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1.
Homing endonucleases are highly specific enzymes, capable of recognizing and cleaving unique DNA sequences in complex genomes. Since such DNA cleavage events can result in targeted allele-inactivation and/or allele-replacement in vivo, the ability to engineer homing endonucleases matched to specific DNA sequences of interest would enable powerful and precise genome manipulations. We have taken a step-wise genetic approach in analyzing individual homing endonuclease I-CreI protein/DNA contacts, and describe here novel interactions at four distinct target site positions. Crystal structures of two mutant endonucleases reveal the molecular interactions responsible for their altered DNA target specificities. We also combine novel contacts to create an endonuclease with the predicted target specificity. These studies provide important insights into engineering homing endonucleases with novel target specificities, as well as into the evolution of DNA recognition by this fascinating family of proteins.  相似文献   

2.
Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.  相似文献   

3.
Homing endonucleases have great potential as tools for targeted gene therapy and gene correction, but identifying variants of these enzymes capable of cleaving specific DNA targets of interest is necessary before the widespread use of such technologies is possible. We identified homologues of the LAGLIDADG homing endonuclease I-AniI and their putative target insertion sites by BLAST searches followed by examination of the sequences of the flanking genomic regions. Amino acid substitutions in these homologues that were located close to the target site DNA, and thus potentially conferring differences in target specificity, were grafted onto the I-AniI scaffold. Many of these grafts exhibited novel and unexpected specificities. These findings show that the information present in genomic data can be exploited for endonuclease specificity redesign.  相似文献   

4.
Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4 bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications.  相似文献   

5.
Homing endonucleases are microbial DNA-cleaving enzymes that mobilize their own reading frames by generating double strand breaks at specific genomic invasion sites. These proteins display an economy of size, and yet recognize long DNA sequences (typically 20 to 30 base pairs). They exhibit a wide range of fidelity at individual nucleotide positions in a manner that is strongly influenced by host constraints on the coding sequence of the targeted gene. The activity of these proteins leads to site-specific recombination events that can result in the insertion, deletion, mutation, or correction of DNA sequences. Over the past fifteen years, the crystal structures of representatives from several homing endonuclease families have been solved, and methods have been described to create variants of these enzymes that cleave novel DNA targets. Engineered homing endonucleases proteins are now being used to generate targeted genomic modifications for a variety of biotech and medical applications.  相似文献   

6.
Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hamper their applications. Here we report the development of a highly sensitive selection method for the directed evolution of homing endonucleases that couples enzymatic DNA cleavage with the survival of host cells. Using I-SceI as a model homing endonuclease, we have demonstrated that cells with wild-type I-SceI showed a high cell survival rate of 80–100% in the presence of the original I-SceI recognition site, whereas cells without I-SceI showed a survival rate <0.003%. This system should also be readily applicable for directed evolution of other DNA cleavage enzymes.  相似文献   

7.
Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.  相似文献   

8.
9.
10.
Homing endonuclease structure and function   总被引:14,自引:0,他引:14  
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.  相似文献   

11.
The presence of a homing endonuclease gene (HEG) within a microbial intron or intein empowers the entire element with the ability to invade genomic targets. The persistence of a homing endonuclease lineage depends in part on conservation of its DNA target site. One such rDNA sequence has been invaded both in archaea and in eukarya, by LAGLIDADG and His–Cys box homing endonucleases, respectively. The bases encoded by this target include a universally conserved ribosomal structure, termed helix 69 (H69) in the large ribosomal subunit. This region forms the ‘B2a’ intersubunit bridge to the small ribosomal subunit, contacts bound tRNA in the A- and P-sites, and acts as a trigger for ribosome disassembly through its interactions with ribosome recycling factor. We have determined the DNA-bound structure and specificity profile of an archaeal LAGLIDADG homing endonuclease (I-Vdi141I) that recognizes this target site, and compared its specificity with the analogous eukaryal His–Cys box endonuclease I-PpoI. These homodimeric endonuclease scaffolds have arrived at similar specificity profiles across their common biological target and analogous solutions to the problem of accommodating conserved asymmetries within the DNA sequence, but with differences at individual base pairs that are fine-tuned to the sequence conservation of archaeal versus eukaryal ribosomes.  相似文献   

12.
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   

13.
Posey KL  Gimble FS 《Biochemistry》2002,41(7):2184-2190
Target sites for homing endonucleases occur infrequently in complex genomes. As a consequence, these enzymes can be used in mammalian systems to introduce double-strand breaks at recognition sites inserted within defined loci to study DNA repair by homologous and nonhomologous recombination. Using homing endonucleases for gene targeting in vivo would be more feasible if temporal or spatial regulation of their enzymatic activity were possible. Here, we show that the DNA cleavage activity of the yeast PI-SceI homing endonuclease can be turned on and off using a redox switch. Two cysteine pairs (Cys-64/Cys-344 and Cys-67/Cys-365) were separately inserted into flexible DNA binding loop(s) to create disulfide bonds that lock the endonuclease into a nonproductive conformation. The cleavage activities of the reduced Cys-64/Cys-344 and Cys-67/Cys-365 variants are similar or slightly lower than that of the control protein, but the activities of the proteins in the oxidized state are decreased more than 30-fold. Modulating the activity of the proteins is easily accomplished by adding or removing the reducing agent. We show that defects in DNA binding account for the decreased DNA cleavage activities of the proteins containing disulfide bonds. Interestingly, the Cys-67/Cys-365 variant toggles between two different DNA binding conformations under reducing and oxidizing conditions, which may permit the identification of structural differences between the two states. These studies demonstrate that homing endonuclease activity can be controlled using a molecular switch.  相似文献   

14.
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   

15.
Mutations altering the cleavage specificity of a homing endonuclease   总被引:10,自引:9,他引:1       下载免费PDF全文
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.  相似文献   

16.
Novel family of putative homing endonuclease genes was recently discovered during analyses of metagenomic and genomic sequence data. One such protein is encoded within a group I intron that resides in the recA gene of the Bacillus thuringiensis 03058-36 bacteriophage. Named I-Bth0305I, the endonuclease cleaves a DNA target in the uninterrupted recA gene at a position immediately adjacent to the intron insertion site. The enzyme displays a multidomain, homodimeric architecture and footprints a DNA region of ~60 bp. Its highest specificity corresponds to a 14-bp pseudopalindromic sequence that is directly centered across the DNA cleavage site. Unlike many homing endonucleases, the specificity profile of the enzyme is evenly distributed across much of its target site, such that few single base pair substitutions cause a significant decrease in cleavage activity. A crystal structure of its C-terminal domain confirms a nuclease fold that is homologous to very short patch repair (Vsr) endonucleases. The domain architecture and DNA recognition profile displayed by I-Bth0305I, which is the prototype of a homing lineage that we term the 'EDxHD' family, are distinct from previously characterized homing endonucleases.  相似文献   

17.
Coevolution of a homing endonuclease and its host target sequence   总被引:2,自引:0,他引:2  
We have determined the specificity profile of the homing endonuclease I-AniI and compared it to the conservation of its host gene. Homing endonucleases are encoded within intervening sequences such as group I introns. They initiate the transfer of such elements by cleaving cognate alleles lacking the intron, leading to their transfer via homologous recombination. Each structural homing endonuclease family has arrived at an appropriate balance of specificity and fidelity that avoids toxicity while maximizing target recognition and invasiveness. I-AniI recognizes a strongly conserved target sequence in a host gene encoding apocytochrome B and has fine-tuned its specificity to correlate with wobble versus nonwobble positions across that sequence and to the amount of degeneracy inherent in individual codons. The physiological target site in the host gene is not the optimal substrate for recognition and cleavage: at least one target variant identified during a screen is bound more tightly and cleaved more rapidly. This is a result of the periodic cycle of intron homing, which at any time can present nonoptimal combinations of endonuclease specificity and insertion site sequences in a biological host.  相似文献   

18.
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   

19.
We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution.  相似文献   

20.
The PI-SceI protein from Saccharomyces cerevisiae is a member of the LAGLIDADG family of homing endonucleases that have been used in genomic engineering. To assess the flexibility of the PI-SceI-binding interaction and to make progress towards the directed evolution of homing endonucleases that cleave specified DNA targets, we applied a two-hybrid method to select PI-SceI variants from a randomized expression library that bind to different DNA substrates. In particular, the codon for Arg94, which is located in the protein splicing domain and makes essential contacts to two adjacent base-pairs, and the codons for four proximal residues were randomized. There is little conservation of the wild-type amino acid residues at the five randomized positions in the variants that were selected to bind to the wild-type site, yet one of the purified derivatives displays DNA-binding specificity and DNA endonuclease activity that is similar to that of the wild-type enzyme. A spectrum of DNA-binding behaviors ranging from partial relaxation of specificity to marked shifts in target site recognition are present in variants selected to bind to sites containing mutations at the two base-pairs. Our results illustrate the inherent plasticity of the PI-SceI/DNA interface and demonstrate that selection based on DNA binding is an effective means of altering the DNA cleavage specificity of homing endonucleases. Furthermore, it is apparent that homing endonuclease target specificity derives, in part, from constraints on the flexibility of DNA contacts imposed by hydrogen bonds to proximal residues.  相似文献   

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