Biol. 12, 696–705. endonuclease. Proc. Natl. Acad. Sci. USA. 106, 5099–5104.

Goodrich-Blair, H., and Shub, D.A. (1996). Beyond homing: competition Mehta, P., Katta, K., and Krishnaswamy, S. (2004). HNH family subclassifica- between intron endonucleases confers a selective advantage on flanking tion leads to identification of commonality in the His-Me endonuclease super- genetic markers. Cell 84, 211–221. family. Protein Science 13, 295–300.

Grishin, N.V. (2001). Mh1 domain of Smad is a degraded homing endonu- Metzger, M.J., McConnell-Smith, A., Stoddard, B.L., and Miller, A.D. (2010). clease. J. Mol. Biol. 307, 31–37. Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV template. Nucleic Acids Res., in Grizot, S., Smith, J., Daboussi, F., Prieto, J., Redondo, P., Merino, N., Villate, press. Published online September 28, 2010. 10.1093/nar/gkq826. M., Thomas, S., Lemaire, L., Montoya, G., et al. (2009). Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Moure, C.M., Gimble, F., and Quiocho, F. (2002). Crystal structure of the intein Acids Res. 37, 5405–5419. homing endonuclease PI-SceI bound to its recognition sequence. Nature Struct. Biol. 9, 764–770. Gruen, M., Chang, K., Serbanescu, I., and Liu, D.R. (2002). An in vivo selection system for homing endonuclease activity. Nucleic Acids Res. 30, e29. Moure, C.M., Gimble, F.S., and Quiocho, F.A. (2003). The crystal structure of the gene targeting homing endonuclease I-SceI reveals the origins of its target Henke, R.M., Butow, R.A., and Perlman, P.S. (1995). Maturase and endonu- site specificity. J. Mol. Biol. 334, 685–696. clease functions depend on separate conserved domains of the bifunctional protein encoded by the group I intron aI4 alpha of yeast mitochondrial DNA. Moure, C.M., Gimble, F.S., and Quiocho, F.A. (2008). Crystal structures of EMBO J. 14, 5094–5099. I-SceI complexed to nicked DNA substrates: snapshots of intermediates along the DNA cleavage reaction pathway. Nucleic Acids Res. 36, 3287–3296. Ho, Y., Kim, S.J., and Waring, R.B. (1997). A protein encoded by a group I intron in Aspergillus nidulans directly assists RNA splicing and is a DNA endo- Netter, P., Petrochilo, E., Slonimski, P.P., Bolotin-Fukuhara, M., Coen, D., nuclease. Proc. Natl. Acad. Sci. USA 94, 8994–8999. Deutsch, J., and Dujon, B. (1974). Mitochondrial genetics. VII. Allelism and mapping studies of ribosomal mutants resistant to chloramphenicol, erythro- Jacquier, A., and Dujon, B. (1985). An intron-encoded protein is active in mycin and spiramycin in S. cerevisiae. Genetics 78, 1063–1100. a gene conversion process that spreads an intron into a mitochondrial gene. Cell 41, 383–394. Niu, Y., Tenney, K., Li, H., and Gimble, F.S. (2008). Engineering variants of the I-SceI homing endonuclease with strand-specific and site-specific DNA- Jarjour, J., West-Foyle, H., Certo, M.T., Hubert, C.G., Doyle, L., Getz, M.M., nicking activity. J. Mol. Biol. 382, 188–202. Stoddard, B.L., and Scharenberg, A.M. (2009). High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface Pabo, C.O., and Sauer, R.T. (1992). Transcription factors: Structural families display. Nucleic Acids Res. 37, 6871–6880. and principles of DNA recognition. Ann. Rev. Biochem. 61, 1053–1095.

Jin, Y., Binkowski, G., Simon, L.D., and Norris, D. (1997). Ho endonuclease Paques, F., and Duchateau, P. (2007). Meganucleases and DNA double-strand cleaves MAT DNA in vitro by an inefﬁcient stoichiometric reaction mechanism. break-induced recombination: perspectives for gene therapy. Current Gene J. Biol. Chem. 272, 7352–7359. Therapy 7, 49–66.

Jurica, M.S., Monnat, R.J., Jr., and Stoddard, B.L. (1998). DNA recognition Roberts, R.J., Belfort, M., Bestor, T., Bhagwat, A.S., Bickle, T.A., Bitinaite, J., and cleavage by the LAGLIDADG homing endonuclease I- CreI. Mol. Cell 2, Blumenthal, R.M., Degtyarev, S., Dryden, D.T., Dybvig, K., et al. (2003). A 469–476. nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res. 31, 1805–1812. Kaiser, B.K., Clifton, M.C., Shen, B.W., and Stoddard, B.L. (2009). The structure of a bacterial DUF199/WhiA protein: domestication of an invasive endonu- Rosen, L.E., Morrison, H.A., Masri, S., Brown, M.J., Springstubb, B., Sussman, clease. Structure 17, 1368–1376. D., Stoddard, B.L., and Seligman, L.M. (2006). Homing endonuclease I-CreI derivatives with novel DNA target specificities. Nucleic Acids Res. 34, Knizewski, L., and Ginalski, K. (2007). Bacterial DUF199/COG1481 proteins 4791–4800. including sporulation regulator WhiA are distant homologs of LAGLIDADG homing endonucleases that retained only DNA binding. Cell Cycle 6, Rouet, P., Smih, F., and Jasin, M. (1994). Introduction of double-strand breaks 1666–1670. into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell Biol. 14, 8096–8106. Kuhlmann, U.C., Moore, G.R., James, R., Kleanthous, C., and Hemmings, A.M. (1999). Structural parsimony in endonuclease active sites: should the Scalley-Kim, M., McConnell-Smith, A., and Stoddard, B.L. (2007). Coevolution number of homing endonuclease families be redefined? FEBS Letters 463, of homing endonuclease specificity and its host target sequence. J. Mol. Biol. 1–2. 372, 1305–1319.

Seligman, L.M., Chisholm, K.M., Chevalier, B.S., Chadsey, M.S., Edwards, Stoddard, B.L., Monnat, R.J., Jr., and Scharenberg, A.M. (2007). Advances in S.T., Savage, J.H., and Veillet, A.L. (2002). Mutations altering the cleavage engineering homing endonucleases for gene targeting: ten years after struc- specificity of a homing endonuclease. Nucleic Acids Res. 30, 3870–3879. tures. In Progress in Gene Therapy: Autologous and cancer stem cell gene therapy, R. Bertolotti and K. Ozawa, eds. (Singapore: World Scientific eBooks), Sethuraman, J., Majer, A., Friedrich, N.C., Edgell, D.R., and Hausner, G. pp. 135–167. (2009). Genes within genes: multiple LAGLIDADG homing endonucleases target the ribosomal protein S3 gene encoded within an rnl group I intron of Sussman, D., Chadsey, M., Fauce, S., Engel, A., Bruett, A., Monnat, R.J., Jr., Ophiostoma and related taxa. Mol. Biol. Evol. 26, 2299–2315. Stoddard, B.L., and Seligman, L.M. (2004). Isolation and characterization of new homing endonuclease specificities at individual target site positions. Shen, B.W., Landthaler, M., Shub, D.A., and Stoddard, B.L. (2004). DNA J. Mol. Biol. 342, 31–41. binding and cleavage by the HNH homing endonuclease I-HmuI. J. Mol. Biol. 342, 43–56. Szczepanek, T., and Lazowska, J. (1996). Replacement of two non-adjacent amino acids in the S.cerevisiae bi2 intron-encoded RNA maturase is sufficient Shen, B.W., Heiter, D.F., Chan, S.-H., Wang, H., Xu, S.-Y., Morgan, R.D., to gain a homing-endonuclease activity. EMBO J. 15, 3758–3767. Wilson, G.G., and Stoddard, B.L. (2010). Unusual target site disruption by the rare-cutting HNH restriction endonuclease PacI. Structure 18, 734–743. Thyme, S.B., Jarjour, J., Takeuchi, R., Havranek, J.J., Ashworth, J., Scharen- Silva, G.H., and Belfort, M. (2004). Analysis of the LAGLIDADG interface of the berg, A.M., Stoddard, B.L., and Baker, D. (2009). Exploitation of binding monomeric homing endonuclease I-DmoI. Nucleic Acids Res. 32, 3156–3168. energy for catalysis and design. Nature 461, 1300–1304.

Silva, G.H., Belfort, M., Wende, W., and Pingoud, A. (2006). From monomeric Turmel, M., Cote, V., Otis, C., Mercier, J.P., Gray, M.W., Lonergan, K.M., and to homodimeric endonucleases and back: engineering novel speciﬁcity of Lemieux, C. (1995). Evolutionary transfer of ORF-containing group I introns LAGLIDADG enzymes. J. Mol. Biol. 361, 744–754. between different subcellular compartments (chloroplast and mitochondrion). Mol. Biol. Evol. 12, 533–545. Smith, J., Grizot, S., Arnould, S., Duclert, A., Epinat, J.C., Chames, P., Prieto, J., Redondo, P., Blanco, F.J., Bravo, J., et al. (2006). A combinatorial approach Van Roey, P., Waddling, C.A., Fox, K.M., Belfort, M., and Derbyshire, V. (2001). to create artiﬁcial homing endonucleases cleaving chosen sequences. Nucleic Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI Acids Res. 34, e149. with its substrate. EMBO J. 20, 3631–3637.

Sokolowska, M., Czapinska, H., and Bochtler, M. (2009). Crystal structure of Van Roey, P., Meehan, L., Kowalski, J.C., Belfort, M., and Derbyshire, V. the beta beta alpha-Me type II restriction endonuclease Hpy99I with target (2002). Catalytic domain structure and hypothesis for function of GIY-YIG DNA. Nucleic Acids Res. 37, 3799–3810. intron endonuclease I-TevI. Nat. Struct. Biol. 9, 806–811.

Sokolowska, M., Czapinska, H., and Bochtler, M. (2010). Hpy188I-DNA pre- Wenzlau, J.M., Saldanha, R.J., Butow, R.A., and Perlman, P.S. (1989). A latent and post-cleavage complexes–snapshots of the GIY-YIG nuclease mediated intron-encoded maturase is also an endonuclease needed for intron mobility. catalysis. Nucleic Acids Res., in press. Published online October 8, 2010. Cell 56, 421–430. 10.1093/nar/gkq821.

Steuer, S., Pingoud, V., Pingoud, A., and Wende, W. (2004). Chimeras of the Xie, Z., Li, W., Tian, Y., Liu, G., and Tan, H. (2007). Identification and character- homing endonuclease PI-SceI and the homologous Candida tropicalis intein: ization of sawC, a whiA-like gene, essential for sporulation in Streptomyces a study to explore the possibility of exchanging DNA-binding modules to ansochromogenes. Arch Microbiol. 188, 575–582. obtain highly speciric endonucleases with altered specificity. Chembiochem 5, 206–213. Zhao, L., Bonocora, R.P., Shub, D.A., and Stoddard, B.L. (2007). The restriction fold turns to the dark side: a bacterial homing endonuclease with a Stoddard, B.L. (2005). Homing endonuclease structure and function. Quarterly PD-(D/E)-XK motif. Embo J. 26, 2432–2442. Reviews of Biophysics 38, 49–95. Zhao, L., Pellenz, S., and Stoddard, B.L. (2008). Activity and Specificity of Stoddard, B., and Belfort, M. (2010). Social networking between mobile introns the Bacterial PD-(D/E)XK Homing Endonuclease I-Ssp6803I. J. Mol. Biol. and their host genes. Mol. Microbiol. 78, 1–4. 385, 1498–1510.