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1.
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.  相似文献   

2.
Homing endonucleases (HEs) promote the evolutionary persistence of selfish DNA elements by catalyzing element lateral transfer into new host organisms. The high site specificity of this lateral transfer reaction, termed homing, reflects both the length (14–40 bp) and the limited tolerance of target or homing sites for base pair changes. In order to better understand molecular determinants of homing, we systematically determined the binding and cleavage properties of all single base pair variant target sites of the canonical LAGLIDADG homing endonucleases I-CreI and I-MsoI. These Chlorophyta algal HEs have very similar three-dimensional folds and recognize nearly identical 22 bp target sites, but use substantially different sets of DNA-protein contacts to mediate site-specific recognition and cleavage. The site specificity differences between I-CreI and I-MsoI suggest different evolutionary strategies for HE persistence. These differences also provide practical guidance in target site finding, and in the generation of HE variants with high site specificity and cleavage activity, to enable genome engineering applications.  相似文献   

3.
Divalent metal ions play a crucial role in forming the catalytic centres of DNA endonucleases. Substitution of Mg2+ ions by Fe2+ ions in two archaeal intron-encoded homing endonucleases, I-DmoI and I-PorI, yielded functional enzymes and enabled the generation of reactive hydroxyl radicals within the metal ion binding sites. Specific hydroxyl radical-induced cleavage was observed within, and immediately after, two conserved LAGLIDADG motifs in both proteins and at sites at, and near, the scissile phosphates of the corresponding DNA substrates. Titration of Fe2+-containing protein-DNA complexes with Ca2+ ions, which are unable to support endonucleolytic activity, was performed to distinguish between the individual metal ions in the complex. Mutations of single amino acids in this region impaired catalytic activity and caused the preferential loss of a subset of hydroxyl radical cleavages in both the protein and the DNA substrate, suggesting an active role in metal ion coordination for these amino acids. The data indicate that the endonucleases cleave their DNA substrates as monomeric enzymes, and contain a minimum of four divalent metal ions located at or near the catalytic centres of each endonuclease. The metal ions involved in cleaving the coding and the non-coding strand are positioned immediately after the N- and C-terminally located LAGLIDADG motifs, respectively. The dual protein/nucleic acid footprinting approach described here is generally applicable to other protein-nucleic acid complexes when the natural metal ion can be replaced by Fe2+.  相似文献   

4.
The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding. Here, we show that a semi-rational approach can be used to derive hundreds of novel proteins from I-CreI, a homing endonuclease from the LAGLIDADG family. These novel endonucleases display a wide range of cleavage patterns in yeast and mammalian cells that in most cases are highly specific and distinct from I-CreI. Second, rules for protein/DNA interaction can be inferred from statistical analysis. Third, novel endonucleases can be combined to create heterodimeric protein species, thereby greatly enhancing the number of potential targets. These results describe a straightforward approach for engineering novel endonucleases with tailored specificities, while preserving the activity and specificity of natural homing endonucleases, and thereby deliver new tools for genome engineering.  相似文献   

5.
The archaeal intron-encoded homing enzymes I-PorI and I-DmoI belong to a family of endonucleases that contain two copies of a characteristic LAGLIDADG motif. These endonucleases cleave their intron- or intein- alleles site-specifically, and thereby facilitate homing of the introns or inteins which encode them. The protein structure and the mechanism of DNA recognition of these homing enzymes is largely unknown. Therefore, we examined these properties of I-PorI and I-DmoI by protein footprinting. Both proteins were susceptible to proteolytic cleavage within regions that are equidistant from each of the two LAGLIDADG motifs. When complexed with their DNA substrates, a characteristic subset of the exposed sites, located in regions immediately after and 40-60 amino acids after each of the LAGLIDADG motifs, were protected. Our data suggest that the enzymes are structured into two, tandemly repeated, domains, each containing both the LAGLIDADG motif and two putative DNA binding regions. The latter contains a potentially novel DNA binding motif conserved in archaeal homing enzymes. The results are consistent with a model where the LAGLIDADG endonucleases bind to their non-palindromic substrates as monomeric enzymes, with each of the two domains recognizing one half of the DNA substrate.  相似文献   

6.
7.
Meganucleases are sequence-specific endonucleases with large cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These enzymes open novel perspectives for genome engineering in a wide range of fields, including gene therapy. A new crystal structure of the I-CreI dimer without DNA has allowed the comparison with the DNA-bound protein. The C-terminal loop displays a different conformation, which suggests its implication in DNA binding. A site-directed mutagenesis study in this region demonstrates that whereas the C-terminal helix is negligible for DNA binding, the final C-terminal loop is essential in DNA binding and cleavage. We have identified two regions that comprise the Ser138–Lys139 and Lys142–Thr143 pairs whose double mutation affect DNA binding in vitro and abolish cleavage in vivo. However, the mutation of only one residue in these sites allows DNA binding in vitro and cleavage in vivo. These findings demonstrate that the C-terminal loop of I-CreI endonuclease plays a fundamental role in its catalytic mechanism and suggest this novel site as a region to take into account for engineering new endonucleases with tailored specificity.  相似文献   

8.
The monomeric homing endonuclease PI-SceI cleaves the two strands of its DNA substrate in a concerted manner, which raises the question of whether this enzyme harbours one or two catalytic centres. If PI-SceI has only one catalytic centre, one would expect that cross-linking enzyme and substrate should prevent reorientation of the enzyme required to perform the second cut after having made the first cut: PI-SceI, however, when cross-linked to its substrate, is able to cleave both DNA strands. If PI-SceI has two catalytic centres, one would expect that it should be possible to inactivate one catalytic centre by mutation and obtain a variant with preference for a substrate nicked in one strand; such variants have been found. The structural homology between the catalytic domain of PI-SceI having a pseudo 2-fold symmetry, and I-CreI, a homodimeric homing endonuclease, suggests that in PI-SceI active site I, which attacks the top strand, comprises Asp218, Asp229 and Lys403, while Asp326, Thr341 and Lys301 make up active site II, which cleaves the bottom strand. Cleavage experiments with modified oligodeoxynucleotides and metal ion mapping experiments demonstrate that PI-SceI interacts differently with the two strands at the cleavage position, supporting a model of two catalytic centres.  相似文献   

9.
Homing endonucleases recognize and generate a DNA double-strand break, which has been used to promote gene targeting. These enzymes recognize long DNA stretches; they are highly sequence-specific enzymes and display a very low frequency of cleavage even in complete genomes. Although a large number of homing endonucleases have been identified, the landscape of possible target sequences is still very limited to cover the complexity of the whole eukaryotic genome. Therefore, the finding and molecular analysis of homing endonucleases identified but not yet characterized may widen the landscape of possible target sequences. The previous characterization of protein-DNA interaction before the engineering of new homing endonucleases is essential for further enzyme modification. Here we report the crystal structure of I-CvuI in complex with its target DNA and with the target DNA of I-CreI, a homologue enzyme widely used in genome engineering. To characterize the enzyme cleavage mechanism, we have solved the I-CvuI DNA structures in the presence of non-catalytic (Ca2+) and catalytic ions (Mg2+). We have also analyzed the metal dependence of DNA cleavage using Mg2+ ions at different concentrations ranging from non-cleavable to cleavable concentrations obtained from in vitro cleavage experiments. The structure of I-CvuI homing endonuclease expands the current repertoire for engineering custom specificities, both by itself as a new scaffold alone and in hybrid constructs with other related homing endonucleases or other DNA-binding protein templates.  相似文献   

10.
Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.  相似文献   

11.
We have developed and used a genetic selection system in Escherichia coli to study functional requirements for homing site recognition and cleavage by a representative eukaryotic mobile intron endonuclease. The homing endonuclease, I-CreI, was originally isolated from the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. I-CreI homing site mutants contained base pair substitutions or single base deletions that altered the rate of homing site cleavage and/or product release. I-CreI endonuclease mutants fell into six phenotypic classes that differed in in vivo activity, toxicity or genetic dominance. Inactivating mutations clustered in the N-terminal 60% of the I-CreI amino acid sequence, and two frameshift mutations were isolated that resulted in premature translation termination though retained partial activity. These mutations indicate that the N-terminal two-thirds of the I-CreI endonuclease is sufficient for homing site recognition and cleavage. Substitution mutations altered in four potential active site residues were examined: D20N, Q47H or R70A substitutions inactivated endonuclease activity, whereas S22A did not. The genetic approach we have taken complements phylogenetic and structural studies of mobile intron endonucleases and has provided new information on the mechanistic basis of I-CreI homing site recognition and cleavage.  相似文献   

12.
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.  相似文献   

13.
Homing endonucleases (HEs) cut long DNA target sites with high specificity to initiate and target the lateral transfer of mobile introns or inteins. This high site specificity of HEs makes them attractive reagents for gene targeting to promote DNA modification or repair. We have generated several hundred catalytically active, monomerized versions of the well-characterized homodimeric I-CreI and I-MsoI LAGLIDADG family homing endonuclease (LHE) proteins. Representative monomerized I-CreI and I-MsoI proteins (collectively termed mCreIs or mMsoIs) were characterized in detail by using a combination of biochemical, biophysical and structural approaches. We also demonstrated that both mCreI and mMsoI proteins can promote cleavage-dependent recombination in human cells. The use of single chain LHEs should simplify gene modification and targeting by requiring the expression of a single small protein in cells, rather than the coordinate expression of two separate protein coding genes as is required when using engineered heterodimeric zinc finger or homing endonuclease proteins.  相似文献   

14.
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.  相似文献   

15.
Mycobacterium leprae recA harbors an in‐frame insertion sequence that encodes an intein homing endonuclease (PI‐MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG‐type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI‐MleI is distinctive from other members of the family of LAGLIDADG‐type HEases for its modular structure with functionally separable domains for DNA‐binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI‐MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active‐site residues essential for DNA target site recognition and double‐stranded DNA cleavage, we performed site‐directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild‐type PI‐MleI and its variants disclosed that the two amino acid residues, Asp122 (in Block C) and Asp193 (in functional Block E), are crucial to the double‐stranded DNA endonuclease activity, whereas Asp218 (in pseudo‐Block E) is not. However, despite the reduced catalytic activity, the PI‐MleI variants, like the wild‐type PI‐MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA‐binding affinities, but abolished the double‐stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double‐stranded DNA cleavage activity, compared with the wild‐type PI‐MleI. These results provide compelling evidence that Asp122 and Asp193 in DOD motif I and II, respectively, are bona fide active‐site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.  相似文献   

16.
I-DmoI is a 22 kDa endonuclease encoded by an intron in the 23 S rRNA gene of the hyperthermophilic archaeon Desulfurococcus mobilis. The structure of I-DmoI has been determined to 2.2 A resolution using multi-wavelength anomalous diffraction techniques. I-DmoI, a protein of the LAGLIDADG motif family, represents the first structure of a freestanding endonuclease with two LAGLIDADG motifs, and the first of a thermostable homing endonuclease. I-DmoI consists of two similar alpha/beta domains (alphabetabetaalphabetabetaalpha) related by pseudo 2-fold symmetry. The LAGLIDADG motifs are located at the carboxy-terminal end of the first alpha-helix of each domain. These helices form a two-helix bundle at the interface between the domains and are perpendicular to a saddle-shaped DNA binding surface, formed by two four-stranded antiparallel beta-sheets. Despite substantially different sequences, the overall fold of I-DmoI is similar to that of two other LAGLIDADG proteins for which the structures are known, I-CreI and the endonuclease domain of PI-SceI. The three structures differ most in the loops connecting the beta-strands, relating to the respective DNA target site sizes and geometries. In addition, the absence of conserved residues surrounding the active site, other than those within the LAGLIDADG motif, is of mechanistic importance. Finally, the carboxy-terminal domain of I-DmoI is smaller and has a more irregular fold than the amino-terminal domain, which is more similar to I-CreI, a symmetric homodimeric endonuclease. This is reversed compared to PI-SceI, where the amino-terminal domain is more similar to carboxy-terminal domain of I-DmoI and to I-CreI, with interesting evolutionary implications.  相似文献   

17.
Controversy surrounds the metal-dependent mechanism of H-N-H endonucleases, enzymes involved in a variety of biological functions, including intron homing and DNA repair. To address this issue we determined the crystal structures for complexes of the H-N-H motif containing bacterial toxin colicin E9 with Zn(2+), Zn(2+).DNA, and Mg(2+).DNA. The structures show that the rigid V-shaped architecture of the active site does not undergo any major conformational changes on binding to the minor groove of DNA and that the same interactions are made to the nucleic acid regardless of which metal ion is bound to the enzyme. The scissile phosphate contacts the single metal ion of the motif through distortion of the DNA brought about by the insertion of the Arg-96-Glu-100 salt bridge into the minor groove and a network of contacts to the DNA phosphate backbone that straddle the metal site. The Mg(2+)-bound structure reveals an unusual coordination scheme involving two H-N-H histidine residues, His-102 and His-127. The mechanism of DNA cleavage is likely related to that of other single metal ion-dependent endonucleases, such as I-PpoI and Vvn, although in these enzymes the single alkaline earth metal ion is coordinated by oxygen-bearing amino acids. The structures also provide a rationale as to why H-N-H endonucleases are inactive in the presence of Zn(2+) but active with other transition metal ions such as Ni(2+). This is because of coordination of the Zn(2+) ion through a third histidine, His-131. "Active" transition metal ions are those that bind more weakly to the H-N-H motif because of the disengagement of His-131, which we suggest allows a water molecule to complete the catalytic cycle.  相似文献   

18.
以已公布的114种真菌线粒体基因组数据为依据,对cob内含子及其编码的Ⅱ型LAGLIDADG归巢内切酶进行全面分析,以揭示其进化规律。在cob内含子中共发现27个Ⅱ型LAGLIDADG归巢内切酶基因,其中18个位于S433内含子插入位点,其余9个散布在另外8个插入位点。结合Pfam数据,将Ⅱ型LAGLIDADG归巢内切酶分成10个主要类群,其中4个类群存在不同生物界物种间的水平迁移。S433位点的18个归巢内切酶均属于类群1,它们与宿主内含子可能从共同祖先垂直遗传而来,并在传递过程中伴有水平迁移;其他归巢内切酶及宿主内含子则应是水平迁移的结果。类群1中的归巢内切酶可分为两个亚类,两亚类识别的靶序列存在明显差异;保守模体氨基酸序列分析显示它们大多数具有潜在内切酶活性。全面呈现了真菌线粒体cob内含子及其编码的Ⅱ型LAGLIDADG归巢内切酶的存在状态和进化模式,为归巢内切酶的改造和设计提供了新素材。  相似文献   

19.
We have determined the crystal structure of the PvuII endonuclease in the presence of Mg(2+). According to the structural data, divalent metal ion binding in the PvuII subunits is highly asymmetric. The PvuII-Mg(2+) complex has two distinct metal ion binding sites, one in each monomer. One site is formed by the catalytic residues Asp58 and Glu68, and has extensive similarities to a catalytically important site found in all structurally examined restriction endonucleases. The other binding site is located in the other monomer, in the immediate vicinity of the hydroxyl group of Tyr94; it has no analogy to metal ion binding sites found so far in restriction endonucleases. To assign the number of metal ions involved and to better understand the role of Mg(2+) binding to Tyr94 for the function of PvuII, we have exchanged Tyr94 by Phe and characterized the metal ion dependence of DNA cleavage of wild-type PvuII and the Y94F variant. Wild-type PvuII cleaves both strands of the DNA in a concerted reaction. Mg(2+) binding, as measured by the Mg(2+) dependence of DNA cleavage, occurs with a Hill coefficient of 4, meaning that at least two metal ions are bound to each subunit in a cooperative fashion upon formation of the active complex. Quenched-flow experiments show that DNA cleavage occurs about tenfold faster if Mg(2+) is pre-incubated with enzyme or DNA than if preformed enzyme-DNA complexes are mixed with Mg(2+). These results show that Mg(2+) cannot easily enter the active center of the preformed enzyme-DNA complex, but that for fast cleavage the metal ions must already be bound to the apoenzyme and carried with the enzyme into the enzyme-DNA complex. The Y94F variant, in contrast to wild-type PvuII, does not cleave DNA in a concerted manner and metal ion binding occurs with a Hill coefficient of 1. These results indicate that removal of the Mg(2+) binding site at Tyr94 completely disrupts the cooperativity in DNA cleavage. Moreover, in quenched-flow experiments Y94F cleaves DNA about ten times more slowly than wild-type PvuII, regardless of the order of mixing. From these results we conclude that wild-type PvuII cleaves DNA in a fast and concerted reaction, because the Mg(2+) required for catalysis are already bound at the enzyme, one of them at Tyr94. We suggest that this Mg(2+) is shifted to the active center during binding of a specific DNA substrate. These results, for the first time, shed light on the pathway by which metal ions as essential cofactors enter the catalytic center of restriction endonucleases.  相似文献   

20.
Monomeric homing endonucleases of the LAGLIDADG family recognize DNA in a bipartite manner, reflecting the underlying structural assembly of two protein domains (A and B) related by pseudo 2-fold symmetry. This architecture allows for changes in DNA specificity via the distinct combination of these half-site domains. The key to engineering such hybrid proteins lies in the LAGLIDADG two-helix bundle that forms both the domain interface and the endonuclease active site. In this study, we utilize domain A of the monomeric I-DmoI to demonstrate the feasibility of generating functional homodimeric endonucleases that recognize palindromic DNA sequences derived from the original, non-palindromic target. Wild-type I-DmoI domain A is capable of forming a homodimer (H-DmoA) that binds tightly to, but does not cleave efficiently, its anticipated DNA target. Partial restoration of DNA cleavage ability was obtained by re-engineering the LAGLIDADG dimerization interface (H-DmoC). Upon fusing two copies of H-DmoC via a short peptide linker, a novel, site-specific DNA endonuclease was created (H-DmoC2). Like I-DmoI, H-DmoC2 is thermostable and cleaves the new target DNA to generate the predicted 4 nt 3'-OH overhangs but, unlike I-DmoI, H-DmoC2 retains stringent cleavage specificity when substituting Mn2+ for Mg2+ as co-factor. This novel endonuclease allows speculation regarding specificity of monomeric LAGLIDADG proteins, while it supports the evolutionary genesis of these proteins by a gene duplication event.  相似文献   

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