Homing endonuclease I-CreI derivatives with novel DNA target specificities

Homing endonucleases are highly specific enzymes, capable of recognizing and cleaving unique DNA sequences in complex genomes. Since such DNA cleavage events can result in targeted allele-inactivation and/or allele-replacement in vivo, the ability to engineer homing endonucleases matched to specific DNA sequences of interest would enable powerful and precise genome manipulations. We have taken a step-wise genetic approach in analyzing individual homing endonuclease I-CreI protein/DNA contacts, and describe here novel interactions at four distinct target site positions. Crystal structures of two mutant endonucleases reveal the molecular interactions responsible for their altered DNA target specificities. We also combine novel contacts to create an endonuclease with the predicted target specificity. These studies provide important insights into engineering homing endonucleases with novel target specificities, as well as into the evolution of DNA recognition by this fascinating family of proteins.     

Mutations altering the cleavage specificity of a homing endonuclease

The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein–DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences.     

Isolation and characterization of new homing endonuclease specificities at individual target site positions

Homing endonucleases are highly specific DNA endonucleases, encoded within mobile introns or inteins, that induce targeted recombination, double-strand repair and gene conversion of their cognate target sites. Due to their biological function and high level of target specificity, these enzymes are under intense investigation as tools for gene targeting. These studies require that naturally occurring enzymes be redesigned to recognize novel target sites. Here, we report studies in which the homodimeric LAGLIDADG homing endonuclease I-CreI is altered at individual side-chains corresponding to contact points to distinct base-pairs in its target site. The resulting enzyme constructs drive specific elimination of selected DNA targets in vivo and display shifted specificities of DNA binding and cleavage in vitro. Crystal structures of two of these constructs demonstrate that substitution of individual side-chain/DNA contact patterns can occur with almost no structural deformation or rearrangement of the surrounding complex, facilitating an isolated, modular redesign strategy for homing endonuclease activity and specificity.     

The structure of I-CeuI homing endonuclease: Evolving asymmetric DNA recognition from a symmetric protein scaffold

Homing endonucleases are highly specific catalysts of DNA strand breaks, leading to the transfer of mobile intervening sequences containing the endonuclease ORF. We have determined the structure and DNA recognition behavior of I-CeuI, a homodimeric LAGLIDADG endonuclease from Chlamydomonas eugametos. This symmetric endonuclease displays unique structural elaborations on its core enzyme fold, and it preferentially cleaves a highly asymmetric target site. This latter property represents an early step, prior to gene fusion, in the generation of asymmetric DNA binding platforms from homodimeric ancestors. The divergence of the sequence, structure, and target recognition behavior of homing endonucleases, as illustrated by this study, leads to the invasion of novel genomic sites by mobile introns during evolution.     

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 ΔH_binding 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 ΔS_binding.     

Reactions of BglI and other type II restriction endonucleases with discontinuous recognition sites

Type II restriction enzymes generally recognize continuous sequences of 4-8 consecutive base pairs on DNA, but some recognize discontinuous sites where the specified sequence is interrupted by a defined length of nonspecific DNA. To date, a mechanism has been established for only one type II endonuclease with a discontinuous site, SfiI at GGCCNNNNNGGCC (where N is any base). In contrast to orthodox enzymes such as EcoRV, dimeric proteins that act at a single site, SfiI is a tetramer that interacts with two sites before cleaving DNA. BglI has a similar recognition sequence (GCCNNNNNGGC) to SfiI but a crystal structure like EcoRV. BglI and several other endonucleases with discontinuous sites were examined to see if they need two sites for their DNA cleavage reactions. The enzymes included some with sites containing lengthy segments of nonspecific DNA, such as XcmI (CCANNNNNNNNNTGG). In all cases, they acted at individual sites. Elongated recognition sites do not necessitate unusual reaction mechanisms. Other experiments on BglI showed that it bound to and cleaved DNA in the same manner as EcoRV, thus further delineating a distinct group of restriction enzymes with similar structures and a common reaction mechanism.     

I-Ssp6803I: the first homing endonuclease from the PD-(D/E)XK superfamily exhibits an unusual mode of DNA recognition

MOTIVATION: Restriction endonucleases (REases) and homing endonucleases (HEases) are biotechnologically important enzymes. Nearly all structurally characterized REases belong to the PD-(D/E)XK superfamily of nucleases, while most HEases belong to an unrelated LAGLIDADG superfamily. These two protein folds are typically associated with very different modes of protein-DNA recognition, consistent with the different mechanisms of action required to achieve high specificity. REases recognize short DNA sequences using multiple contacts per base pair, while HEases recognize very long sites using a few contacts per base pair, thereby allowing for partial degeneracy of the target sequence. Thus far, neither REases with the LAGLIDADG fold, nor HEases with the PD-(D/E)XK fold, have been found. RESULTS: Using protein fold recognition, we have identified the first member of the PD-(D/E)XK superfamily among homing endonucleases, a cyanobacterial enzyme I-Ssp6803I. We present a model of the I-Ssp6803I-DNA complex based on the structure of Type II restriction endonuclease R.BglI and predict the active site and residues involved in specific DNA sequence recognition by I-Ssp6803I. Our finding reveals a new unexpected evolutionary link between HEases and REases and suggests how PD-(D/E)XK nucleases may develop a 'HEase-like' way of interacting with the extended DNA sequence. This in turn may be exploited to study the evolution of DNA sequence specificity and to engineer nucleases with new substrate specificities.     

The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites

Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family.     

Homing endonucleases: from microbial genetic invaders to reagents for targeted DNA modification

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.     

Crystal Structure of the Homing Endonuclease I-CvuI Provides a New Template for Genome Modification

Homing endonucleases recognize and generate a DNA double-strand break, which has been used to promote gene targeting. These enzymes recognize long DNA stretches; they are highly sequence-specific enzymes and display a very low frequency of cleavage even in complete genomes. Although a large number of homing endonucleases have been identified, the landscape of possible target sequences is still very limited to cover the complexity of the whole eukaryotic genome. Therefore, the finding and molecular analysis of homing endonucleases identified but not yet characterized may widen the landscape of possible target sequences. The previous characterization of protein-DNA interaction before the engineering of new homing endonucleases is essential for further enzyme modification. Here we report the crystal structure of I-CvuI in complex with its target DNA and with the target DNA of I-CreI, a homologue enzyme widely used in genome engineering. To characterize the enzyme cleavage mechanism, we have solved the I-CvuI DNA structures in the presence of non-catalytic (Ca²⁺) and catalytic ions (Mg²⁺). We have also analyzed the metal dependence of DNA cleavage using Mg²⁺ ions at different concentrations ranging from non-cleavable to cleavable concentrations obtained from in vitro cleavage experiments. The structure of I-CvuI homing endonuclease expands the current repertoire for engineering custom specificities, both by itself as a new scaffold alone and in hybrid constructs with other related homing endonucleases or other DNA-binding protein templates.     

Creation of a type IIS restriction endonuclease with a long recognition sequence

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.     

A highly sensitive selection method for directed evolution of homing endonucleases

Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hamper their applications. Here we report the development of a highly sensitive selection method for the directed evolution of homing endonucleases that couples enzymatic DNA cleavage with the survival of host cells. Using I-SceI as a model homing endonuclease, we have demonstrated that cells with wild-type I-SceI showed a high cell survival rate of 80–100% in the presence of the original I-SceI recognition site, whereas cells without I-SceI showed a survival rate <0.003%. This system should also be readily applicable for directed evolution of other DNA cleavage enzymes.     

Structures of the rare-cutting restriction endonuclease NotI reveal a unique metal binding fold involved in DNA binding

The structure of the rare-cutting restriction endonuclease NotI, which recognizes the 8 bp target 5'-GCGGCCGC-3', has been solved with and without bound DNA. Because of its specificity (recognizing a site that occurs once per 65 kb), NotI is used to generate large genomic fragments and to map DNA methylation status. NotI contains a unique metal binding fold, found in a variety of putative endonucleases, occupied by an iron atom coordinated within a tetrahedral Cys4 motif. This domain positions nearby protein elements for DNA recognition, and serves a structural role. While recognition of the central six base pairs of the target is accomplished via a saturated hydrogen bond network typical of restriction enzymes, the most peripheral base pairs are engaged in a single direct contact in the major groove, reflecting reduced pressure to recognize those positions. NotI may represent an evolutionary intermediate between mobile endonucleases (which recognize longer target sites) and canonical restriction endonucleases.     

Assessing the plasticity of DNA target site recognition of the PI-SceI homing endonuclease using a bacterial two-hybrid selection system

The PI-SceI protein from Saccharomyces cerevisiae is a member of the LAGLIDADG family of homing endonucleases that have been used in genomic engineering. To assess the flexibility of the PI-SceI-binding interaction and to make progress towards the directed evolution of homing endonucleases that cleave specified DNA targets, we applied a two-hybrid method to select PI-SceI variants from a randomized expression library that bind to different DNA substrates. In particular, the codon for Arg94, which is located in the protein splicing domain and makes essential contacts to two adjacent base-pairs, and the codons for four proximal residues were randomized. There is little conservation of the wild-type amino acid residues at the five randomized positions in the variants that were selected to bind to the wild-type site, yet one of the purified derivatives displays DNA-binding specificity and DNA endonuclease activity that is similar to that of the wild-type enzyme. A spectrum of DNA-binding behaviors ranging from partial relaxation of specificity to marked shifts in target site recognition are present in variants selected to bind to sites containing mutations at the two base-pairs. Our results illustrate the inherent plasticity of the PI-SceI/DNA interface and demonstrate that selection based on DNA binding is an effective means of altering the DNA cleavage specificity of homing endonucleases. Furthermore, it is apparent that homing endonuclease target specificity derives, in part, from constraints on the flexibility of DNA contacts imposed by hydrogen bonds to proximal residues.     

Non-specific protein-DNA interactions control I-CreI target binding and cleavage

Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4 bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications.     

Coevolution of a homing endonuclease and its host target sequence

We have determined the specificity profile of the homing endonuclease I-AniI and compared it to the conservation of its host gene. Homing endonucleases are encoded within intervening sequences such as group I introns. They initiate the transfer of such elements by cleaving cognate alleles lacking the intron, leading to their transfer via homologous recombination. Each structural homing endonuclease family has arrived at an appropriate balance of specificity and fidelity that avoids toxicity while maximizing target recognition and invasiveness. I-AniI recognizes a strongly conserved target sequence in a host gene encoding apocytochrome B and has fine-tuned its specificity to correlate with wobble versus nonwobble positions across that sequence and to the amount of degeneracy inherent in individual codons. The physiological target site in the host gene is not the optimal substrate for recognition and cleavage: at least one target variant identified during a screen is bound more tightly and cleaved more rapidly. This is a result of the periodic cycle of intron homing, which at any time can present nonoptimal combinations of endonuclease specificity and insertion site sequences in a biological host.     

The MmeI family: type II restriction–modification enzymes that employ single-strand modification for host protection

The type II restriction endonucleases form one of the largest families of biochemically-characterized proteins. These endonucleases typically share little sequence similarity, except among isoschizomers that recognize the same sequence. MmeI is an unusual type II restriction endonuclease that combines endonuclease and methyltransferase activities in a single polypeptide. MmeI cuts DNA 20 bases from its recognition sequence and modifies just one DNA strand for host protection. Using MmeI as query we have identified numerous putative genes highly similar to MmeI in database sequences. We have cloned and characterized 20 of these MmeI homologs. Each cuts DNA at the same distance as MmeI and each modifies a conserved adenine on only one DNA strand for host protection. However each enzyme recognizes a unique DNA sequence, suggesting these enzymes are undergoing rapid evolution of DNA specificity. The MmeI family thus provides a rich source of novel endonucleases while affording an opportunity to observe the evolution of DNA specificity. Because the MmeI family enzymes employ modification of only one DNA strand for host protection, unlike previously described type II systems, we propose that such single-strand modification systems be classified as a new subgroup, the type IIL enzymes, for Lone strand DNA modification.     

Structure-Function Studies of Two Yeast Homing Endonucleases that Evolved to Cleave Identical Targets with Dissimilar Rates and Specificities

The LAGLIDADG family of homing endonucleases (LHEs) bind to and cleave their DNA recognition sequences with high specificity. Much of our understanding for how these proteins evolve their specificities has come from studying LHE homologues. To gain insight into the molecular basis of LHE specificity, we characterized I-WcaI, the homologue of the Saccharomyces cerevisiae I-SceI LHE found in Wickerhamomyces canadensis. Although I-WcaI and I-SceI cleave the same recognition sequence, expression of I-WcaI, but not I-SceI, is toxic in bacteria. Toxicity suppressing mutations frequently occur at I-WcaI residues critical for activity and I-WcaI cleaves many more non-cognate sequences in the Escherichia coli genome than I-SceI, suggesting I-WcaI endonuclease activity is the basis of toxicity. In vitro, I-WcaI is a more active and a less specific endonuclease than I-SceI, again accounting for the observed toxicity in vivo. We determined the X-ray crystal structure of I-WcaI bound to its cognate target site and found that I-WcaI and I-SceI use residues at different positions to make similar base-specific contacts. Furthermore, in some regions of the DNA interface where I-WcaI specificity is lower, the protein makes fewer DNA contacts than I-SceI. Taken together, these findings demonstrate the plastic nature of LHE site recognition and suggest that I-WcaI and I-SceI are situated at different points in their evolutionary pathways towards acquiring target site specificity.     

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.     

Homing endonuclease structure and function

Homing endonucleases are encoded by open reading frames that are embedded within group I, group II and archael introns, as well as inteins (intervening sequences that are spliced and excised post-translationally). These enzymes initiate transfer of those elements (and themselves) by generating strand breaks in cognate alleles that lack the intervening sequence, as well as in additional ectopic sites that broaden the range of intron and intein mobility. Homing endonucleases can be divided into several unique families that are remarkable in several respects: they display extremely high DNA-binding specificities which arise from long DNA target sites (14-40 bp), they are tolerant of a variety of sequence variations in these sites, and they display disparate DNA cleavage mechanisms. A significant number of homing endonucleases also act as maturases (highly specific cofactors for the RNA splicing reactions of their cognate introns). Of the known homing group I endonuclease families, two (HNH and His-Cys box enzymes) appear to be diverged from a common ancestral nuclease. While crystal structures of several representatives of the LAGLIDADG endonuclease family have been determined, only structures of single members of the HNH (I-HmuI), His-Cys box (I-PpoI) and GIY-YIG (I-TevI) families have been elucidated. These studies provide an important source of information for structure-function relationships in those families, and are the centerpiece of this review. Finally, homing endonucleases are significant targets for redesign and selection experiments, in hopes of generating novel DNA binding and cutting reagents for a variety of genomic applications.