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Barry L Stoddard - One of the best experts on this subject based on the ideXlab platform.
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Homing Endonucleases from mobile group I introns: discovery to genome engineering
2014Co-Authors: Barry L StoddardAbstract:Homing Endonucleases are highly specific DNA cleaving enzymes that are encoded within genomes of all forms of microbial life including phage and eukaryotic organelles. These proteins drive the mobility and persistence of their own reading frames. The genes that encode homing Endonucleases are often embedded within self-splicing elements such as group I introns, group II introns and inteins. This combination of molecular functions is mutually advantageous: the endonuclease activity allows surrounding introns and inteins to act as invasive DNA elements, while the splicing activity allows the endonuclease gene to invade a coding sequence without disrupting its product. Crystallographic analyses of representatives from all known homing endonuclease families have illustrated both their mechanisms of action and their evolutionary relationships to a wide range of host proteins. Several homing Endonucleases have been completely redesigned and used for a variety of genome engineering applications. Recent efforts to augment homing Endonucleases with auxiliary DNA recognition elements and/or nucleic acid processing factors has further accelerated their use for applications that demand exceptionally high specificity and activity.
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activity specificity and structure of i bth0305i a representative of a new homing endonuclease family
2011Co-Authors: Gregory K Taylor, Daniel F Heiter, Shmuel Pietrokovski, Barry L StoddardAbstract:Homing endonuclease are proteins that drive the dominant, non-Mendelian inheritance of their own reading frames by catalyzing a double-strand break (DSB) at specific DNA target sites in a recipient genome (1). The DSB is repaired via homologous recombination, using an allele of the target gene that contains the homing endonuclease gene (HEG) as a repair template; this copies the HEG into the site of DNA cleavage. HEGs are often embedded within self-splicing introns or inteins. The inclusion of a self-splicing genetic element as part of the mobile DNA allows invasion of highly conserved regions in crucial host genes without disrupting their essential functions. The coevolution of a homing endonuclease, its surrounding intron or intein, and the host gene results in an intricate network of genetic and physical interactions that affect the expression, specificity and invasiveness of the mobile element (2). To succeed as mobile genetic elements, homing Endonucleases must balance competing requirements for high DNA cleavage specificity (to avoid host toxicity) versus the need for reduced fidelity at various base pairs in their target site (to facilitate genetic mobility in the face of sequence drift within potential DNA target sites). Homing Endonucleases and associated mobile introns and inteins that have successfully achieved this balance are encoded in genomes of bacteria, organelles of fungi and algae, single cell protists and in the bacteriophage and viruses that accompany and infect those organisms. There are five well-characterized families of homing Endonucleases, which are each classified according to their unique protein folds and distinct catalytic active sites and DNA cleavage mechanisms (1). Members of the ‘LADLIDADG’ family, so named on the basis of their most conserved protein motif, are found in eukaryotic organellar and archaeal genomes, and are the most specific of the known homing Endonucleases (3). They exist both as homodimers that are limited to recognition of palindromic and near-palindromic target sites, and as pseudosymmetric monomers (where two structurally similar domains are tethered together on a single protein chain) that can target completely asymmetric targets. Members of the ‘His-Cys box’ and the ‘PD…(D/E)-xK’ families (found in protists and in cyanobacteria, respectively) also form multimeric protein complexes that recognize symmetric target sequences (4,5). In contrast, members of the HNH and GIY-YIG families (usually found in bacteriophage) display multidomain structures (corresponding to separate DNA binding and catalytic regions) and adopt highly elongated conformations when bound to DNA (6–8). As a result, those proteins usually recognize long non-palindromic sequences with significantly reduced fidelity (9,10). Recently, a novel type of fractured gene structure, containing separately encoded halves of self-splicing inteins that interrupt individual host genes in the same locus, was discovered during an analysis of environmental metagenomic sequence data collected by the Global Ocean Sampling (GOS) project (11). These split intein sequences are found in a diverse set of host genes that are primarily involved in DNA synthesis and repair. The inteins are themselves often interrupted either by open reading frames (ORFs) that encode members of the GIY-YIG homing endonuclease family, or by novel ORFs that do not exhibit significant sequence similarity to previously characterized homing endonuclease families. Homologs of those uncharacterized ORFs were also found associated with introns or as free-standing genes. In total, 15 members of the newly discovered gene family were described, including two within previously annotated recA genes in the NCBI sequence database. The C-terminal region of this newly identified protein family displays limited sequence homology [typically corresponding to e-values from a BLASTP (12) <10−3] to the catalytic domain of the very short patch repair (‘Vsr’) Endonucleases (enzymes that generate a 5′ nick at T:G mismatches in newly replicated DNA and thus stimulate DNA nucleotide excision repair) (13,14). Several catalytic residues from Vsr Endonucleases are conserved across all members of the new gene family, and form the composite sequence motif EDxHD. These residues include an essential aspartate that coordinates a catalytic magnesium ion, a histidine believed to act as a general base and a neighboring aspartate residue. Based on the presence of a recognizable endonuclease catalytic domain within these intron- and intein-associated microbial ORFs and the conservation of catalytic residues within that domain, this gene family was therefore hypothesized to encode a novel lineage of homing Endonucleases. These ORFs also display sequence signatures in their N-terminal regions that are similar to those found in several nuclease associated modular DNA-binding motifs (‘NUMODs’) (15). NUMODs are frequently found in other homing Endonucleases from bacteriophage, such as the GIY-YIG endonuclease I-TevI (8) and the HNH endonuclease I-HmuI (6). In those cases, the NUMODs are found at the C-terminal end of those proteins (a reversed domain organization compared to the metagenomic ORFs described above). The extended conformation that NUMOD regions adopt upon DNA binding dictates that they make relatively sparse contacts across their long target sites. A representative member of this novel homing endonuclease family, which we have named I-Bth0305I, was identified in the NCBI sequence database during the same genomic analysis (11). This ORF is located within a group I intron that interrupts the RecA gene of Bacillus thuringiensis 0305ϕ8–36 bacteriophage. Experiments described in this manuscript describe the binding site, cleavage pattern and specificity of I-Bth0305I, and the crystal structure of its catalytic domain. These experiments demonstrate that I-Bth0305I is a site-specific endonuclease that forms a homodimer and contacts a region of DNA up to 60 bp in length. Unlike many bacteriophage homing Endonucleases (which tether relatively nonspecific catalytic nuclease domains to sequence-specific DNA-binding domains, and therefore display significant specificity for DNA base pairs that are located some distance from the site of cleavage), I-Bth0305I displays its greatest specificity across the central residues of its recognition site (spanning the positions of DNA cleavage and intron insertion), and little additional sequence specificity at positions more distant from the cleavage site. The crystal structure of the I-Bth0305I catalytic domain confirms that members of this putative homing endonuclease family share a common ancestor with the Vsr mismatch repair endonuclease, and supports a similar mechanism for DNA strand cleavage.
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Tapping natural reservoirs of homing Endonucleases for targeted gene modification
2011Co-Authors: Ryo Takeuchi, David R Edgell, Abigail R Lambert, Amanda Nga-sze Mak, Kyle Jacoby, Russell J. Dickson, Gregory B. Gloor, Andrew M. Scharenberg, Barry L StoddardAbstract:Homing Endonucleases mobilize their own genes by generating double-strand breaks at individual target sites within potential host DNA. Because of their high specificity, these proteins are used for “genome editing” in higher eukaryotes. However, alteration of homing endonuclease specificity is quite challenging. Here we describe the identification and phylogenetic analysis of over 200 naturally occurring LAGLIDADG homing Endonucleases (LHEs). Biochemical and structural characterization of Endonucleases from one clade within the phylogenetic tree demonstrates strong conservation of protein structure contrasted against highly diverged DNA target sites and indicates that a significant fraction of these proteins are sufficiently stable and active to serve as engineering scaffolds. This information was exploited to create a targeting enzyme to disrupt the endogenous monoamine oxidase B gene in human cells. The ubiquitous presence and diversity of LHEs described in this study may facilitate the creation of many tailored nucleases for genome editing.
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fractured genes a novel genomic arrangement involving new split inteins and a new homing endonuclease family
2009Co-Authors: Bareket Dassa, Barry L Stoddard, N London, Ora Schuelerfurman, Shmuel PietrokovskiAbstract:Inteins are genetic elements, inserted in-frame into protein-coding genes, whose products catalyze their removal from the protein precursor via a protein-splicing reaction. Intein domains can be split into two fragments and still ligate their flanks by a trans-protein-splicing reaction. A bioinformatic analysis of environmental metagenomic data revealed 26 different loci with a novel genomic arrangement. In each locus, a conserved enzyme coding region is broken in two by a split intein, with a free-standing endonuclease gene inserted in between. Eight types of DNA synthesis and repair enzymes have this ‘fractured’ organization. The new types of naturally split-inteins were analyzed in comparison to known split-inteins. Some loci include apparent gene control elements brought in with the endonuclease gene. A newly predicted homing endonuclease family, related to very-short patch repair (Vsr) Endonucleases, was found in half of the loci. These putative homing Endonucleases also appear in group-I introns, and as stand-alone inserts in the absence of surrounding intervening sequences. The new fractured genes organization appears to be present mainly in phage, shows how Endonucleases can integrate into inteins, and may represent a missing link in the evolution of gene breaking in general, and in the creation of split-inteins in particular.
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the restriction fold turns to the dark side a bacterial homing endonuclease with a pd d e xk motif
2007Co-Authors: Lei Zhao, Richard P. Bonocora, David A. Shub, Barry L StoddardAbstract:The homing endonuclease I-Ssp6803I causes the insertion of a group I intron into a bacterial tRNA gene—the only example of an invasive mobile intron within a bacterial genome. Using a computational fold prediction, mutagenic screen and crystal structure determination, we demonstrate that this protein is a tetrameric PD-(D/E)-XK endonuclease—a fold normally used to protect a bacterial genome from invading DNA through the action of restriction Endonucleases. I-Ssp6803I uses its tetrameric assembly to promote recognition of a single long target site, whereas restriction endonuclease tetramers facilitate cooperative binding and cleavage of two short sites. The limited use of the PD-(D/E)-XK nucleases by mobile introns stands in contrast to their frequent use of LAGLIDADG and HNH Endonucleases—which in turn, are rarely incorporated into restriction/modification systems.
Robert A. Bambara - One of the best experts on this subject based on the ideXlab platform.
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acetylation of dna2 endonuclease helicase and flap endonuclease 1 by p300 promotes dna stability by creating long flap intermediates
2010Co-Authors: Lata Balakrishnan, Jason A Stewart, Piotr Polaczek, Judith L Campbell, Robert A. BambaraAbstract:Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5′–3′ endonuclease, the 5′–3′ helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two Endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase α.
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AP endonuclease 1 coordinates flap endonuclease 1 and DNA ligase I activity in long patch base excision repair.
2002Co-Authors: Tamara A. Ranalli, Samson Tom, Robert A. BambaraAbstract:Base loss is common in cellular DNA, resulting from spontaneous degradation and enzymatic removal of damaged bases. Apurinic/apyrimidinic (AP) Endonucleases recognize and cleave abasic (AP) sites during base excision repair (BER). APE1 (REF1, HAP1) is the predominant AP endonuclease in mammalian cells. Here we analyzed the influences of APE1 on the human BER pathway. Specifically, APE1 enhanced the enzymatic activity of both flap endonuclease1 (FEN1) and DNA ligase I. FEN1 was stimulated on all tested substrates, regardless of flap length. Interestingly, we have found that APE1 can also inhibit the activities of both enzymes on substrates with a tetrahydrofuran (THF) residue on the 5'-downstream primer of a nick, simulating a reduced abasic site. However once the THF residue was displaced at least a single nucleotide, stimulation of FEN1 activity by APE1 resumes. Stimulation of DNA ligase I required the traditional nicked substrate. Furthermore, APE1 was able to enhance overall product formation in reconstitution of BER steps involving FEN1 cleavage followed by ligation. Overall, APE1 both stimulated downstream components of BER and prevented a futile cleavage and ligation cycle, indicating a far-reaching role in BER.
Ralph A D Williams - One of the best experts on this subject based on the ideXlab platform.
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two thermostable type ii restriction Endonucleases from icelandic strains of the genus thermus tsp4c i acn gt a novel type ii restriction endonuclease and tsp8e i an isoschizomer of the mesophilic enzyme bgl i gccnnnn nggc
1995Co-Authors: Simon G Welch, Ralph A D WilliamsAbstract:Sixteen isolates of thermophilic bacteria from the genus Thermus, isolated from neutral and alkaline hot water springs in the southwest region of Iceland, were tested for the presence of restriction Endonucleases. Extracts from five of the isolates showed evidence of the presence of restriction endonuclease activity by producing discrete nucleotide fragments when incubated at 65 degrees C with lambda phage DNA. Two of the isolates (Tsp4C and Tsp8E) were found to have particularly high levels of restriction endonuclease activity, and the respective enzymes from these two Thermus isolates were partially purified and characterized and their recognition and cleavage sites were determined. Enzyme Tsp4C I is a novel Type II restriction endonuclease recognizing the interrupted palindromic tetranucleotide sequence ACNGT, where N can be any one of the four bases in DNA. Tsp4C I, which retains full enzyme activity when incubated for 10 min at temperatures up to 76 degrees C, hydrolyses the phosphodiester bond in both strands of a double-stranded DNA substrate between the third and fourth bases of the recognition sequence (ACN/GT), generating fragments with a single base 3'-OH overhang. Enzyme Tsp8E I is a thermostable isoschizomer of the mesophilic Type II restriction endonuclease Bgl I (GCCNNNN/NGGC) [Lee, Clanton and Chirikjiam (1979) Fed. Proc. 28, 294], generating fragments with a three base 3'-OH overhang. However, unlike Bgl I, Tsp8E I exhibits considerable thermal stability, retaining full enzyme activity when incubated for 10 min at temperatures up to 78 degrees C. Both Tsp4C I and Tsp8E I represent significant additions to the small but expanding list of the extremely thermostable restriction Endonucleases.
David A. Shub - One of the best experts on this subject based on the ideXlab platform.
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the restriction fold turns to the dark side a bacterial homing endonuclease with a pd d e xk motif
2007Co-Authors: Lei Zhao, Richard P. Bonocora, David A. Shub, Barry L StoddardAbstract:The homing endonuclease I-Ssp6803I causes the insertion of a group I intron into a bacterial tRNA gene—the only example of an invasive mobile intron within a bacterial genome. Using a computational fold prediction, mutagenic screen and crystal structure determination, we demonstrate that this protein is a tetrameric PD-(D/E)-XK endonuclease—a fold normally used to protect a bacterial genome from invading DNA through the action of restriction Endonucleases. I-Ssp6803I uses its tetrameric assembly to promote recognition of a single long target site, whereas restriction endonuclease tetramers facilitate cooperative binding and cleavage of two short sites. The limited use of the PD-(D/E)-XK nucleases by mobile introns stands in contrast to their frequent use of LAGLIDADG and HNH Endonucleases—which in turn, are rarely incorporated into restriction/modification systems.
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DNA binding and cleavage by the HNH homing endonuclease I-HmuI.
2004Co-Authors: Betty W. Shen, David A. Shub, Markus Landthaler, Barry L StoddardAbstract:The structure of I-HmuI, which represents the last family of homing Endonucleases without a defining crystallographic structure, has been determined in complex with its DNA target. A series of diverse protein structural domains and motifs, contacting sequential stretches of nucleotide bases, are distributed along the DNA target. I-HmuI contains an N-terminal domain with a DNA-binding surface found in the I-PpoI homing endonuclease and an associated HNH/N active site found in the bacterial colicins, and a C-terminal DNA-binding domain previously observed in the I-TevI homing endonuclease. The combination and exchange of these features between protein families indicates that the genetic mobility associated with homing Endonucleases extends to the level of independent structural domains. I-HmuI provides an unambiguous structural connection between the His-Cys box Endonucleases and the bacterial colicins, supporting the hypothesis that these enzymes diverged from a common ancestral nuclease.
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group i intron homing in bacillus phages spo1 and sp82 a gene conversion event initiated by a nicking homing endonuclease
2004Co-Authors: Markus Landthaler, Nelson C Lau, David A. ShubAbstract:Many group I introns encode Endonucleases that promote intron homing by initiating a double-stranded break-mediated homologous recombination event. In this work we describe intron homing in Bacillus subtilis phages SPO1 and SP82. The introns encode the DNA Endonucleases I-HmuI and I-HmuII, respectively, which belong to the H-N-H endonuclease family and possess nicking activity in vitro. Coinfections of B. subtilis with intron-minus and intron-plus phages indicate that I-HmuI and I-HmuII are required for homing of the SPO1 and SP82 introns, respectively. The homing process is a gene conversion event that does not require the major B. subtilis recombination pathways, suggesting that the necessary functions are provided by phage-encoded factors. Our results provide the first examples of H-N-H endonuclease-mediated intron homing and the first demonstration of intron homing initiated by a nicking endonuclease.
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the nicking homing endonuclease i basi is encoded by a group i intron in the dna polymerase gene of the bacillus thuringiensis phage bastille
2003Co-Authors: Markus Landthaler, David A. ShubAbstract:Here we describe the discovery of a group I intron in the DNA polymerase gene of Bacillus thuringiensis phage Bastille. Although the intron insertion site is identical to that of the Bacillus subtilis phages SPO1 and SP82 introns, the Bastille intron differs from them substantially in primary and secondary structure. Like the SPO1 and SP82 introns, the Bastille intron encodes a nicking DNA endonuclease of the H-N-H family, I-BasI, with a cleavage site identical to that of the SPO1-encoded enzyme I-HmuI. Unlike I-HmuI, which nicks both intronminus and intron-plus DNA, I-BasI cleaves only intron-minus alleles, which is a characteristic of typical homing Endonucleases. Interestingly, the C-terminal portions of these H-N-H phage Endonucleases contain a conserved sequence motif, the intron-encoded endonuclease repeat motif (IENR1) that also has been found in Endonucleases of the GIY-YIG family, and which likely comprises a small DNA-binding module with a globular bbaab fold, suggestive of module shuffling between different homing endonuclease families.
Raymond J Monnat - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterization of new homing endonuclease specificities at individual target site positions
2004Co-Authors: Django Sussman, Meggen S Chadsey, Barry L Stoddard, Raymond J Monnat, Steve Fauce, Alex Engel, Anna Bruett, Lenny M SeligmanAbstract: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.
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Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI.
2003Co-Authors: Brett Chevalier, Raymond J Monnat, Monique Turmel, Claude Lemieux, Barry L StoddardAbstract:Homing Endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing Endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of Endonucleases at an early stage of divergence in their target specificity.
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design activity and structure of a highly specific artificial endonuclease
2002Co-Authors: Tanja Kortemme, David Baker, Raymond J MonnatAbstract:Abstract 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.
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The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites
2001Co-Authors: Brett S. Chevalier, Raymond J Monnat, Barry L StoddardAbstract: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.