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Barry L Stoddard - One of the best experts on this subject based on the ideXlab platform.

  • Rapid Determination of Homing Endonuclease DNA Binding Specificity Profile
    Methods in molecular biology (Clifton N.J.), 2014
    Co-Authors: Lei Zhao, Barry L Stoddard
    Abstract:

    Evaluating the binding specificity and identifying the most preferred target sequence for a Homing Endonuclease often represents a key step during its characterization, engineering, and application for genome engineering. This chapter describes a high-throughput, fluorescence-based, competition-binding assay which can be used to measure the relative binding affinities of the Homing Endonuclease to a large number of DNA target site variants in a single experiment. The base preference at each position of the target sequence can be quantitated based on this assay, and the overall binding specificity of the enzyme can thereby be determined and compared with its cleavage specificity.

  • lahedes the laglidadg Homing Endonuclease database and engineering server
    Nucleic Acids Research, 2012
    Co-Authors: Gregory K Taylor, Lucas H Petrucci, Abigail R Lambert, Sarah K Baxter, Jordan Jarjour, Barry L Stoddard
    Abstract:

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

  • activity specificity and structure of i bth0305i a representative of a new Homing Endonuclease family
    Nucleic Acids Research, 2011
    Co-Authors: Gregory K Taylor, Daniel F Heiter, Shmuel Pietrokovski, Barry L Stoddard
    Abstract:

    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.

  • Activity, specificity and structure of I-Bth0305I: a representative of a new Homing Endonuclease family
    Nucleic acids research, 2011
    Co-Authors: Gregory K Taylor, Daniel F Heiter, Shmuel Pietrokovski, Barry L Stoddard
    Abstract:

    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)

  • high resolution profiling of Homing Endonuclease binding and catalytic specificity using yeast surface display
    Nucleic Acids Research, 2009
    Co-Authors: Jordan Jarjour, Barry L Stoddard, Hoku Westfoyle, Michael T Certo, Christopher G Hubert, Lindsey Doyle, Melissa M Getz, Andrew M Scharenberg
    Abstract:

    Experimental analysis and manipulation of protein–DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG Homing Endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein–DNA interactions.

Georg Hausner - One of the best experts on this subject based on the ideXlab platform.

  • Insertion of Group II Intron-Based Ribozyme Switches into Homing Endonuclease Genes
    In Vitro Mutagenesis, 2017
    Co-Authors: Tuhin Kumar Guha, Georg Hausner
    Abstract:

    Fungal mitochondrial genomes act as “reservoirs” for Homing Endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing, targeted mutagenesis and gene therapy applications. Herein, we present strategies where Homing Endonuclease open reading frames (HEases ORFs) are interrupted with group II intron sequences. The ultimate goal is to achieve in vivo expression of HEases that can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. That addition of exogenous magnesium chloride (MgCl2) appears to stimulate splicing of nonnative group II introns in Escherichia coli and the addition of cobalt chloride (CoCl2) to the growth medium antagonizes the expression of HEase activity (i.e., splicing). Group II introns are potentially autocatalytic self-splicing elements and thus can be used as molecular switches that allow for temporal regulated HEase expression. This should be useful in precision genome engineering, mutagenesis, and minimizing off-target activities.

  • Insertion of Group II Intron-Based Ribozyme Switches into Homing Endonuclease Genes
    In Vitro Mutagenesis, 2017
    Co-Authors: Tuhin Kumar Guha, Georg Hausner
    Abstract:

    Fungal mitochondrial genomes act as “reservoirs” for Homing Endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing, targeted mutagenesis and gene therapy applications. Herein, we present strategies where Homing Endonuclease open reading frames (HEases ORFs) are interrupted with group II intron sequences. The ultimate goal is to achieve in vivo expression of HEases that can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. That addition of exogenous magnesium chloride (MgCl2) appears to stimulate splicing of nonnative group II introns in Escherichia coli and the addition of cobalt chloride (CoCl2) to the growth medium antagonizes the expression of HEase activity (i.e., splicing). Group II introns are potentially autocatalytic self-splicing elements and thus can be used as molecular switches that allow for temporal regulated HEase expression. This should be useful in precision genome engineering, mutagenesis, and minimizing off-target activities.

  • using group ii introns for attenuating the in vitro and in vivo expression of a Homing Endonuclease
    PLOS ONE, 2016
    Co-Authors: Tuhin Kumar Guha, Georg Hausner
    Abstract:

    In Chaetomium thermophilum (DSM 1495) within the mitochondrial DNA (mtDNA) small ribosomal subunit (rns) gene a group IIA1 intron interrupts an open reading frame (ORF) encoded within a group I intron (mS1247). This arrangement offers the opportunity to examine if the nested group II intron could be utilized as a regulatory element for the expression of the Homing Endonuclease (HEase). Constructs were generated where the codon-optimized ORF was interrupted with either the native group IIA1 intron or a group IIB type intron. This study showed that the expression of the HEase (in vivo) in Escherichia coli can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. Exogenous magnesium chloride (MgCl2) stimulated the expression of a functional HEase but the addition of cobalt chloride (CoCl2) to growth media antagonized the expression of HEase activity. Ultimately the ability to attenuate HEase activity might be useful in precision genome engineering, minimizing off target activities, or where pathways have to be altered during a specific growth phase.

  • A Homing Endonuclease with a switch: characterization of a twintron encoded Homing Endonuclease.
    Fungal genetics and biology : FG & B, 2014
    Co-Authors: Tuhin Kumar Guha, Georg Hausner
    Abstract:

    The small ribosomal subunit gene residing in the mitochondrial DNA of the thermophilic fungus Chaetomium thermophilum var. thermophilum La Touche DSM 1495 is interrupted by a twintron at position mS1247. The mS1247 twintron represents the first mixed twintron found in fungal mtDNA, composed of an external group I intron encoding a LAGLIDADG open reading frame that is interrupted by an internal group II intron. Splicing of the internal group II intron reconstitutes the open reading frame and thus facilitates the expression of the encoded Homing Endonuclease. The cleavage assays suggest that the twintron encodes an active Homing Endonuclease that could potentially mobilize the twintron to rns genes that have not yet been invaded by this mobile composite element.

  • Evolutionary Dynamics of the mS952 Intron: A Novel Mitochondrial Group II Intron Encoding a LAGLIDADG Homing Endonuclease Gene
    Journal of Molecular Evolution, 2011
    Co-Authors: Sahra-taylor Mullineux, Karla Willows, Georg Hausner
    Abstract:

    Examination of the mitochondrial small subunit ribosomal RNA ( rns ) gene of five species of the fungal genus Leptographium revealed that the gene has been invaded at least once at position 952 by a group II intron encoding a LAGLIDADG Homing Endonuclease gene. Phylogenetic analyses of the intron and Homing Endonuclease sequences indicated that each element in Leptographium species forms a single clade and is closely related to the group II intron/Homing Endonuclease gene composite element previously reported at position 952 of the mitochondrial rns gene of Cordyceps species and of Cryphonectria parasitica . The results of an intron survey of the mt rns gene of Leptographium species superimposed onto the phylogenetic analysis of the host organisms suggest that the composite element was transmitted vertically in Leptographium lundbergii . However, its stochastic distribution among strains of L. wingfieldii , L. terebrantis , and L. truncatum suggests that it has been horizontally transmitted by lateral gene transfer among these species, although the random presence of the intron may reflect multiple random loss events. A model is proposed describing the initial invasion of the group II intron in the rns gene of L. lundbergii by a LAGLIDADG Homing Endonuclease gene and subsequent evolution of this gene to recognize a novel DNA target site, which may now promote the mobility of the intron and Homing Endonuclease gene as a composite element.

David R. Edgell - One of the best experts on this subject based on the ideXlab platform.

  • Mitochondrial gene content and schematic representation of group I intron and Homing Endonuclease genes found in Scleractinia.
    2017
    Co-Authors: Juan Sebastián Celis, David R. Edgell, Björn Stelbrink, Daniel Wibberg, Torsten Hauffe, Jochen Blom, Jörn Kalinowski, Thomas Wilke
    Abstract:

    (A) Schematic map of the P. rus (complex Scleractinia) mitochondrial genome showing gene content and group I introns inserted in the COXI and NAD5 genes. The width of each box is proportional to gene size. (B) Representation of intron 884 (complex Scleractinia and Corallimorpharia) and intron 720 (robust Scleractinia) showing 5´and 3´ exons, and the putative Homing Endonuclease gene (HEG) of the LAGLIDADG family. The black arrows indicate intron insertion position in the COXI gene.

  • Perpetuating the Homing Endonuclease life cycle: identification of mutations that modulate and change I-TevI cleavage preference
    Nucleic acids research, 2016
    Co-Authors: Alexander C. Roy, Geoffrey G. Wilson, David R. Edgell
    Abstract:

    Homing Endonucleases are sequence-tolerant DNA Endonucleases that act as mobile genetic elements. The ability of Homing Endonucleases to cleave substrates with multiple nucleotide substitutions suggests a high degree of adaptability in that changing or modulating cleavage preference would require relatively few amino acid substitutions. Here, using directed evolution experiments with the GIY-YIG Homing Endonuclease I-TevI that targets the thymidylate synthase gene of phage T4, we readily isolated variants that dramatically broadened I-TevI cleavage preference, as well as variants that fine-tuned cleavage preference. By combining substitutions, we observed an ∼10 000-fold improvement in cleavage on some substrates not cleaved by the wild-type enzyme, correlating with a decrease in readout of information content at the cleavage site. Strikingly, we were able to change the cleavage preference of I-TevI to that of the isoschizomer I-BmoI which targets a different cleavage site in the thymidylate synthase gene, recapitulating the evolution of cleavage preference in this family of Homing Endonucleases. Our results define a strategy to isolate GIY-YIG nuclease domains with distinct cleavage preferences, and provide insight into how Homing Endonucleases may escape a dead-end life cycle in a population of saturated target sites by promoting transposition to different target sites.

  • Divalent Metal Ion Differentially Regulates the Sequential Nicking Reactions of the GIY-YIG Homing Endonuclease I-BmoI
    PloS one, 2011
    Co-Authors: Benjamin P Kleinstiver, Andrew D Fernandes, Wesley Bérubé-janzen, David R. Edgell
    Abstract:

    Homing Endonucleases are site-specific DNA Endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of Homing Endonucleases, the modular GIY-YIG Endonucleases are least understood in terms of mechanism. The GIY-YIG Homing Endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.

  • a unified genetic computational and experimental framework identifies functionally relevant residues of the Homing Endonuclease i bmoi
    Nucleic Acids Research, 2010
    Co-Authors: Benjamin P Kleinstiver, Andrew D Fernandes, Gregory B Gloor, David R. Edgell
    Abstract:

    Insight into protein structure and function is best obtained through a synthesis of experimental, structural and bioinformatic data. Here, we outline a framework that we call MUSE (mutual information, unigenic evolution and structure-guided elucidation), which facilitated the identification of previously unknown residues that are relevant for function of the GIY-YIG Homing Endonuclease I-BmoI. Our approach synthesizes three types of data: mutual information analyses that identify co-evolving residues within the GIY-YIG catalytic domain; a unigenic evolution strategy that identifies hyper- and hypo-mutable residues of I-BmoI; and interpretation of the unigenic and co-evolution data using a homology model. In particular, we identify novel positions within the GIY-YIG domain as functionally important. Proof-of-principle experiments implicate the non-conserved I71 as functionally relevant, with an I71N mutant accumulating a nicked cleavage intermediate. Moreover, many additional positions within the catalytic, linker and C-terminal domains of I-BmoI were implicated as important for function. Our results represent a platform on which to pursue future studies of I-BmoI and other GIY-YIG-containing proteins, and demonstrate that MUSE can successfully identify novel functionally critical residues that would be ignored in a traditional structure-function analysis within an extensively studied small domain of approximately 90 amino acids.

  • Phage T4 mobE promotes trans Homing of the defunct Homing Endonuclease I-TevIII
    Nucleic acids research, 2009
    Co-Authors: Gavin W. Wilson, David R. Edgell
    Abstract:

    Homing Endonucleases are site-specific DNA Endonucleases that typically function as mobile genetic elements by introducing a double-strand break (DSB) in genomes that lack the Endonuclease, resulting in a unidirectional gene conversion event that mobilizes the Homing Endonuclease gene and flanking DNA. Here, we characterize phage T4encoded mobE, a predicted free-standing HNH family Homing Endonuclease. We show that mobE is promoterless and dependent on upstream transcription for expression, and that an internal intrinsic terminator regulates mobE transcript levels. Crucially, in vivo mapping experiments revealed a MobE-dependent, strand-specific nick in the non-coding strand of the nrdB gene of phage T2. An internal deletion of the predicted HNH catalytic motif of MobE abolishes nicking, and reduces high-frequency inheritance of mobE. Sequence polymorphisms of progeny phage that inherit mobE are consistent with DSB repair pathways. Significantly, we found that mobility of the neighboring I-TevIII, a defunct Homing Endonuclease encoded within a group I intron interrupting the nrdB gene of phage T4, was dependent on an intact mobE gene. Thus, our data indicate that the stagnant nrdB intron and I-TevIII are mobilized in trans as a consequence of a MobE-dependent gene conversion event, facilitating persistence of genetic elements that have no inherent means of promoting their own mobility.

Raymond J Monnat - One of the best experts on this subject based on the ideXlab platform.

  • a synthetic Homing Endonuclease based gene drive system in the human malaria mosquito
    Nature, 2011
    Co-Authors: Nikolai Windbichler, Raymond J Monnat, Miriam Menichelli, Philippos Aris Papathanos, Summer B Thyme, Umut Y Ulge, Blake T Hovde, David Baker, Austin Burt, Andrea Crisanti
    Abstract:

    Genetic approaches to manipulating or eradicating disease vectors have been proposed as alternatives to malaria eradication. The success of this approach depends on efficient spread of a genetic modification in field populations. Windbichler et al. show that a synthetic genetic element consisting of mosquito regulatory elements and the Homing Endonuclease gene I-SceI can spread from a small number of individual Anopheles gambiae mosquitoes into large receptive populations in just a few generations. This is the first demonstration of a synthetic gene drive system in the main human malaria vector — and a similar approach should be applicable to many other pest species. Genetic methods of manipulating or eradicating disease vector populations have long been discussed as an attractive alternative to existing control measures because of their potential advantages in terms of effectiveness and species specificity1,2,3. The development of genetically engineered malaria-resistant mosquitoes has shown, as a proof of principle, the possibility of targeting the mosquito’s ability to serve as a disease vector4,5,6,7. The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations8. We have suggested previously that Homing Endonuclease genes (HEGs), a class of simple selfish genetic elements, could be exploited for this purpose9. Here we demonstrate that a synthetic genetic element, consisting of mosquito regulatory regions10 and the Homing Endonuclease gene I-SceI11,12,13, can substantially increase its transmission to the progeny in transgenic mosquitoes of the human malaria vector Anopheles gambiae. We show that the I-SceI element is able to invade receptive mosquito cage populations rapidly, validating mathematical models for the transmission dynamics of HEGs. Molecular analyses confirm that expression of I-SceI in the male germline induces high rates of site-specific chromosomal cleavage and gene conversion, which results in the gain of the I-SceI gene, and underlies the observed genetic drive. These findings demonstrate a new mechanism by which genetic control measures can be implemented. Our results also show in principle how sequence-specific genetic drive elements like HEGs could be used to take the step from the genetic engineering of individuals to the genetic engineering of populations.

  • Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG Homing Endonuclease
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Audrey Mcconnell Smith, Raymond J Monnat, Ryo Takeuchi, Stefan Pellenz, Luther Davis, Nancy Maizels, Barry L Stoddard
    Abstract:

    Homing Endonucleases stimulate gene conversion by generating site-specific DNA double-strand breaks that are repaired by homologous recombination. These enzymes are potentially valuable tools for targeted gene correction and genome engineering. We have engineered a variant of the I-AniI Homing Endonuclease that nicks its cognate target site. This variant contains a mutation of a basic residue essential for proton transfer and solvent activation in one active site. The cleavage mechanism, DNA-binding affinity, and substrate specificity profile of the nickase are similar to the wild-type enzyme. I-AniI nickase stimulates targeted gene correction in human cells, in cis and in trans, at ≈1/4 the efficiency of the wild-type enzyme. The development of sequence-specific nicking enzymes like the I-AniI nickase will facilitate comparative analyses of DNA repair and mutagenesis induced by single- or double-strand breaks.

  • crystallization and preliminary x ray studies of i ppoi a nuclear intron encoded Homing Endonuclease from physarum polycephalum
    Protein Science, 2008
    Co-Authors: Karen E. Flick, Raymond J Monnat, D Mchugh, J D Heath, Kathryn M Stephens, Barry L Stoddard
    Abstract:

    The Homing Endonuclease I-PpoI is encoded by an op- tional third intron, Pp LSU 3, found in nuclear, extrachromosomal copies of the Physarum polycephalum 26s rRNA gene. This en- donuclease promotes the lateral transfer or "Homing" of its encod- ing intron by recognizing and cleaving a partially symmetric, 15 bp Homing site in 26s rDNA alleles that lack the Pp LSU 3 intron. The open reading frame encoding I-PpoI has been subcloned, and the Endonuclease has been overproduced in E. coli. Purified recombi- nant I-PpoI has been co-crystallized with a 21 bp Homing site DNA duplex. The crystals belong to space group P3,21, with unit cell dimensions a = b = 114 A, c = 89 A. The results of initial X-ray diffraction experiments indicate that the asymmetric unit contains an enzyme homodimer and one duplex DNA molecule, and that the unit cell has a specific volume of 3.4 A,'/dalton. These experiments also provide strong evidence that I-PpoI contains several bound zinc ions as part of its structure.

  • altered target site specificity variants of the i ppoi his cys box Homing Endonuclease
    Nucleic Acids Research, 2007
    Co-Authors: Jennifer L Eklund, Umut Y Ulge, Jennifer H Eastberg, Raymond J Monnat
    Abstract:

    We used a yeast one-hybrid assay to isolate and characterize variants of the eukaryotic Homing Endonuclease I-PpoI that were able to bind a mutant, cleavage-resistant I-PpoI target or ‘Homing’ site DNA in vivo. Native I-PpoI recognizes and cleaves a semi-palindromic 15-bp target site with high specificity in vivo and in vitro. This target site is present in the 28S or equivalent large subunit rDNA genes of all eukaryotes. I-PpoI variants able to bind mutant target site DNA had from 1 to 8 amino acid substitutions in the DNA–protein interface. Biochemical characterization of these proteins revealed a wide range of site–binding affinities and site discrimination. One-third of variants were able to cleave target site DNA, but there was no systematic relationship between site-binding affinity and site cleavage. Computational modeling of several variants provided mechanistic insight into how amino acid substitutions that contact, or are adjacent to, specific target site DNA base pairs determine I-PpoI site-binding affinity and site discrimination, and may affect cleavage efficiency.

  • isolation and characterization of new Homing Endonuclease specificities at individual target site positions
    Journal of Molecular Biology, 2004
    Co-Authors: Django Sussman, Meggen S Chadsey, Barry L Stoddard, Raymond J Monnat, Steve Fauce, Alex Engel, Anna Bruett, Lenny M Seligman
    Abstract:

    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.

David A. Shub - One of the best experts on this subject based on the ideXlab platform.

  • a Homing Endonuclease and the 50 nt ribosomal bypass sequence of phage t4 constitute a mobile dna cassette
    Proceedings of the National Academy of Sciences of the United States of America, 2011
    Co-Authors: Richard P. Bonocora, Qinglu Zeng, Ethan V Abel, David A. Shub
    Abstract:

    Since its initial description more than two decades ago, the ribosome bypass (or “hop”) sequence of phage T4 stands out as a uniquely extreme example of programmed translational frameshifting. The gene for a DNA topoisomerase subunit of T4 has been split by a 1-kb insertion into two genes that retain topoisomerase function. A second 50-nt insertion, beginning with an in-phase stop codon, is inserted near the start of the newly created downstream gene 60. Instead of terminating at this stop codon, approximately half of the ribosomes skip 50 nucleotides and continue translation in a new reading frame. However, no functions, regulatory or otherwise, have been imputed for the truncated peptide that results from termination at codon 46 or for the bypass sequence itself. Moreover, how this unusual mRNA organization arose and why it is maintained have never been explained. We show here that a Homing Endonuclease (MobA) is encoded in the insertion that created gene 60, and the mobA gene together with the bypass sequence constitute a mobile DNA cassette. The bypass sequence provides protection against self-cleavage by the nuclease, whereas the nuclease promotes horizontal spread of the entire cassette to related bacteriophages. Group I introns frequently provide protection against self-cleavage by associated Homing Endonucleases. We present a scenario by which the bypass sequence, which is otherwise a unique genetic element, might have been derived from a degenerate group I intron.

  • A Free-Standing Homing Endonuclease Targets an Intron Insertion Site in the psbA Gene of Cyanophages
    Current biology : CB, 2009
    Co-Authors: Qinglu Zeng, Richard P. Bonocora, David A. Shub
    Abstract:

    Homing Endonuclease genes are mobile elements that promote their duplication into cognate sites that lack the Endonuclease gene [1, 2]. The Homing Endonuclease initiates this event through site-specific DNA cleavage. Copying of the Endonuclease gene follows as a consequence of DNA repair. A genome containing a Homing Endonuclease gene is subject to self-cleavage. Protection is accomplished through DNA sequence polymorphisms, as is the case in intronless Homing of free-standing Endonuclease genes [3, 4], or by disruption of the recognition site by a group I intron (or intein) into which the Endonuclease ORF is embedded. We describe here a novel free-standing Homing Endonuclease from cyanobacteriophage S-PM2, which is similar to the DNA resolvase of bacteriophage T4 and is encoded adjacent to an intron-containing psbA gene [5, 6]. The Endonuclease makes a specific double-strand cut near the intron insertion site (IIS), its DNA recognition site spans the IIS, and it is unable to cleave intron-containing psbA genes. This interdependence of a free-standing Endonuclease gene and a group I intron, which we denote "collaborative Homing," has not been reported previously and gives support to a hypothesis of formation of composite mobile introns by independent convergence of an intron and an Endonuclease gene on the same target sequence.

  • the restriction fold turns to the dark side a bacterial Homing Endonuclease with a pd d e xk motif
    The EMBO Journal, 2007
    Co-Authors: Lei Zhao, Richard P. Bonocora, David A. Shub, Barry L Stoddard
    Abstract:

    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.

  • DNA binding and cleavage by the HNH Homing Endonuclease I-HmuI.
    Journal of molecular biology, 2004
    Co-Authors: Betty W. Shen, David A. Shub, Markus Landthaler, Barry L Stoddard
    Abstract:

    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.

  • the nicking Homing Endonuclease i basi is encoded by a group i intron in the dna polymerase gene of the bacillus thuringiensis phage bastille
    Nucleic Acids Research, 2003
    Co-Authors: Markus Landthaler, David A. Shub
    Abstract:

    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.