Non-specific Nuclease Research Articles

Gene Correction by Homologous Recombination With Zinc Finger Nucleases in Primary Cells From a Mouse Model of a Generic Recessive Genetic Disease

Zinc Finger nucleases (ZFNs) have been used to create precise genome modifications at frequencies that might be therapeutically useful in gene therapy. We created a mouse model of a generic recessive genetic disease to establish a preclinical system to develop the use of ZFN-mediated gene correction for gene therapy. We knocked a mutated GFP gene into the ROSA26 locus in murine embryonic stem (ES) cells and used these cells to create a transgenic mouse. We used ZFNs to determine the frequency of gene correction by gene targeting in different primary cells from this model. We achieved targeting frequencies from 0.17 to 6% in different cell types, including primary fibroblasts and astrocytes. We demonstrate that ex vivo gene-corrected fibroblasts can be transplanted back into a mouse where they retained the corrected phenotype. In addition, we achieved targeting frequencies of over 1% in ES cells, and the targeted ES cells retained the ability to differentiate into cell types from all three germline lineages. In summary, potentially therapeutically relevant frequencies of ZFN-mediated gene targeting can be achieved in a variety of primary cells and these cells can then be transplanted back into a recipient.

The Enzyme and the cDNA Sequence of a Thermolabile and Double-Strand Specific DNase from Northern Shrimps (Pandalus borealis)

BackgroundWe have previously isolated a thermolabile nuclease specific for double-stranded DNA from industrial processing water of Northern shrimps (Pandalus borealis) and developed an application of the enzyme in removal of contaminating DNA in PCR-related technologies.Methodology/Principal FindingsA 43 kDa nuclease with a high specific activity of hydrolysing linear as well as circular forms of DNA was purified from hepatopancreas of Northern shrimp (Pandalus borealis). The enzyme displayed a substrate preference that was shifted from exclusively double-stranded DNA in the presence of magnesium to also encompass significant activity against single-stranded DNA when calcium was added. No activity against RNA was detected. Although originating from a cold-environment animal, the shrimp DNase has only minor low-temperature activity. Still, the enzyme was irreversibly inactivated by moderate heating with a half-life of 1 min at 65°C. The purified protein was partly sequenced and derived oligonucleotides were used to prime amplification of the encoding cDNA. This cDNA sequence revealed an open reading frame encoding a 404 amino acid protein containing a signal peptide. By sequence similarity the enzyme is predicted to belong to a family of DNA/RNA non-specific nucleases even though this shrimp DNase lacks RNase activity and is highly double-strand specific in some respects. These features are in agreement with those previously established for endonucleases classified as similar to the Kamchatka crab duplex-specific nuclease (Par_DSN). Sequence comparisons and phylogenetic analyses confirmed that the Northern shrimp nuclease resembles the Par_DSN-like nucleases and displays a more distant relationship to the Serratia family of nucleases.Conclusions/SignificanceThe shrimp nuclease contains enzyme activity that may be controlled by temperature or buffer compositions. The double-stranded DNA specificity, as well as the thermolabile feature, strengthens its potential for in vitro applications.

Fluorescent probes for the analysis of DNA strand scission in base excision repair.

We have developed fluorescent probes for the detection of strand scission in the excision repair of oxidatively damaged bases. They were hairpin-shaped oligonucleotides, each containing an isomer of thymine glycol or 5,6-dihydrothymine as a damaged base in the center, with a fluorophore and a quencher at the 5′- and 3′-ends, respectively. Fluorescence was detected when the phosphodiester linkage at the damage site was cleaved by the enzyme, because the short fragment bearing the fluorophore could not remain in a duplex form hybridized to the rest of the molecule at the incubation temperature. The substrate specificities of Escherichia coli endonuclease III and its human homolog, NTH1, determined by using these probes agreed with those determined previously by gel electrophoresis using 32P-labeled substrates. Kinetic parameters have also been determined by this method. Since different fluorophores were attached to the oligonucleotides containing each lesion, reactions with two types of substrates were analyzed separately in a single tube, by changing the excitation and detection wavelengths. These probes were degraded during an incubation with a cell extract. Therefore, phosphorothioate linkages were incorporated to protect the probes from nonspecific nucleases, and the base excision repair activity was successfully detected in HeLa cells.

Redesign of High-Affinity Nonspecific Nucleases with Altered Sequence Preference

It is of crucial importance to elucidate the underlying principles that govern the binding affinity and selectivity between proteins and DNA. Here we use the nuclease domain of Colicin E7 (nColE7) as a model system to generate redesigned nucleases with improved DNA-binding affinities. ColE7 is a bacterial toxin, bearing a nonspecific endonuclease domain with a preference for hydrolyzing DNA phosphodiester bonds at the 3'O-side after thymine and adenine; i.e., it prefers Thy and Ade at the -1 site. Using systematic computational screening, six nColE7 mutants were predicted to bind DNA with high affinity. Five of the redesigned single-point mutants were constructed and purified, and four mutants had a 3- to 5-fold higher DNA binding affinity than wild-type nColE7 as measured by fluorescence kinetic assays. Moreover, three of the designed mutants, D493N, D493Q, and D493R, digested DNA with an increased preference for guanine at +3 sites compared to the wild-type enzyme, as shown by DNA footprint assays. X-ray structure determination of the ColE7 mutant D493Q-DNA complex in conjunction with structural and free energy decomposition analyses provides a physical basis for the improved protein-DNA interactions: Replacing D493 at the protein-DNA interface with an amino acid residue that can maintain the native hydrogen bonds removes the unfavorable electrostatic repulsion between the negatively charged carboxylate and DNA phosphate groups. These results show that computational screening combined with biochemical, structural, and free energy analyses provide a useful means for generating redesigned nucleases with a higher DNA-binding affinity and altered sequence preferences in DNA cleavage.

Sub-cellular trafficking and functionality of 2′- O -methyl and 2′- O -methyl-phosphorothioate molecular beacons

Molecular beacons (MBs) have shown great potential for the imaging of RNAs within single living cells; however, the ability to perform accurate measurements of RNA expression can be hampered by false-positives resulting from nonspecific interactions and/or nuclease degradation. These false-positives could potentially be avoided by introducing chemically modified oligonucleotides into the MB design. In this study, fluorescence microscopy experiments were performed to elucidate the subcellular trafficking, false-positive signal generation, and functionality of 2′-O-methyl (2Me) and 2′-O-methyl-phosphorothioate (2MePS) MBs. The 2Me MBs exhibited rapid nuclear sequestration and a gradual increase in fluorescence over time, with nearly 50% of the MBs being opened nonspecifically within 24 h. In contrast, the 2MePS MBs elicited an instantaneous increase in false-positives, corresponding to ∼5–10% of the MBs being open, but little increase was observed over the next 24 h. Moreover, trafficking to the nucleus was slower. After 24 h, both MBs were localized in the nucleus and lysosomal compartments, but only the 2MePS MBs were still functional. When the MBs were retained in the cytoplasm, via conjugation to NeutrAvidin, a significant reduction in false-positives and improvement in functionality was observed. Overall, these results have significant implications for the design and applications of MBs for intracellular RNA measurement.

A nonradioactive plate-based assay for stimulators of nonspecific DNA nicking by HIV-1 integrase and other nucleases

Retroviral integrase enzymes have a nonspecific endonuclease activity that is stimulated by certain compounds, suggesting that integrase could be manipulated to damage viral DNA. To identify integrase stimulator (IS) compounds as potential antiviral agents, we have developed a nonradioactive assay that is suitable for high-throughput screening. The assay uses a 49-mer oligonucleotide that is 5′-labeled with a fluorophore, 3′-tagged with a quencher, and designed to form a hairpin that mimics radioactive double-stranded substrates in gel-based nicking assays. Reactions in 384-well plates are analyzed on a real-time PCR machine after a single heat denaturation and subsequent cooling to a point between the melting temperatures of unnicked substrate and nicked products (no cycling is required). Under these conditions, unnicked DNA reforms the hairpin and quenches fluorescence, whereas completely nicked DNA yields a large signal. The assay was linear with time, stimulator concentration, and amount of integrase, and 20% concentrations of the solvent used for many chemical libraries did not interfere with the assay. The assay had an excellent Z′ factor, and it reliably detected known IS compounds. This assay, which is adaptable to other nonspecific nucleases, will be useful for identifying additional IS compounds to develop the novel antiviral strategy of stimulating integrase to destroy retroviral DNA.

Polymerase chain reaction, nuclease digestion, and mass spectrometry based assay for the trinucleotide repeat status of the fragile X mental retardation 1 gene.

CGG repeat expansions in the 5' noncoding region of the fragile X mental retardation 1 gene (FMR1) give rise to both neurodevelopmental and neurodegenerative human diseases depending on the length of the expansion. Expansions beyond 200 repeats (full mutation) generally result in gene silencing and fragile X syndrome (FXS), the leading heritable form of cognitive impairment and autism. Smaller expansions (55-200 CGG repeats; "premutation") give rise to the neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS) through an entirely distinct, toxic mRNA gain-of-function mechanism. A rapid means for both high-risk and newborn screening for allele size would provide a greater opportunity for early intervention and family counseling as well as furnish critical data on repeat size distribution and expanded allele frequencies. In the current work, we propose a novel mass spectrometry (MS) based method for the rapid identification of expanded CGG repeats to complement a recently described polymerase chain reaction (PCR) method for large population screening. In this combined approach, the optimized PCR method is used to amplify the relevant region of FMR1, followed by extensive nonspecific nuclease digestion. The resulting oligonucleotides are analyzed by MS in a manner that provides the relative proportion of triplet repeat oligonucleotides in seconds per sample. This assay enables swift and reproducible detection of expanded CGG alleles using a single blood spot and in principle is suitable for large scale studies and newborn screening. Moreover, this analytical scheme establishes a unique new intersection of MS with molecular biology, with potential for significant interdisciplinary impact.

In vitro comet assay for DNA repair: a warning concerning application to cultured cells

The comet assay (single-cell gel electrophoresis) is a sensitive and simple method for measuring DNA damage. An early modification of the assay involved the application of specific repair endonucleases to convert lesions to breaks; thus, for example, endonuclease III was used to measure oxidized pyrimidines. This concept has now been extended to produce an in vitro assay for DNA repair activity in a cell-free extract, for example from lymphocytes. The extract is incubated with substrate DNA containing specific base damage, and repair incision is detected as breaks in this DNA. We have recently been studying effects of phytochemicals in cultured cells, whether as antioxidants or as potential modulators of DNA repair. We realized that there is a need to check that observed effects that appear as an enhancement of repair (i.e. increased breaks in substrate DNA) are not simply due to a direct damaging effect of the phytochemical or to induction of non-specific nucleases. Here, we describe a rigorous approach to testing for this possibility, which we recommend to anyone carrying out similar experiments.

Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN)

BackgroundCustomized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells.Methodology/Principal FindingsHere we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts.Conclusions and SignificanceIn summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight) and suggests that OPEN provides a reliable method for introducing targeted mutations in nearly any zebrafish gene of interest.

Enhanced resistance of DNA nanostructures to enzymatic digestion

The ability of nucleases to perform their catalytic functions depends on the sequence and structural features of target DNA substrates. Due to their size and shape, several DNA tetrahedra are resistant to the action of specific and non-specific nucleases. Such enhanced stability is a key requirement for DNA nanostructures to be useful as delivery vehicles.

Expanding or Restricting the Target Site Repertoire of Zinc-finger Nucleases: The Inter-domain Linker as a Major Determinant of Target Site Selectivity

Precise manipulations of complex genomes by zinc-finger nucleases (ZFNs) depend on site-specific DNA cleavage, which requires two ZFN subunits to bind to two target half-sites separated by a spacer of 6 base pairs (bp). ZFN subunits consist of a specific DNA-binding domain and a nonspecific cleavage domain, connected by a short inter-domain linker. In this study, we conducted a systematic analysis of 11 candidate-based linkers using episomal and chromosomal targets in two human cell lines. We achieved gene targeting in up to 20% of transfected cells and identified linker variants that enforce DNA cleavage at narrowly defined spacer lengths and linkers that expand the repertoire of potential target sites. For instance, a nine amino acid (aa) linker induced efficient gene conversion at chromosomal sites with 7- or 16-bp spacers, whereas 4-aa linkers had activity optima at 5- and 6-bp spacers. Notably, single aa substitutions in the 4-aa linker affected the ZFN activity significantly, and both gene conversion and ZFN-associated toxicity depended on the linker/spacer combination and the cell type. In summary, both sequence and length of the inter-domain linker determine ZFN activity and target-site specificity, and are therefore important parameters to account for when designing ZFNs for genome editing.

Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab

BackgroundNucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking.ResultsUsing degenerative primers and the rapid amplification of cDNA ends (RACE) procedure, we cloned the Duplex-Specific Nuclease (DSN) gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 – 7.5) and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact.ConclusionWe describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate specificity of DSN should prove valuable in certain molecular biology applications.

481. Sleeping Beauty Transposase Exhibits Sequence Non-Specific Nuclease Activity in the Absence of Host Cell-Harbored Co-Factors

481. Sleeping Beauty Transposase Exhibits Sequence Non-Specific Nuclease Activity in the Absence of Host Cell-Harbored Co-Factors

Is crab duplex-specific nuclease a member of the Serratia family of non-specific nucleases?

Kamchatka crab duplex-specific nuclease (Par_DSN) has been classified as a member of the family of DNA/RNA non-specific beta-beta-alpha metal finger (bba-Me-finger) nucleases, the archetype of which is the nuclease from Serratia marcescens. Although the enzyme under investigation seems to belong to the family of S. marcescens nucleases, Par_DSN exhibits a marked preference for double-stranded DNA as a substrate and this property is unusual for other members of this family. We have searched other Arthropod species and identified a number of novel Par_DSN homologs. A phylogenetic analysis demonstrates that the Par_DSN-like enzymes constitute a separate branch in the evolutionary tree of bba-Me-finger nucleases. Combining sequence analysis and site-directed mutagenesis, we found that Par_DSN and its homologs possess the nuclease domain that is slightly longer than that of classic Serratia relatives. The active site composition of Par_DSN is similar but not identical to that of classic Serratia nucleases. Based on these findings, we proposed a new classification of Par_DSN-like nucleases.

Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1

BackgroundThermostable enzymes from thermophiles have attracted extensive studies. In this investigation, a nuclease-encoding gene (designated as GBSV1-NSN) was obtained from a thermophilic bacteriophage GBSV1 for the first time.ResultsAfter recombinant expression in Escherichia coli, the purified GBSV1-NSN exhibited non-specific nuclease activity, being able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Based on sequence analysis, the nuclease shared no homology with any known nucleases, suggesting that it was a novel nuclease. The characterization of the recombinant GBSV1-NSN showed that its optimal temperature and pH were 60°C and 7.5, respectively. The results indicated that the enzymatic activity was inhibited by enzyme inhibitors or detergents, such as ethylene diamine tetraacetic acid, citrate, dithiothreitol, β-mercaptoethanol, guanidine hydrochloride, urea and SDS. In contrast, the nuclease activity was enhanced by TritonX-100, Tween-20 or chaps to approximately 124.5% – 141.6%. The Km of GBSV1-NSN nuclease was 231, 61 and 92 μM, while its kcat was 1278, 241 and 300 s-1 for the cleavage of dsDNA, ssDNA and RNA, respectively.ConclusionOur study, therefore, presented a novel thermostable non-specific nuclease from thermophilic bacteriophage and its overexpression and purification for scientific research and applications.

Ectopic Expression of Mitochondria Endonuclease Pnu1p from Schizosaccharomyces pombe Induces Cell Death of the Yeast

Endonuclease G (EndoG) is a mitochondrial non-specific nuclease that is highly conserved among the eukaryotes. Although the precise role of EndoG in mitochondria is not yet known, the enzyme is released from the mitochondria and digests nuclear DNA during apoptosis in mammalian cells. Schizosaccharomyces pombe has an EndoG homolog Pnu1p (previously named SpNuc1) that is produced as a precursor protein with a mitochondrial targeting sequence. During the sorting into mitochondria the signal sequence is cleaved to yield the functionally active endonuclease. From the analogy to EndoG, active extramitochondrial Pnu1p may trigger cell killing by degrading nuclear DNA. Here, we tested this possibility by expressing a truncated Pnu1p lacking the signal sequence in the extramitochondrial region of pnu1-deleted cells. The truncated Pnu1p was localized in the cytosol and nuclei of yeast cells. And ectopic expression of active Pnu1p led to cell death with fragmentation of nuclear DNA. This suggests that the Pnu1p is possibly involved in a certain type of yeast cell death via DNA fragmentation. Although expression of human Bak in S. pombe was lethal, Pnu1p nuclease is not necessary for hBak-induced cell death.

Rational design of a chimeric endonuclease targeted to NotI recognition site

A cleavage-deficient variant of NotI restriction endonuclease (GCGGCCGC) was isolated by random mutagenesis of the notIR gene. The NotI variant D160N was shown to bind DNA and protect plasmid DNA from EagI (CGGCCG) and NotI digestions. The EDTA-resistant BmrI restriction endonuclease cleaves DNA sequence ACTGGG N5/N4. The N-terminal cleavage domain of BmrI (residues 1-198) with non-specific nuclease activity was fused to the NotI variant D160N with a short linker. The engineered chimeric endonuclease (CH-endonuclease) recognizes NotI sites specifically in the presence of high salt (100-150 mM NaCl) and divalent cations Mg++ or Ca++. In contrast to wild-type NotI, which cuts within its recognition sequence, BmrI198-NotI (D160N) cleaves DNA outside of NotI sites, resulting in deletion of the NotI site and the adjacent sequences. The fusion of the BmrI cleavage domain to cleavage-deficient variants of Type II restriction enzymes to generate novel cleavage sites will provide useful tools for DNA manipulation.

Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells

There have been a growing number of studies where molecular beacons (MBs) are used to image RNA expression in living cells; however, the ability to make accurate measurements can be hampered by the generation of false-positive signals resulting from non-specific interactions and/or nuclease degradation. In the present study, we found that such non-specific signals only arise in the nucleus of living cells. When MBs are retained in the cytoplasmic compartment, by linking them to quantum dots (QDs), false-positive signals are reduced to marginal levels. Consequently, MB–QD conjugates were used to measure the expression of the endogenous proto-oncogene c-myc in MCF-7 breast cancer cells by quantifying the total fluorescent signal emanating from individual cells. Upon the addition of tamoxifen, measurements of MB fluorescence indicated a 71% reduction in c-myc expression, which correlated well with RT-PCR measurements. Variations in MB fluorescence resulting from instrumental fluctuations were accounted for by imaging fluorescent calibration standards on a daily basis. Further, it was established that measurements of the total fluorescent signal were not sensitive to the focal plane. Overall, these results provide evidence that accurate measurements of RNA levels can be made when MBs are retained in the cytoplasm.

Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1

Available tools for genetic analysis in the anaerobic rumen bacterium Prevotella bryantii are limited to only two known systems for gene delivery, and no genes, with the exception of plasmid maintenance and selection genes, have been successfully expressed from plasmids in any species of the genus Prevotella until now. It is shown here that nucB, a newly cloned nuclease gene from P. bryantii, can be controllably expressed from shuttle vector pRH3 in P. bryantii strain TC1-1, depending on the tetracycline concentration in the growth medium. nucB expression is also growth-medium dependent and this regulation presumably takes place at the translational level. His-tagged NucB was purified from P. bryantii TC1-1 culture supernatant and was shown to degrade DNA as well as RNA; it is most likely a minor 36 kDa P. bryantii non-specific nuclease.

Restriction endonuclease BpuJI specific for the 5′-CCCGT sequence is related to the archaeal Holliday junction resolvase family

Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5′-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3 nt from the 3′-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.