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Barry L. Stoddard - One of the best experts on this subject based on the ideXlab platform.
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activity specificity and structure of i bth0305i a representative of a new homing Endonuclease family
Nucleic Acids Research, 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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computational redesign of Endonuclease dna binding and cleavage specificity
Nature, 2006Co-Authors: Justin Ashworth, Barry L. Stoddard, Raymond J Monnat, James J Havranek, Carlos M Duarte, Django Sussman, David BakerAbstract:Altering the specificity of DNA-cleaving enzymes could be useful in many medical or biotechnological applications, but it is quite a challenge in terms of computational protein design. Ashwell et al. have used computational redesign to alter the target-site specificity of the I-MsoI homing Endonuclease, while maintaining wild-type binding affinity. The redesigned enzyme binds and cleaves the new DNA recognition site about 10,000 times more effectively than the wild-type enzyme, with target discrimination comparable to the original Endonuclease. These results suggest that computational protein design methods can be used to create novel and highly specific Endonucleases for gene therapy and other applications. Redesign of the I-MsoI Endonuclease binds and cleaves the new recognition site ∼10,000-fold more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original Endonuclease. The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein–DNA recognition, and has considerable practical relevance for biotechnology and medicine1,2,3,4,5,6. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing Endonuclease I-MsoI7 using a physically realistic atomic-level forcefield8,9. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling10. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site ∼10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original Endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific Endonucleases for gene therapy and other applications.
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Homing Endonuclease structure and function.
Quarterly reviews of biophysics, 2005Co-Authors: Barry L. StoddardAbstract:Homing Endonucleases are encoded by open reading frames that are embedded within group I, group II and archael introns, as well as inteins (intervening sequences that are spliced and excised post-translationally). These enzymes initiate transfer of those elements (and themselves) by generating strand breaks in cognate alleles that lack the intervening sequence, as well as in additional ectopic sites that broaden the range of intron and intein mobility. Homing Endonucleases can be divided into several unique families that are remarkable in several respects: they display extremely high DNA-binding specificities which arise from long DNA target sites (14-40 bp), they are tolerant of a variety of sequence variations in these sites, and they display disparate DNA cleavage mechanisms. A significant number of homing Endonucleases also act as maturases (highly specific cofactors for the RNA splicing reactions of their cognate introns). Of the known homing group I Endonuclease families, two (HNH and His-Cys box enzymes) appear to be diverged from a common ancestral nuclease. While crystal structures of several representatives of the LAGLIDADG Endonuclease family have been determined, only structures of single members of the HNH (I-HmuI), His-Cys box (I-PpoI) and GIY-YIG (I-TevI) families have been elucidated. These studies provide an important source of information for structure-function relationships in those families, and are the centerpiece of this review. Finally, homing Endonucleases are significant targets for redesign and selection experiments, in hopes of generating novel DNA binding and cutting reagents for a variety of genomic applications.
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The LAGLIDADG Homing Endonuclease Family
Homing Endonucleases and Inteins, 2005Co-Authors: Brett Chevalier, Raymond J Monnat, Barry L. StoddardAbstract:The LAGLIDADG protein family includes the first identified and biochemically characterized intron-encoded proteins (Dujon 1980; Lazowska et al. 1980; Jacquier and Dujon 1985), as described in this volume by Dujon. It has been variously termed the ‘DOD’, ‘dodecapeptide’, ‘dodecamer’, and ‘decapeptide’ Endonuclease family, based on the conservation of a ten-residue sequence motif (Dujon 1989; Dujon et al. 1989; Belfort et al. 1995; Belfort and Roberts 1997; Dalgaard et al. 1997; Chevalier and Stoddard 2001). The LAGLIDADG Endonucleases are the most diverse of the homing Endonuclease families. Their host range includes the genomes of plant and algal chloroplasts, fungal and protozoan mitochondria, bacteria and Archaea (Dalgaard et al. 1997). One reason for the wide phylogenetic distribution of LAGLIDADG genes appears to be their remarkable ability to invade unrelated types of intervening sequences, including group I introns, archaeal introns and inteins (Belfort and Roberts 1997; Chevalier and Stoddard 2001). Descendents of LAGLIDADG homing Endonucleases also include the yeast HO mating type switch Endonuclease (Jin et al. 1997), which is encoded by an independent reading frame rather than within an intron, but does carry remnants of an inactive intein domain (Haber and Wolfe, this Vol.), and maturases that assist in RNA splicing (Delahodde et al. 1989; Lazowska et al. 1989; Schafer et al. 1994; Geese and Waring 2001; Caprara and Waring, this Vol.).
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Catalytic Mechanisms of Restriction and Homing Endonucleases
Biochemistry, 2002Co-Authors: Eric A. Galburt, Barry L. StoddardAbstract:The catalytic mechanisms of type II restriction Endonucleases and homing Endonucleases are discussed and compared. Brief reviews of the chemistry of phosphoryl transfers and canonical one-metal and two-metal endonucleolytic mechanisms are provided along with possible future directions in the study of Endonuclease active sites. The discussion of type II restriction Endonucleases is comprised of a description of the general architecture of the canonical active site structural motif followed by more in-depth examples of one- and two-metal mechanisms. The homing Endonuclease section is comprised of four sections describing what is known regarding the cleavage mechanisms of the four group I intron homing Endonuclease families: LAGLIDADG, His-Cys box, H-N-H, and GIY-YIG.
K D Bloch - One of the best experts on this subject based on the ideXlab platform.
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Digestion of DNA with restriction Endonucleases.
Current protocols in molecular biology, 2001Co-Authors: K D Bloch, B GrossmannAbstract:Restriction Endonucleases recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction Endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. The first method presented in this unit is the cleavage of a single DNA sample with a single restriction Endonuclease. A number of common applications of this technique are also described. These include digesting a given DNA sample with more than one Endonuclease, digesting multiple DNA samples with the same Endonuclease, and partially digesting DNA such that cleavage only occurs at a subset of the restriction sites. A protocol for methylating specific DNA sequences and protecting them from restriction Endonuclease cleavage is also presented. A collection of tables describing restriction Endonucleases and their properties (including information about recognition sequences, types of termini produced, buffer conditions, and conditions for thermal inactivation) is given at the end of the unit.
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Digestion of DNA with restriction Endonucleases.
Current protocols in neuroscience, 2001Co-Authors: K D BlochAbstract:Restriction Endonucleases recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction Endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. This appendix describes restriction Endonucleases and their properties.
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APPENDIX 4I Digestion of DNA with Restriction Endonucleases
Current protocols in protein science, 1998Co-Authors: K D BlochAbstract:Restriction Endonucleases recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction Endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. This appendix describes restriction Endonucleases and their properties.
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Digestion of DNA with restriction Endonucleases.
Current Protocols in Immunology, 1992Co-Authors: K D BlochAbstract:Restriction Endonucleases recognize short DNA sequences and catalyze the cleavage of double-stranded DNA at specific sites within or adjacent to the recognition sequences. This unit describes how to cleave DNA and refers the investigator to frequently updated manuals by Endonuclease suppliers for the precise optimized conditions. Alternate procedures are given for digesting a DNA sample with several different enzymes and digesting multiple DNA samples with the same Endonuclease.
Brett Chevalier - One of the best experts on this subject based on the ideXlab platform.
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The LAGLIDADG Homing Endonuclease Family
Homing Endonucleases and Inteins, 2005Co-Authors: Brett Chevalier, Raymond J Monnat, Barry L. StoddardAbstract:The LAGLIDADG protein family includes the first identified and biochemically characterized intron-encoded proteins (Dujon 1980; Lazowska et al. 1980; Jacquier and Dujon 1985), as described in this volume by Dujon. It has been variously termed the ‘DOD’, ‘dodecapeptide’, ‘dodecamer’, and ‘decapeptide’ Endonuclease family, based on the conservation of a ten-residue sequence motif (Dujon 1989; Dujon et al. 1989; Belfort et al. 1995; Belfort and Roberts 1997; Dalgaard et al. 1997; Chevalier and Stoddard 2001). The LAGLIDADG Endonucleases are the most diverse of the homing Endonuclease families. Their host range includes the genomes of plant and algal chloroplasts, fungal and protozoan mitochondria, bacteria and Archaea (Dalgaard et al. 1997). One reason for the wide phylogenetic distribution of LAGLIDADG genes appears to be their remarkable ability to invade unrelated types of intervening sequences, including group I introns, archaeal introns and inteins (Belfort and Roberts 1997; Chevalier and Stoddard 2001). Descendents of LAGLIDADG homing Endonucleases also include the yeast HO mating type switch Endonuclease (Jin et al. 1997), which is encoded by an independent reading frame rather than within an intron, but does carry remnants of an inactive intein domain (Haber and Wolfe, this Vol.), and maturases that assist in RNA splicing (Delahodde et al. 1989; Lazowska et al. 1989; Schafer et al. 1994; Geese and Waring 2001; Caprara and Waring, this Vol.).
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design activity and structure of a highly specific artificial Endonuclease
Molecular Cell, 2002Co-Authors: Brett Chevalier, Tanja Kortemme, Meggen S Chadsey, David Baker, Raymond J Monnat, Barry L. StoddardAbstract: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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mutations altering the cleavage specificity of a homing Endonuclease
Nucleic Acids Research, 2002Co-Authors: Lenny M Seligman, Brett Chevalier, Meggen S Chadsey, Karen M Chisholm, Samuel T Edwards, Jeremiah Savage, Adeline L VeilletAbstract:The homing Endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein–DNA contacts. We test these predictions by analyzing a set of Endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such Endonucleases demonstrate that our genetic approach is effective in identifying homing Endonucleases that recognize and cleave novel target sequences.
Lynn Harrison - One of the best experts on this subject based on the ideXlab platform.
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Repair of oxidative damage to DNA: enzymology and biology.
Annual Review of Biochemistry, 1994Co-Authors: Bruce Demple, Lynn HarrisonAbstract:DNA GL YCOSYLASES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 921 Thymine Glycol Glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 921 ForfTUlmidopyrimidine Glycosylase (Fpg/MutM) . . . . . . . . . . . . .. . . . . . . . 924 MutY: A DNA MisfTUltch Glycosylase for Oxidative DafTUlge . . . . . . . . . . . . . 927 Hypoxanthine-DNA Glycosylase 927 5-Hydroxymethyluracil and 5-Hydroxymethylcytosine DNA Glycosylases . . . . . . 928 UV Endonucleases 929 REPAIR OF DEOXYRIBOSE DAMAGES: AP EndonucleaseS AND 3'-TRIMMING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931 Exonuclease III of E. coli 931 Eukaryotic AP Endonucleases Related to Exonuclease III . . . . . . . . . . . . . . . 933 E. coli Endonuclease IV 937 S. cerevisiae ApnI Protein 938 AP Endonucleases as Metalloproteins 939
Marlene Belfort - One of the best experts on this subject based on the ideXlab platform.
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catalytic domain structure and hypothesis for function of giy yig intron Endonuclease i tevi
Nature Structural & Molecular Biology, 2002Co-Authors: Patrick Van Roey, Marlene Belfort, Lisa Meehan, Joseph C Kowalski, Victoria DerbyshireAbstract:I-TevI, a member of the GIY-YIG family of homing Endonucleases, consists of an N-terminal catalytic domain and a C-terminal DNA-binding domain joined by a flexible linker. The GIY-YIG motif is in the N-terminal domain of I-TevI, which corresponds to a phylogenetically widespread catalytic cartridge that is often associated with mobile genetic elements. The crystal structure of the catalytic domain of I-TevI, the first of any GIY-YIG Endonuclease, reveals a novel α/β-fold with a central three-stranded antiparallel β-sheet flanked by three helices. The most conserved and putative catalytic residues are located on a shallow, concave surface and include a metal coordination site. Similarities in the three-dimensional arrangement of the catalytically important residues and the cation-binding site with those of the His-Cys box Endonuclease I-PpoI suggest the possibility of mechanistic relationships among these different families of homing Endonucleases despite completely different folds.
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A site-specific Endonuclease encoded by a typical archaeal intron
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Jacob Z. Dalgaard, R A Garrett, Marlene BelfortAbstract:Abstract The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing Endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron Endonucleases in that it is thermostable and is expressed only from linear and cyclized intron species and not from the precursor RNA. However, in analogy to its eukaryotic counterparts, but unlike the bacteriophage enzymes, I-Dmo I makes a staggered double-strand cut that generates 4-nt 3' extensions. Additionally, although the archaeal and group I introns have entirely different structural properties and splicing pathways, I-Dmo I shares sequence similarity, in the form of the LAGLI-DADG motif, with group I intron Endonucleases of eukaryotes. These observations support the independent evolutionary origin of Endonucleases and intron core elements and are consistent with the invasive potential of Endonuclease genes.
