Crystal structure of the thermostable archaeal intron-encoded endonuclease I-DmoI

I-DmoI is a 22 kDa endonuclease encoded by an intron in the 23 S rRNA gene of the hyperthermophilic archaeon Desulfurococcus mobilis. The structure of I-DmoI has been determined to 2.2 A resolution using multi-wavelength anomalous diffraction techniques. I-DmoI, a protein of the LAGLIDADG motif family, represents the first structure of a freestanding endonuclease with two LAGLIDADG motifs, and the first of a thermostable homing endonuclease. I-DmoI consists of two similar alpha/beta domains (alphabetabetaalphabetabetaalpha) related by pseudo 2-fold symmetry. The LAGLIDADG motifs are located at the carboxy-terminal end of the first alpha-helix of each domain. These helices form a two-helix bundle at the interface between the domains and are perpendicular to a saddle-shaped DNA binding surface, formed by two four-stranded antiparallel beta-sheets. Despite substantially different sequences, the overall fold of I-DmoI is similar to that of two other LAGLIDADG proteins for which the structures are known, I-CreI and the endonuclease domain of PI-SceI. The three structures differ most in the loops connecting the beta-strands, relating to the respective DNA target site sizes and geometries. In addition, the absence of conserved residues surrounding the active site, other than those within the LAGLIDADG motif, is of mechanistic importance. Finally, the carboxy-terminal domain of I-DmoI is smaller and has a more irregular fold than the amino-terminal domain, which is more similar to I-CreI, a symmetric homodimeric endonuclease. This is reversed compared to PI-SceI, where the amino-terminal domain is more similar to carboxy-terminal domain of I-DmoI and to I-CreI, with interesting evolutionary implications.     

Design,activity, and structure of a highly specific artificial endonuclease

We have generated an artificial highly specific endonuclease by fusing domains of homing endonucleases I-DmoI and I-CreI and creating a new 1400 A(2) protein interface between these domains. Protein engineering was accomplished by combining computational redesign and an in vivo protein-folding screen. The resulting enzyme, E-DreI (Engineered I-DmoI/I-CreI), binds a long chimeric DNA target site with nanomolar affinity, cleaving it precisely at a rate equivalent to its natural parents. The structure of an E-DreI/DNA complex demonstrates the accuracy of the protein interface redesign algorithm and reveals how catalytic function is maintained during the creation of the new endonuclease. These results indicate that it may be possible to generate novel highly specific DNA binding proteins from homing endonucleases.     

Protein footprinting approach to mapping DNA binding sites of two archaeal homing enzymes: evidence for a two-domain protein structure.

The archaeal intron-encoded homing enzymes I-PorI and I-DmoI belong to a family of endonucleases that contain two copies of a characteristic LAGLIDADG motif. These endonucleases cleave their intron- or intein- alleles site-specifically, and thereby facilitate homing of the introns or inteins which encode them. The protein structure and the mechanism of DNA recognition of these homing enzymes is largely unknown. Therefore, we examined these properties of I-PorI and I-DmoI by protein footprinting. Both proteins were susceptible to proteolytic cleavage within regions that are equidistant from each of the two LAGLIDADG motifs. When complexed with their DNA substrates, a characteristic subset of the exposed sites, located in regions immediately after and 40-60 amino acids after each of the LAGLIDADG motifs, were protected. Our data suggest that the enzymes are structured into two, tandemly repeated, domains, each containing both the LAGLIDADG motif and two putative DNA binding regions. The latter contains a potentially novel DNA binding motif conserved in archaeal homing enzymes. The results are consistent with a model where the LAGLIDADG endonucleases bind to their non-palindromic substrates as monomeric enzymes, with each of the two domains recognizing one half of the DNA substrate.     

Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI

The general structural fold of the LAGLIDADG endonuclease family consists of two similar α/β domains (αββαββα) that assemble either as homodimers or monomers with the domains related by pseudo-two-fold symmetry. At the center of this symmetry is the closely packed LAGLIDADG two-helix bundle that forms the main inter- or intra-molecular contact region between the domains of single- or double-motif proteins, respectively. In this work, we further examine the role of the LAGLIDADG residues involved in the helix–helix interaction. The interchangeability of the LAGLIDADG helix interaction was explored by grafting interfacial residues from the homodimeric I-CreI into the corresponding positions in the monomeric I-DmoI. The resulting LAGLIDADG exchange mutant is partially active, preferring to nick dsDNA rather than making the customary double-strand break. A series of partial revertants within the mutated LAGLIDADG region are shown to restore cleavage activity to varying degrees resulting in one I-DmoI mutant that is more active than wild-type I-DmoI. The phenotype of some of these mutants was reconciled on the basis of similarity to the GxxxG helix interaction found in transmembrane proteins. Additionally, a split variant of I-DmoI was created, demonstrating that the LAGLIDADG helices of I-DmoI are capable of forming and maintaining the protein–protein interface in trans to create an active heterodimer.     

Mapping metal ions at the catalytic centres of two intron-encoded endonucleases.

Divalent metal ions play a crucial role in forming the catalytic centres of DNA endonucleases. Substitution of Mg2+ ions by Fe2+ ions in two archaeal intron-encoded homing endonucleases, I-DmoI and I-PorI, yielded functional enzymes and enabled the generation of reactive hydroxyl radicals within the metal ion binding sites. Specific hydroxyl radical-induced cleavage was observed within, and immediately after, two conserved LAGLIDADG motifs in both proteins and at sites at, and near, the scissile phosphates of the corresponding DNA substrates. Titration of Fe2+-containing protein-DNA complexes with Ca2+ ions, which are unable to support endonucleolytic activity, was performed to distinguish between the individual metal ions in the complex. Mutations of single amino acids in this region impaired catalytic activity and caused the preferential loss of a subset of hydroxyl radical cleavages in both the protein and the DNA substrate, suggesting an active role in metal ion coordination for these amino acids. The data indicate that the endonucleases cleave their DNA substrates as monomeric enzymes, and contain a minimum of four divalent metal ions located at or near the catalytic centres of each endonuclease. The metal ions involved in cleaving the coding and the non-coding strand are positioned immediately after the N- and C-terminally located LAGLIDADG motifs, respectively. The dual protein/nucleic acid footprinting approach described here is generally applicable to other protein-nucleic acid complexes when the natural metal ion can be replaced by Fe2+.     

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.     

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.     

Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets

The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding. Here, we show that a semi-rational approach can be used to derive hundreds of novel proteins from I-CreI, a homing endonuclease from the LAGLIDADG family. These novel endonucleases display a wide range of cleavage patterns in yeast and mammalian cells that in most cases are highly specific and distinct from I-CreI. Second, rules for protein/DNA interaction can be inferred from statistical analysis. Third, novel endonucleases can be combined to create heterodimeric protein species, thereby greatly enhancing the number of potential targets. These results describe a straightforward approach for engineering novel endonucleases with tailored specificities, while preserving the activity and specificity of natural homing endonucleases, and thereby deliver new tools for genome engineering.     

Evolution from DNA to RNA recognition by the bI3 LAGLIDADG maturase

LAGLIDADG endonucleases bind across adjacent major grooves via a saddle-shaped surface and catalyze DNA cleavage. Some LAGLIDADG proteins, called maturases, facilitate splicing by group I introns, raising the issue of how a DNA-binding protein and an RNA have evolved to function together. In this report, crystallographic analysis shows that the global architecture of the bI3 maturase is unchanged from its DNA-binding homologs; in contrast, the endonuclease active site, dispensable for splicing facilitation, is efficiently compromised by a lysine residue replacing essential catalytic groups. Biochemical experiments show that the maturase binds a peripheral RNA domain 50 A from the splicing active site, exemplifying long-distance structural communication in a ribonucleoprotein complex. The bI3 maturase nucleic acid recognition saddle interacts at the RNA minor groove; thus, evolution from DNA to RNA function has been mediated by a switch from major to minor groove interaction.     

Mapping of a DNA binding region of the PI-sceI homing endonuclease by affinity cleavage and alanine-scanning mutagenesis.

The PI-SceI protein is a member of the LAGLIDADG family of homing endonucleases that is generated by a protein splicing reaction. PI-SceI has a bipartite domain structure, and the protein splicing and endonucleolytic reactions are catalyzed by residues in domains I and II, respectively. Structural and mutational evidence indicates that both domains mediate DNA binding. Treatment of the protein with trypsin breaks a peptide bond within a disordered region of the endonuclease domain situated between residues Val-270 and Leu-280 and interferes with the ability of this domain to bind DNA. To identify specific residues in this region that are involved in DNA binding and/or catalysis, alanine-scanning mutagenesis was used to create a set of PI-SceI mutant proteins that were assayed for activity. One of these mutants, N281A, was >300-fold less active than wild-type PI-SceI, and two other proteins, R277A and N284A, were completely inactive. These decreases in cleavage activity parallel similar decreases in substrate binding by the endonuclease domains of these mutant proteins. We mapped the approximate position of the disordered region to one of the ends of the 31 base pair PI-SceI recognition sequence using mutant proteins that were substituted with cysteine at residues Asn-274 and Glu-283 and tethered to the chemical nuclease FeBABE. These mutational and affinity cleavage data strongly support a model of PI-SceI docked to its DNA substrate that suggests that one or more residues identified here are responsible for contacting base pair A/T(-)(9), which is essential for substrate binding.     

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein,a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

Mycobacterium leprae recA harbors an in‐frame insertion sequence that encodes an intein homing endonuclease (PI‐MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG‐type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI‐MleI is distinctive from other members of the family of LAGLIDADG‐type HEases for its modular structure with functionally separable domains for DNA‐binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI‐MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active‐site residues essential for DNA target site recognition and double‐stranded DNA cleavage, we performed site‐directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild‐type PI‐MleI and its variants disclosed that the two amino acid residues, Asp¹²² (in Block C) and Asp¹⁹³ (in functional Block E), are crucial to the double‐stranded DNA endonuclease activity, whereas Asp²¹⁸ (in pseudo‐Block E) is not. However, despite the reduced catalytic activity, the PI‐MleI variants, like the wild‐type PI‐MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA‐binding affinities, but abolished the double‐stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double‐stranded DNA cleavage activity, compared with the wild‐type PI‐MleI. These results provide compelling evidence that Asp¹²² and Asp¹⁹³ in DOD motif I and II, respectively, are bona fide active‐site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.     

Statistical modeling and analysis of the LAGLIDADG family of site-specific endonucleases and identification of an intein that encodes a site-specific endonuclease of the HNH family.

The LAGLIDADG and HNH families of site-specific DNA endonucleases encoded by viruses, bacteriophages as well as archaeal, eucaryotic nuclear and organellar genomes are characterized by the sequence motifs 'LAGLIDADG' and 'HNH', respectively. These endonucleases have been shown to occur in different environments: LAGLIDADG endonucleases are found in inteins, archaeal and group I introns and as free standing open reading frames (ORFs); HNH endonucleases occur in group I and group II introns and as ORFs. Here, statistical models (hidden Markov models, HMMs) that encompass both the conserved motifs and more variable regions of these families have been created and employed to characterize known and potential new family members. A number of new, putative LAGLIDADG and HNH endonucleases have been identified including an intein-encoded HNH sequence. Analysis of an HMM-generated multiple alignment of 130 LAGLIDADG family members and the three-dimensional structure of the I- Cre I endonuclease has enabled definition of the core elements of the repeated domain (approximately 90 residues) that is present in this family of proteins. A conserved negatively charged residue is proposed to be involved in catalysis. Phylogenetic analysis of the two families indicates a lack of exchange of endonucleases between different mobile elements (environments) and between hosts from different phylogenetic kingdoms. However, there does appear to have been considerable exchange of endonuclease domains amongst elements of the same type. Such events are suggested to be important for the formation of elements of new specficity.     

Generation and analysis of mesophilic variants of the thermostable archaeal I-DmoI homing endonuclease

The hyperthermophilic archaeon Desulfurococcus mobilis I-DmoI protein belongs to the family of proteins known as homing endonucleases (HEs). HEs are highly specific DNA-cleaving enzymes that recognize long stretches of DNA and are powerful tools for genome engineering. Because of its monomeric nature, I-DmoI is an ideal scaffold for generating mutant enzymes with novel DNA specificities, similarly reported for homodimeric HEs, but providing single chain endonucleases instead of dimers. However, this would require the use of a mesophilic variant cleaving its substrate at temperatures of 37 degrees C and below. We have generated mesophilic mutants of I-DmoI, using a single round of directed evolution that relies on a functional assay in yeast. The effect of mutations identified in the novel proteins has been investigated. These mutations are located distant to the DNA-binding site and cause changes in the size and polarity of buried residues, suggesting that they act by destabilizing the protein. Two of the novel proteins have been produced and analyzed in vitro. Their overall structures are similar to that of the parent protein, but they are destabilized against thermal and chemical denaturation. The temperature-dependent activity profiles for the mutants shifted toward lower temperatures with respect to the wild-type activity profile. However, the most destabilized mutant was not the most active at low temperatures, suggesting that other effects, like local structural distortions and/or changes in the protein dynamics, also influence their activity. These mesophilic I-DmoI mutants form the basis for generating new variants with tailored DNA specificities.     

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.     

Mva1269I: a monomeric type IIS restriction endonuclease from Micrococcus varians with two EcoRI- and FokI-like catalytic domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.     

Mining endonuclease cleavage determinants in genomic sequence data

Homing endonucleases have great potential as tools for targeted gene therapy and gene correction, but identifying variants of these enzymes capable of cleaving specific DNA targets of interest is necessary before the widespread use of such technologies is possible. We identified homologues of the LAGLIDADG homing endonuclease I-AniI and their putative target insertion sites by BLAST searches followed by examination of the sequences of the flanking genomic regions. Amino acid substitutions in these homologues that were located close to the target site DNA, and thus potentially conferring differences in target specificity, were grafted onto the I-AniI scaffold. Many of these grafts exhibited novel and unexpected specificities. These findings show that the information present in genomic data can be exploited for endonuclease specificity redesign.     

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.     

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.     

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.     

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.