Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gene targeting by homologous recombination is a powerful method to manipulate the genome precisely and could be exploited to correct genetic defects. Zinc finger nucleases are designed proteins that fuse a zinc finger DNA binding domain to the nuclease domain from the FokI restriction endonuclease. Zinc finger nucleases were generated that stimulated gene targeting from half-site sequences from the human beta-globin gene and the human common gamma-chain gene. Zinc finger nucleases were also generated that stimulated gene targeting at full sites from the green fluorescent protein gene and the human CD8alpha gene. This work built on the prior zinc finger design work of others and in targeting these four genes had a 100% success rate at designing nucleases to the consensus half-site 5'-GNNGNNGNN-3' and the consensus full site 5'-NNCNNCNNCNNNNNNGNNGNNGNN-3', suggesting that zinc finger nucleases can be empirically designed to stimulate gene targeting in a large portion of the mammalian genome.
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PMID:Mammalian gene targeting with designed zinc finger nucleases. 1616 74

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting--the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys2His2) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for 'directed mutagenesis' and targeted 'gene editing' of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.
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PMID:Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. 1625 1

GIY-YIG homing endonucleases are modular proteins, with conserved N-terminal catalytic domains connected by linkers to C-terminal DNA-binding domains. I-TevI, the T4 phage GIY-YIG intron endonuclease, functions both in promoting td intron homing, and in acting as a transcriptional autorepressor. Repression is achieved by binding to an operator, which is cleaved at 100-fold reduced efficiency relative to the intronless homing site. The linker includes a zinc finger, which functions in distance determination, to constrain the catalytic domain to cleave the homing site at a fixed position. Here we show that I-BmoI, a related GIY-YIG endonuclease lacking a zinc finger, also possesses some cleavage distance discrimination. Furthermore, hybrid endonucleases constructed by swapping the domains of I-BmoI and I-TevI are active, precise and demonstrate that features other than the zinc finger facilitate distance determination. Most importantly, I-TevI zinc finger mutants cleave the operator more efficiently than the homing site, the converse of wild-type protein. These results are consistent with the zinc finger acting as a measuring device, directing efficient cleavage of the homing site to promote intron mobility, while reducing cleavage at the operator to ensure transcriptional autorepression and phage viability.
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PMID:Distance determination by GIY-YIG intron endonucleases: discrimination between repression and cleavage functions. 1658 1

The oligonucleotide/oligosaccharide-binding (OB) fold is central to the architecture of single-stranded- DNA-binding proteins, which are polypeptides essential for diverse cellular processes, including DNA replication, repair, and recombination. In archaea, single-stranded DNA-binding proteins composed of multiple OB folds and a zinc finger domain, in a single polypeptide, have been described. The OB folds of these proteins were more similar to their eukaryotic counterparts than to their bacterial ones. Thus, the archaeal protein is called replication protein A (RPA), as in eukaryotes. Unlike most organisms, Methanosarcina acetivorans harbors multiple functional RPA proteins, and it was our interest to determine whether the different proteins play different roles in DNA transactions. Of particular interest was lagging-strand DNA synthesis, where recently RPA has been shown to regulate the size of the 5' region cleaved during Okazaki fragment processing. We report here that M. acetivorans RPA1 (MacRPA1), a protein composed of four OB folds in a single polypeptide, inhibits cleavage of a long flap (20 nucleotides) by M. acetivorans flap endonuclease 1 (MacFEN1). To gain a further insight into the requirement of the different regions of MacRPA1 on its inhibition of MacFEN1 endonuclease activity, N-terminal and C-terminal truncated derivatives of the protein were made and were biochemically and biophysically analyzed. Our results suggested that MacRPA1 derivatives with at least three OB folds maintained the properties required for inhibition of MacFEN1 endonuclease activity. Despite these interesting observations, further biochemical and genetic analyses are required to gain a deeper understanding of the physiological implications of our findings.
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PMID:Methanosarcina acetivorans flap endonuclease 1 activity is inhibited by a cognate single-stranded-DNA-binding protein. 1692 82

The polyadenylation of messenger RNAs is mediated by a multi-subunit complex that is conserved in eukaryotes. Among the most interesting of these proteins is the 30-kDa-subunit of the Cleavage and Polyadenylation Specificity Factor, or CPSF30. In this study, the Arabidopsis CPSF30 ortholog, AtCPSF30, is characterized. This protein possesses an unexpected endonucleolytic activity that is apparent as an ability to nick and degrade linear as well as circular single-stranded RNA. Endonucleolytic action by AtCPSF30 leaves RNA 3' ends with hydroxyl groups, as they can be labeled by RNA ligase with [32P]-cytidine-3',5'-bisphosphate. Mutations in the first of the three CCCH zinc finger motifs of the protein abolish RNA binding by AtCPSF30 but have no discernible effects on nuclease activity. In contrast, mutations in the third zinc finger motif eliminate the nuclease activity of the protein, and have a modest effect on RNA binding. The N-terminal domain of another Arabidopsis polyadenylation factor subunit, AtFip1(V), dramatically inhibits the nuclease activity of AtCPSF30 but has a slight negative effect on the RNA-binding activity of the protein. These results indicate that AtCPSF30 is a probable processing endonuclease, and that its action is coordinated through its interaction with Fip1.
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PMID:A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor. 1757 67

Custom-designed zinc finger nucleases (ZFNs)--proteins designed to cut at specific DNA sequences--combine the non-specific cleavage domain (N) of Fok I restriction endonuclease with zinc finger proteins (ZFPs). Because the recognition specificities of the ZFPs can be easily manipulated experimentally, ZFNs offer a general way to deliver a targeted site-specific double-strand break (DSB) to the genome. They have become powerful tools for enhancing gene targeting--the process of replacing a gene within a genome of cells via homologous recombination (HR)--by several orders of magnitude. ZFN-mediated gene targeting thus confers molecular biologists with the ability to site-specifically and permanently alter not only plant and mammalian genomes but also many other organisms by stimulating HR via a targeted genomic DSB. Site-specific engineering of the plant and mammalian genome in cells so far has been hindered by the low frequency of HR. In ZFN-mediated gene targeting, this is circumvented by using designer ZFNs to cut at the desired chromosomal locus inside the cells. The DNA break is then patched up using the new investigator-provided genetic information and the cells' own repair machinery. The accuracy and high efficiency of the HR process combined with the ability to design ZFNs that target most DNA sequences (if not all) makes ZFN technology not only a powerful research tool for site-specific manipulation of the plant and mammalian genomes, but also potentially for human therapeutics in the future, in particular for targeted engineering of the human genome of clinically transplantable stem cells.
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PMID:Custom-designed zinc finger nucleases: what is next? 1776 26

We describe two uncommon roles for Zn2+ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg2+. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg2+, Mn2+, or Ca2+. Among these metal ions, Mg2+ and Mn2+ induce promiscuous cleavage by the enzyme, whereas Ca2+-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase.
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PMID:Dual role for Zn2+ in maintaining structural integrity and inducing DNA sequence specificity in a promiscuous endonuclease. 1778 55

The Arabidopsis CPSF30 ortholog (AtCPSF30) is an RNA-binding endonuclease that is part of the plant polyadenylation complex. Previous work (B. Addepalli, A.G. Hunt, Nucleic Acids Res. 35 (2007) 4453-4463) demonstrated that different zinc finger motifs in the protein were responsible for RNA-binding and nuclease activity, respectively. In this study, a more detailed functional map of AtCPSF30 is presented, a map that includes descriptions of novel biochemical activities. Elevated temperatures, the specific zinc chelator 1,10-phenanthroline, and the sulfhydryl reagent dithiothreitol all had differential inhibitory effects on the RNA-binding and nuclease activities. The endonuclease activity of AtCPSF30 was inhibited by relatively high (>100muM) concentrations of zinc, and this inhibition required a plant-specific N-terminal domain apart from the zinc finger core of the protein. ATP stimulated the nuclease activity in the presence of zinc, and this stimulation required a plant-specific C-terminal domain, again apart from the zinc finger core. These studies reveal a subtle and unexpected complexity to AtCPSF30, and raise the possibility that multiple avenues of regulation may impinge on this protein through different functional domains.
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PMID:Redox and heavy metal effects on the biochemical activities of an Arabidopsis polyadenylation factor subunit. 1833 19

I-TevI is a modular intron-encoded endonuclease, consisting of an N-terminal catalytic domain and a C-terminal DNA-binding domain, joined by a 75 amino acid linker. This linker can be divided into three regions, starting at the N terminus: the deletion-intolerant (DI) region; the deletion-tolerant (DT) region; and a zinc finger, which acts as a distance determinant for cleavage. To further explore linker function, we generated deletion and substitution mutants that were tested for their preference to cleave at a particular distance or at the correct sequence. Our results demonstrate that the I-TevI linker is multi-functional, a property that sets it apart from junction sequences in most other proteins. First, the linker DI region has a role in I-TevI cleavage activity. Second, the DT linker region participates in distance determination, as evident from DT mutants that display a phenotype similar to that of the zinc-finger mutants in their selection of a cleavage site. Finally, NMR analysis of a freestanding 56 residue linker segment showed an unstructured stretch corresponding to the DI region and a portion of the DT region, followed by a beta-strand corresponding to the remainder of the DT region and containing a key distance-determining arginine, R129. Mutation of this arginine to alanine abolished distance determination and disrupted the beta-strand, indicating that the structure of the DT linker region has a role in cleavage at a fixed distance.
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PMID:Role of the interdomain linker in distance determination for remote cleavage by homing endonuclease I-TevI. 1849 24

The induction of double-strand breaks (DSBs) in plant genomes can lead to increased homologous recombination or site-specific mutagenesis at the repair site. This phenomenon has the potential for use in gene targeting applications in plant cells upon the induction of site-specific genomic DSBs using zinc finger nucleases (ZFNs). Zinc finger nucleases are artificial restriction enzymes, custom-designed to cleave a specific DNA sequence. The tools and methods for ZFN assembly and validation could potentially boost their application for plant gene targeting. Here we report on the design of biochemical and in planta methods for the analysis of newly designed ZFNs. Cloning begins with de novo assembly of the DNA-binding regions of new ZFNs from overlapping oligonucleotides containing modified helices responsible for DNA-triplet recognition, and the fusion of the DNA-binding domain with a FokI endonuclease domain in a dedicated plant expression cassette. Following the transfer of fully assembled ZFNs into Escherichia coli expression vectors, bacterial lysates were found to be most suitable for in vitro digestion analysis of palindromic target sequences. A set of three in planta activity assays was also developed to confirm the nucleic acid digestion activity of ZFNs in plant cells. The assays are based on the reconstruction of GUS expression following transient or stable delivery of a mutated uidA and ZFN-expressing cassettes into target plants cells. Our tools and assays offer cloning flexibility and simple assembly of tested ZFNs and their corresponding target sites into Agrobacterium tumefaciens binary plasmids, allowing efficient implementation of ZFN-validation assays in planta.
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PMID:A toolbox and procedural notes for characterizing novel zinc finger nucleases for genome editing in plant cells. 1898 Jun 51


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