Non-specific Nuclease Research Articles

Detection of Uracil-Excising DNA Glycosylases in Cancer Cell Samples Using a Three-Dimensional DNAzyme Walker.

DNA glycosylase dysregulation is implicated in carcinogenesis and therapeutic resistance of cancers. Thus, various DNA-based detection platforms have been developed by leveraging the base excision activity of DNA glycosylases. However, the efficacy of DNA-based methods is hampered due to nonspecific degradation by nucleases commonly present in cancer cells and during preparations of cell lysates. In this report, we describe a fluorescence-based assay using a specific and nuclease-resistant three-dimensional DNAzyme walker to investigate the activity of DNA glycosylases from cancer cell lysates. We focus on DNA glycosylases that excise uracil from deoxyuridine (dU) lesions, namely, uracil DNA glycosylase (UDG) and single-stranded monofunctional uracil DNA glycosylase (SMUG1). The limits of detection for detecting UDG and SMUG1 in the buffer were 3.2 and 3.0 pM, respectively. The DNAzyme walker detected uracil excision activity in diluted cancer cell lysate from as few as 48 A549 cells. The results of the UDG inhibitor experiments demonstrate that UDG is the predominant uracil-excising glycosylase in A549 cells. Approximately 500 nM of UDG is present in each A549 cell on average. No fluorescence was generated in the samples lacking DNAzyme activation, indicating that there was no nonspecific nuclease interference. The ability of the DNAzyme walker to respond to glycosylase activity illustrates the potential use of DNAzyme walker technology to monitor and study biochemical processes involving glycosylases.

Activation of Csm6 ribonuclease by cyclic nucleotide binding: in an emergency, twist to open.

Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6' ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6' in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the 'jaws' of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.

Duck plague virus pUL15 performs a nonspecial cleavage activity through its C terminal nuclease domain in vitro

Duck plague virus (DPV), also known as anatid herpesvirus, is a double-stranded DNA virus and a member of α herpesvirus. DPV pUL15 is a homolog of herpes simplex virus 1 (HSV-1) pUL15, a terminase large subunit, and plays a key role in the cleavage and packaging of the viral concatemeric genome. However, the sequence similarity between DPV pUL15 and its homologs is low, and it is not sure if DPV pUL15 has the potential to cleave the concatemeric genome as same as its homologs. Here, we expressed the C terminal domain of DPV pUL15 to explore the nuclease function of DPV pUL15. The main results showed that (Ⅰ) DPV pUL15 C-terminal domain possesses nonspecific nuclease activity and lacks the DNA binding ability. (Ⅱ) DPV pUL15 nuclease activity needs to coordinate with divalent metal ions and tends to be more active at high temperatures. (Ⅲ) Even though the structure of DPV pUL15 nuclease domain is relatively conserved, the mutations of conserved amino acids on the nuclease domain do not significantly inhibit the nuclease activity.

Biochemical characterization of the two novel mgCas12a proteins from the human gut metagenome

CRISPR/Cas9 and Cas12a belonging to the Class II CRISPR system are characterized by a single-component effector protein. Despite unique features of Cas12a like DNA cleavage with 5′ staggered ends and a single crRNA, Cas12a has not been adopted in biotechnological applications to the similar extent as Cas9. To better understand the CRISPR/Cas12 systems, we selected two candidates, designated mgCas12a-1 and mgCas12a-2, from an analysis of the human microbiome metagenome (mg) and provided biochemical characterization. These new Cas12a proteins shared about 37% identity in amino acid sequences and shared the same direct repeat sequences in the crRNA with FnCas12a from Francisella novicida. The purification yield of the recombinant proteins was up to 3.6-fold greater than that of FnCas12a. In cell-free DNA cleavage assays, both mgCas12a proteins showed the higher cleavage efficiencies when Mn2+ was provided with KCl (< 100 mM) than tested other divalent ions. They were able to tolerate ranges of pH points and temperature, and showed the highest cleavage efficiencies at pH 8.0 and 50 °C. In addition, mgCas12a proteins showed 51% less crRNA-independent and 56% less crRNA-dependent non-specific nuclease activity upon prolonged incubation than did FnCas12a. Considering their greater yield in protein preparation and reduced non-specific nuclease activity, our findings may expedite the use of Cas12a especially when genome editing needs to be practiced with the form of ribonucleoproteins.

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

A YoeB toxin cleaves both RNA and DNA

Type II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.

Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA.

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.

Evaluation of RNA Interference for Control of the Grape Mealybug Pseudococcus maritimus (Hemiptera: Pseudococcidae).

Simple SummaryRNA interference (RNAi) is a defense mechanism that protects insects from viruses by targeting and degrading RNA. This feature has been exploited to reduce the expression of endogenous RNA for determining functions of various genes and for killing insect pests by targeting genes that are vital for insect survival. When dsRNA matching perfectly to the target RNA is administered, the RNAi machinery dices the dsRNA into ~21 bp fragments (known as siRNAs) and one strand of siRNA is employed by the RNAi machinery to target and degrade the target RNA. In this study we used a cocktail of dsRNAs targeting grape mealybug’s aquaporin and sucrase genes to kill the insect. Aquaporins and sucrases are important genes enabling these insects to maintain water relations indispensable for survival and digest complex sugars in the diet of plant sap-feeding insects, including mealybugs. In our experiments, administration of dsRNA caused a reduction in expression of the target genes and an increase in insect mortality. These results provide support for the application of RNAi to control the grape mealybug.The grape mealybug Pseudococcus maritimus (Ehrhorn, 1900) (Hemiptera: Pseudococcidae) is a significant pest of grapevines (Vitis spp.) and a vector of disease-causing grape viruses, linked to its feeding on phloem sap. The management of this pest is constrained by the lack of naturally occurring resistance traits in Vitis. Here, we obtained proof of concept that RNA interference (RNAi) using double-stranded RNA (dsRNA) molecules against essential genes for phloem sap feeding can depress insect survival. The genes of interest code for an aquaporin (AQP) and a sucrase (SUC) that are required for osmoregulation in related phloem sap-feeding hemipteran insects (aphids and whiteflies). In parallel, we investigated the grape mealybug genes coding non-specific nucleases (NUC), which reduce RNAi efficacy by degrading administered dsRNA. Homologs of AQP and SUC with experimentally validated function in aphids, together with NUC, were identified in the published transcriptome of the citrus mealybug Planococcus citri by phylogenetic analysis, and sequences of the candidate genes were obtained for Ps. maritimus by PCR with degenerate primers. Using this first sequence information for Ps. maritimus, dsRNA was prepared and administered to the insects via an artificial diet. The treatment comprising dsRNA against AQP, SUC and NUC significantly increased insect mortality over three days, relative to dsRNA-free controls. The dsRNA constructs for AQP and NUC were predicted, from sequence analysis to have some activity against other mealybugs, but none of the three dsRNA constructs have predicted activity against aphids. This study provides the basis to develop in planta RNAi strategies against Ps. maritimus and other mealybug pests of grapevines.

Argonaute system of Kordia jejudonensis is a heterodimeric nucleic acid-guided nuclease

We purified and characterized a prokaryotic argonaute (pAgo) (KjMP) and its associated protein (KjAA) from a bacterium Kordia jejudonensis. The two proteins present as a complex were revealed by the copurification of KjAA with His-tagged KjMP by Ni-NTA affinity column. The KjAA/KjMP complex was a heterodimer evaluated from the molecular weight estimated using size exclusion chromatography. The pAgo complex presented a guide-dependent target DNA cleavage. RNA was the preferred guide; however, DNA also functioned, albeit weakly. Additionally, 5′-phosphorylate or non-phosphorylated guide was equally effective. The purified complex exhibited nonspecific nuclease activity on dsDNA and ssDNA. This is the first study to report that short pAgo and its associated protein form a complex, which has a nucleic acid-guided target recognition and cleavage.

Bacterial non-specific nucleases of the phospholipase D superfamily and their biotechnological potential.

Bacterial non-specific nucleases are ubiquitously distributed and involved in numerous intra- and extracellular processes. Although all nucleases share the basic chemistry for the hydrolysis of phosphodiester bonds in nucleic acid molecules, the catalysis comprises diverse modes of action, which offers great potential for versatile biotechnological applications. A major criterium for their differentiation is substrate specificity. Specific endonucleases are widely used as restriction enzymes in molecular biology approaches, whereas the main applications of non-specific nucleases (NSNs) are the removal of nucleic acids from crude extracts in industrial downstream processing and the prevention of cell clumping in microfabricated channels. In nature, the predominant role of NSNs is the acquisition of nutrient sources such as nucleotides and phosphates. The number of extensively characterized NSNs and available structures is limited. Moreover, their applicability is mostly challenged by the presence of metal chelators that impede the hydrolysis of nucleic acids in a metal ion-dependent manner. However, a few metal ion-independent NSNs that tolerate the presence of metal chelators have been characterized in recent years with none being commercially available to date. The classification and biotechnological potential of bacterial NSNs with a special focus on metal ion-independent nucleases are presented and discussed.Key Points • Bacterial phospholipases (PLD-family) exhibit nucleolytic activity. • Bacterial nucleases of the PLD-family are metal ion-independent. • NSNs can be used in downstream processing approaches.

Gp4 is a nuclease required for morphogenesis of T4-like bacteriophages

An essential step in the morphogenesis of tailed bacteriophages is the joining of heads and tails to form infectious virions. Our understanding of the maturation of complete virus particles remains incomplete. Through an unknown mechanism, phage T4 gene product 4 (gp4) plays an essential role in the head-tail joining step of T4-like phages. Alignment of T4 gp4 homologs identified a type II restriction endonuclease motif. Purified gp4 from both T4 and a marine T4-like bacteriophage, YC, have non-specific nuclease activity in vitro. Mutation of a single conserved amino acid residue in the endonuclease fold of T4 and YC gp4 abrogates nuclease activity. When expressed in trans, the wild type T4 gp4, but neither the mutated T4 protein nor the YC homolog, rescues a T4 gene 4 amber mutant phage. Thus the nuclease activity appears essential for morphogenesis, potentially by cleaving packaged DNA to enable the joining of heads to tails.

The Nuclease from Gram-Negative Bacteria Serratia Marcescens is Weakly Cytotoxic at Therapeutic Doses

The nuclease from S.marcescens heads a family of homological nonspecific nucleases. Its cultivation features, structure, mechanism of action, some physical, chemical and biochemical properties are well studied. It is presented by two isoforms, differing by N-terminal fragment and some properties. The nuclease demonstrated a cancer suppressing effect in mice. It is a key component of the anti-rabies composition which increases survival of the infected mice. It is similar by its efficiency to Pulmozyme® that is used for airway cleansing during the treatment of pulmonary diseases. The aim of the study was to analyze a cytotoxic effect of S. marcescens nuclease and its separate isoforms at different concentrations. To determine the cytotoxicity we used MTT assay and human cell cultures of colorectal cancer -HCT 116, breast cancer MCF-7 and normal skin fibroblasts. Results show that the nuclease demonstrates a weak cytotoxic effect at the amounts of 0.2 – 25 µg per 1 ml of the cell culture medium that is close to its therapeutic doses. This effect is not connected with the loss of the enzymatic activity. The isoforms are similar by their IC50.

Optimizing a production strategy for a nonspecific nuclease from Yersinia enterocolitica subsp. palearctica in genetically engineered Escherichia coli.

A nuclease from Yersinia enterocolitica subsp. palearctica (Nucyep) is a newly found thermostable nonspecific nuclease. The heat-resisting ability of this nuclease would be extremely useful in biological research or pharmaceutical production. However, the application of this nuclease is limited because of its poor yield. This research aimed to improve Nucyep productivity by producing a novel genetically engineered Escherichia coli and optimizing the production procedures. After 4 h of induction by lactose, the new genetically engineered E. coli can express a substantial amount of Nucyep in the form of inclusion bodies. The yield was approximately 0.3 g of inclusion bodies in 1 g of bacterial pellets. The inclusion bodies were extracted by sonication and solubilized in an 8 M urea buffer. Protein renaturation was successfully achieved by dilution method. Pure enzyme was obtained after subjecting the protein solution to anion exchange. The Nucyep showed its nonspecific and heat resistant properties as previously reported (Boissinot etal. 2016). Through a quantification method, its activity was determined to be 1.3×10 6 Kunitz units (K.U.)/mg. These results can serve as a reference for increasing Nucyep production.

Characterization of Single Amino Acid Variations in an EDTA-Tolerating Non-specific Nuclease from the Ice-Nucleating Bacterium Pseudomonas syringae.

Non-specific nuclease (NSN) can be applied in industrial downstream processing to remove nucleic acids from crude protein extracts or in cell-sorting systems to degrade nucleic acids derived from lysed cells. PsNuc from the ice-nucleating bacterium Pseudomonas syringae has the ability to decompose double- and single-stranded DNA in linear or circular form and RNA. It is not affected by the presence of metal-ion chelators such as EDTA and tolerates several protease inhibitors and reducing agents. A multiple sequence alignment of PsNuc with closely related enzymes (97-99% identity on the protein level) within the family Pseudomonaceae revealed the presence of only six amino acid residues that are variable in putative NSN from different members of the genus Pseudomonas. Single amino acid variants were produced in recombinant form in Escherichia coli, purified, and characterized. They showed similar activity compared to PsNuc, but a single variant even displayed an improved performance with an activity of > 20,000U/mg at 35°C, while amino acid residues S148 and V161 were found to be essential for enzymatic functionality. These results suggest that homologous nucleases from Pseudomonaceae display high activity levels in a metal-ion-independent manner and are therefore of interest for applications in biotechnology.

Harnessing type I CRISPR-Cas systems for genome engineering in human cells.

Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5-7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8-11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.

Decoding Essential Amino Acid Residues in the Substrate Groove of a Non-Specific Nuclease from Pseudomonas syringae

Non-specific nucleases (NSN) are of interest for biotechnological applications, including industrial downstream processing of crude protein extracts or cell-sorting approaches in microfabricated channels. Bacterial nucleases belonging to the superfamily of phospholipase D (PLD) are featured for their ability to catalyze the hydrolysis of nucleic acids in a metal-ion-independent manner. In order to gain a deeper insight into the composition of the substrate groove of a NSN from Pseudomonas syringae, semi-rational mutagenesis based on a structure homology model was applied to identify amino acid residues on the protein’s surface adjacent to the catalytic region. A collection of 12 mutant enzymes each with a substitution to a positively charged amino acid (arginine or lysine) was produced in recombinant form and biochemically characterized. Mutations in close proximity to the catalytic region (inner ring) either dramatically impaired or completely abolished the enzymatic performance, while amino acid residues located at the border of the substrate groove (outer ring) only had limited or no effects. A K119R substitution mutant displayed a relative turnover rate of 112% compared to the original nuclease. In conclusion, the well-defined outer ring of the substrate groove is a potential target for modulation of the enzymatic performance of NSNs belonging to the PLD superfamily.

Gene regulatory network stabilized by pervasive weak repressions: microRNA functions revealed by the May-Wigner theory.

Food web and gene regulatory networks (GRNs) are large biological networks, both of which can be analyzed using the May–Wigner theory. According to the theory, networks as large as mammalian GRNs would require dedicated gene products for stabilization. We propose that microRNAs (miRNAs) are those products. More than 30% of genes are repressed by miRNAs, but most repressions are too weak to have a phenotypic consequence. The theory shows that (i) weak repressions cumulatively enhance the stability of GRNs, and (ii) broad and weak repressions confer greater stability than a few strong ones. Hence, the diffuse actions of miRNAs in mammalian cells appear to function mainly in stabilizing GRNs. The postulated link between mRNA repression and GRN stability can be seen in a different light in yeast, which do not have miRNAs. Yeast cells rely on non-specific RNA nucleases to strongly degrade mRNAs for GRN stability. The strategy is suited to GRNs of small and rapidly dividing yeast cells, but not the larger mammalian cells. In conclusion, the May–Wigner theory, supplanting the analysis of small motifs, provides a mathematical solution to GRN stability, thus linking miRNAs explicitly to ‘developmental canalization’.

A non-specific nucleolytic enzyme and its application potential in EDTA-containing buffer solutions.

Metal-ion independent non-specific nucleases are of high potential for applications in EDTA-containing bioprocessing workflows. A novel extracellular non-specific nuclease EcNuc from the enterobacterium Escherichia coli has been identified. The recombinant gene was expressed and the protein was purified. Maximum activity of the enzyme was detected at 41.7°C and at an acidic pH of 5.8. EcNuc tolerates EDTA in the reaction buffer at concentrations of up to 20mM and the activity is not impaired by high concentrations of mono- and divalent metal ions in the absence of EDTA. The viscosity of crude protein extracts after cell lysis in EDTA-containing buffers is reduced when supplemented with EcNuc. Proof-of-concept has been demonstrated that a metal-ion independent non-specific nuclease can be applied for removal of nucleic acids in EDTA-containing buffers for the subsequent purification of proteins from crude extracts.

Active sites and thermostability of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica by site-directed mutagenesis

To identify the amino acid residues that are the critical factors contributing to the high catalytic activity and good thermostability of Yersinia enterocolitica subsp. palearctica non-specific nuclease (Y. NSN), in this work, we analyzed the structure and the functional domain of Y. NSN by using bioinformatics software. Disulphide bonds were analyzed by mass spectrometry. Six active site mutants of Y. NSN, including two salt bridge mutants and four disulphide bond mutants, were obtained by site-directed mutagenesis. Their enzyme activity and thermostability were assayed. The results showed that when changing the amino acids of the active site, or breaking the salt bridges and disulphide bonds, the enzyme activity and thermal stability of Y. NSN decreased significantly, compared to those of the wild-type enzyme. It is indicated that the active site, salt bridges and disulphide bonds play an important role in the enzyme activity and thermostability of Y. NSN.

N-glycosylation of tomato nuclease TBN1 produced in N. benthamiana and its effect on the enzyme activity

A unique analysis of an enzyme activity versus structure modification of the tomato nuclease R-TBN1 is presented. R-TBN1, the non-specific nuclease belonging to the S1-P1 nuclease family, was recombinantly produced in N. benthamiana. The native structure is posttranslationally modified by N-glycosylation at three sites. In this work, it was found that this nuclease is modified by high-mannose type N-glycosylation with a certain degree of macro- and microheterogeneity. To monitor the role of N-glycosylation in its activity, hypo- and hyperglycosylated nuclease mutants, R-TBN1 digested by α-mannosidase, and R-TBN1 deglycosylated by PNGase F were prepared. Deglycosylated R-TBN1 and mutant N94D/N112D were virtually inactive. Compared to R-TBN1 wt, both N94D and N112D mutants showed about 60% and 10% of the activity, respectively, while the N186D, D36S, and D36S/E104 N mutants were equally or even more active than R-TBN1 wt. The partial demannosylation of R-TBN1 did not affect the nuclease activity; moreover, a little shift in substrate specificity was observed. The results show two facts: 1) which sites must be occupied by a glycan for the proper folding and stability and 2) how N. benthamiana glycosylates the foreign nuclease. At the same time, the modifications can be interesting in designing the nuclease activity or specificity through its glycosylation.