Non-specific Endonuclease Research Articles

Specific and Sensitive Visual Proviral DNA Detection of Major Pathogenic Avian Leukosis Virus Subgroups Using CRISPR-Associated Nuclease Cas13a.

Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs' eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs.

A putative endonuclease reduces the efficiency of oral RNA interference in Nilaparvata lugens.

The RNA interference (RNAi) efficiency of double-stranded RNA (dsRNA) delivery to insects by various methods is different and the reduced efficacy of feeding dsRNA is partly due to the presence of DNA/RNA non-specific endonuclease in the insect gut. However, the mechanism leading to the low RNAi efficiency of Nilaparvata lugens by feeding remains elusive. In this study, we identified a putatively DNA/RNA non-specific endonuclease gene in the N. lugens genome database that was highly expressed in the first nymphal instar and the midgut. Different expression levels of NldsRNase after feeding and injection suggested that NldsRNase might interfere with oral RNAi in N. lugens. A co-delivery RNAi strategy further revealed that the presence of NldsRNase reduces RNAi efficiency. In vitro dsRNA degradation experiments also showed that the stability of dsRNA was higher in a gut mixture from nymphs injected with dsNldsRNase. These results support the idea that the low oral RNAi response observed in N. lugens is likely due to the presence of NldsRNase. Our study provides insight into the differences in RNAi response between the injection and feeding of dsRNA in N. lugens and sheds light on the mechanisms underlying the reduced efficacy of RNAi via feeding. These findings may help to inform the development of more-effective RNAi-based strategies controlling N. lugens and other insect pests. © 2024 Society of Chemical Industry.

Unified metagenomic method for rapid detection of microorganisms in clinical samples

BackgroundClinical metagenomics involves the genomic sequencing of all microorganisms in clinical samples ideally after depletion of human DNA to increase sensitivity and reduce turnaround times. Current human DNA depletion methods preferentially preserve either DNA or RNA containing microbes, but not both simultaneously. Here we describe and present data using a practical and rapid mechanical host-depletion method allowing simultaneous detection of RNA and DNA microorganisms linked with nanopore sequencing.MethodsThe human cells from respiratory samples are lysed mechanically using 1.4 mm zirconium-silicate spheres and the human DNA is depleted using a nonspecific endonuclease. The RNA is converted to dsDNA to allow the simultaneous sequencing of DNA and RNA.ResultsThe method decreases human DNA concentration by a median of eight Ct values while detecting a broad range of RNA & DNA viruses, bacteria, including atypical pathogens (Legionella, Chlamydia, Mycoplasma) and fungi (Candida, Pneumocystis, Aspergillus). The first automated reports are generated after 30 min sequencing from a 7 h end-to-end workflow. Sensitivity and specificity for bacterial detection are 90% and 100%, respectively, and viral detection are 92% and 100% after 2 h of sequencing. Prospective validation on 33 consecutive lower respiratory tract samples from ventilated patients with suspected pneumonia shows 60% concordance with routine testing, detection of additional pathogens in 21% of samples and pathogen genomic assembly achieve for 42% of viruses and 33% of bacteria.ConclusionsAlthough further workflow refinement and validation on samples containing a broader range of pathogens is required, it holds promise as a clinically deployable workflow suitable for evaluation in routine microbiology laboratories.

Cloning, heterologous expression, and molecular characterization of a highly active and stable non-specific endonuclease from Pseudomonas fluorescens.

Non-specific endonucleases can be used for the digestion of nucleic acids because they hydrolyze DNA/RNA into 3-5 base pairs (bp) length oligonucleotide fragments without strict selectivity. In this work, a novel non-specific endonuclease from Pseudomonas fluorescens (PfNuc) with high activities for both DNA and RNA was successfully cloned and expressed in Escherichia coli. The production of PfNuc in flask scale could be achieved to 1.73 × 106U/L and 4.82 × 106U/L for DNA and RNA by investigation of the culture and induction conditions. The characterization of PfNuc indicated that it was Mg2+-dependent and the catalytic activity was enhanced by 3.74 folds for DNA and 1.06 folds for RNA in the presence of 5mM Mg2+. The specific activity of PfNuc for DNA was 1.44 × 105U/mg at pH 8.0 and 40°C, and 3.93 × 105U/mg for RNA at pH 8.5 and 45°C. The Km of the enzyme for both DNA and RNA was close to 43µM. The Vmax was 6.40 × 105U/mg and 1.11 × 106U/mg for DNA and RNA, respectively. There was no observed activity loss when PfNuc was stored at 4°C and -20°C after 28days or 10 repeated freeze-thaw cycles at -80°C. Molecular docking revealed that PfNuc formed 17 and 19 hydrogen bonds with single-stranded RNA and double-stranded DNA, respectively. These results could explain the high activity and stability of PfNuc, suggesting its great potential applications in the industry and clinic.

Riemerella anatipestifer AS87_RS02955 Acts as a Virulence Factor and Displays Endonuclease Activity.

Riemerella anatipestifer is an important bacterial pathogen in the global duck industry and causes heavy economic losses. In our previous study, we demonstrated that R. anatipestifer type IX secretion system components GldK and GldM, and the secretion protein metallophosphoesterase, acted as virulence factors. In this study, R. anatipestifer AS87_RS02955 was investigated for virulence and enzymatic activity properties. We constructed AS87_RS02955 mutation and complementation strains to assess bacterial virulence. In vivo bacterial loads showed a significantly reduced bacterial loads in the blood of ducks infected with mutant strain Yb2Δ02955, which was recovered in the blood of ducks infected with the complementation strain cYb2Δ02955, demonstrating that AS87_RS02955 was associated with virulence. Further studies showed AS87_RS02955 was a novel nonspecific endonuclease with no functionally conserved domain, but enzymatic activity toward DNA and RNA was indicated. DNase activity was activated by Zn2+, Cu2+, Mg2+, Ca2+, and Mn2+ ions but inhibited by ethylenediaminetetraacetic acid. RNase activity was independent of metal cations, but stimulated by Mg2+, Ca2+, and Mn2+. RAS87_RS02955 enzymatic activity was active across a broad pH and temperature range. Moreover, we identified four sites in rAS87_RS02955, F39, F92, I134, and F145, which were critical for enzymatic activity. In summary, we showed that R. anatipestifer AS87_RS02955 encoded a novel endonuclease with important roles in bacterial virulence. IMPORTANCE R. anatipestifer AS87_RS02955 was identified as a novel T9SS effector and displayed a nonspecific endonuclease activity in this study. The protein did not contain a conserved His-Asn-His motif structure, which is similar to the endonuclease from Prevotella sp. Its mutant strain Yb2Δ02955 demonstrated significantly attenuated virulence, suggesting AS87_RS02955 is an important virulence factor. Moreover, AS87_RS02955 displayed nonspecific endonuclease activity to cleave λ DNA and MS2 RNA, while four protein sites were critical for endonuclease activity. In conclusion, R. anatipestifer AS87_RS02955 plays important roles in bacterial virulence.

A genetic toolkit for efficient production of secretory protein in Bacillus subtilis

Bacillus subtilis is a microbial cell factory widely used to produce recombinant proteins, but the expression of heterologous proteins is often severely hampered. This study constructed a genetic toolkit for improving the secretory efficiency of heterologous proteins in Bacillus subtilis. First, the protease-deficient hosts were reconstructed. Then, two endogenous constitutive promoters, Phag and PspovG, were screened. Next, a method called systemic combinatorial optimization of ribosome binding site (RBS) equipped with signal peptide (SCORES) was designed for optimizing the secretion and translation of the heterologous protein. Finally, Serratia marcescens nonspecific endonuclease (SMNE), which causes cell death by degrading nucleic acids, was expressed. The enzyme activity in the shake flask reached 7.5 × 106 U/L, which was 7.5-times that of the control RBS and signal peptide combination (RS0). This study not only expanded on the synthetic biology toolbox in B. subtilis but also provided strategies to create a prokaryotic protein expression system.

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

In Vitro Study of the Synergistic Effect of an Enzyme Cocktail and Antibiotics against Biofilms in a Prosthetic Joint Infection Model.

Prosthetic joint infections (PJI) are frequent complications of arthroplasties. Their treatment is made complex by the rapid formation of bacterial biofilms, limiting the effectiveness of antibiotic therapy. In this study, we explore the effect of a tri-enzymatic cocktail (TEC) consisting of an endo-1,4-β-d-glucanase, a β-1,6-hexosaminidase, and an RNA/DNA nonspecific endonuclease combined with antibiotics of different classes against biofilms of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli grown on Ti-6Al-4V substrates. Biofilms were grown in Trypticase soy broth (TSB) with 10 g/liter glucose and 20 g/liter NaCl (TGN). Mature biofilms were assigned to a control group or treated with the TEC for 30 min and then either analyzed or reincubated for 24 h in TGN or TGN with antibiotics. The cytotoxicity of the TEC was assayed against MG-63 osteoblasts, primary murine fibroblasts, and J-774 macrophages using the lactate dehydrogenase (LDH) release test. The TEC dispersed 80.3 to 95.2% of the biofilms' biomass after 30 min. The reincubation of the treated biofilms with antibiotics resulted in a synergistic reduction of the total culturable bacterial count (CFU) compared to that of biofilms treated with antibiotics alone in the three tested species (additional reduction from 2 to more than 3 log10 CFU). No toxicity of the TEC was observed against the tested cell lines after 24 h of incubation. The combination of pretreatment with TEC followed by 24 h of incubation with antibiotics had a synergistic effect against biofilms of S. aureus, S. epidermidis, and E. coli Further studies should assess the potential of the TEC as an adjuvant therapy in in vivo models of PJI.

Identification of a double-stranded RNA-degrading nuclease influencing both ingestion and injection RNA interference efficiency in the red flour beetle Tribolium castaneum

RNA interference (RNAi) efficiency dramatically varies among different insects and among administration methods. Numerous studies have revealed that a poor RNAi response is usually associated with a high double-stranded RNA (dsRNA)-degrading activity. Using the red flour beetle Tribolium castaneum, we conducted genome-wide identification of genes encoding dsRNA-degrading nucleases of the DNA/RNA non-specific endonuclease superfamily. To achieve a robust RNAi response in T. castaneum, four dsRNase genes were identified in the genome that seemed to be the potential factors reducing RNAi efficacy. Analysis of biochemical properties revealed that optimal conditions for the dsRNA-degrading activity were alkaline (pH 8.0) in the absence of Mg2+ at 37 °C. The dsRNA-degrading activity was predominantly present in the gut, and via heterologous expression and RNAi experimentation, gut-specific TcdsRNase1 was confirmed as the major nuclease performing dsRNA degradation. After a knockdown of the TcdsRNase1 nuclease activity, RNAi efficiency improved from 38.6% to 58.9% and from 20.9% to 53.9% for injection and ingestion of dsRNA, respectively. Our results contribute to a comprehensive understanding of the mechanisms influencing dsRNA stability and even RNAi efficiency in T. castaneum and point to a good method for improving RNAi efficiency through downregulation of the relevant nuclease activity.

Characterization of a cell death‐inducing endonuclease‐like venom protein from the parasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

Parasitoid wasps are valuable natural enemies for controlling pests. To ensure successful parasitism, these wasps inject venoms along with their eggs that are deposited either into or on their hosts. Parasitoid venoms regulate host behaviors, development, metabolism and immune responses. Pteromalus puparum is a pupal endoparasitoid that parasitizes a number of butterflies, including the worldwide pest cabbage butterfly, Pieris rapae. Venom from P. puparum has a variety of effects on host hemocytes, including alteration of absolute and relative hemocyte counts, and inhibition of hemocyte spreading and encapsulation. In particular, P. puparum venom causes hemocyte cell death in vivo and in vitro. Using assay-guided chromatography, a cell death-inducing venom fraction was identified and defined as P. puparum endonuclease-like venom protein (PpENVP). It belongs to the DNA/RNA nonspecific endonuclease family, which contains two conserved endonuclease activation sites. We analyzed its expression profiles and demonstrated that PpENVP inhibits gene expression in transfected cells relying on two activation sites. However, RNA interference of PpENVP did not significantly reduce P. puparum venom cytotoxicity, suggesting that PpENVP may not be the sole cytotoxic factor present. Our results provide novel insight into the function of the P. puparum venom cocktail and identify a promising insecticide candidate endonuclease that targets insect hemocytes.

Identification and characterization of multiple dsRNases from a lepidopteran insect, the tobacco cutworm, Spodoptera litura (Lepidoptera: Noctuidae)

RNA interference (RNAi) efficiency varies among insects. RNAi is highly efficient and systemic in coleopteran insects but quite variable and inefficient in lepidopteran insects. Degradation of double-stranded RNA (dsRNA) by double-stranded ribonucleases (dsRNases) is thought to contribute to the variability in RNAi efficiency observed among insects. One or two dsRNases involved in dsRNA digestion have been identified in a few insects. To understand the contribution of dsRNases to reduced RNAi efficiency in lepidopteran insects, we searched the transcriptome of Spodoptera litura and identified six genes coding for DNA/RNA non-specific endonucleases. Phylogenetic analysis revealed the evolutionary expansion of dsRNase genes in insects. The mRNA levels of three midgut-specific dsRNases increased during the larval stage, and the highest dsRNA-degrading activity was detected in third-instar larvae. Proteins produced via the expression of three midgut-specific dsRNases, and the widely expressed dsRNase3, in a baculovirus system showed dsRNase activity for four out of five dsRNases tested. In addition, the increase in dsRNA-degrading activity and upregulation of dsRNase1 and 2 in larvae fed on cabbage leaves suggests that the diet of S. litura can influence dsRNase expression, dsRNA stability, and thus probably RNAi efficiency. This is the first report that multiple dsRNases function together in an RNAi-recalcitrant insect. The data included in this paper suggest that multiple dsRNases coded by the S. litura genome might contribute to the lower and variable RNAi efficiency reported in this and other lepidopteran insects.

Functional screening of a soil metagenome for DNA endonucleases by acquired resistance to bacteriophage infection.

Endonucleases play a crucial role as reagents in laboratory research and diagnostics. Here, metagenomics was used to functionally screen a fosmid library for endonucleases. A fosmid library was constructed using metagenomic DNA isolated from soil sampled from the unique environment of the Kogelberg Nature Reserve in the Western Cape of South Africa. The principle of acquired immunity against phage infection was used to develop a plate-based screening technique for the isolation of restriction endonucleases from the library. Using next-generation sequencing and bioinformatics tools, sequence data were generated and analysed, revealing 113 novel open reading frames (ORFs) encoding putative endonuclease genes and ORFs of unknown identity and function. One endonuclease designated Endo52 was selected from the putative endonuclease ORFs and was recombinantly produced in Escherichia coli Rosetta™ (DE3) pLysS. Endo52 was purified by immobilised metal affinity chromatography and yielded 0.437g per litre of cultivation volume. Its enzyme activity was monitored by cleaving lambda DNA and pUC19 plasmid as substrates, and it demonstrated non-specific endonuclease activity. In addition to endonuclease-like genes, the screen identified several unknown genes. These could present new phage resistance mechanisms and are an opportunity for future investigations.

Hantavirus RdRp Requires a Host Cell Factor for Cap Snatching.

The hantavirus RNA-dependent RNA polymerase (RdRp) snatches 5' capped mRNA fragments from the host cell transcripts and uses them as primers to initiate transcription and replication of the viral genome in the cytoplasm of infected cells. Hantavirus nucleocapsid protein (N protein) binds to the 5' caps of host cell mRNA and protects them from the attack of cellular decapping machinery. N protein rescues long capped mRNA fragments in cellular P bodies that are later processed by an unknown mechanism to generate 10- to 14-nucleotide-long capped RNA primers with a 3' G residue. Hantavirus RdRp has an N-terminal endonuclease domain and a C-terminal uncharacterized domain that harbors a binding site for the N protein. The purified endonuclease domain of RdRp nonspecifically degraded RNA in vitro It is puzzling how such nonspecific endonuclease activity generates primers of appropriate length and specificity during cap snatching. We fused the N-terminal endonuclease domain with the C-terminal uncharacterized domain of the RdRp. The resulting NC mutant, with the assistance of N protein, generated capped primers of appropriate length and specificity from a test mRNA in cells. Bacterially expressed and purified NC mutant and N protein required further incubation with the lysates of human umbilical vein endothelial cells (HUVECs) for the specific endonucleolytic cleavage of a test mRNA to generate capped primers of appropriate length and defined 3' terminus in vitro Our results suggest that an unknown host cell factor facilitates the interaction between N protein and NC mutant and brings the N protein-bound capped RNA fragments in close proximity to the endonuclease domain of the RdRp for specific cleavage at a precise length from the 5' cap. These studies provide critical insights into the cap-snatching mechanism of cytoplasmic viruses and have revealed potential new targets for their therapeutic intervention.IMPORTANCE Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. Hantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with mortality rates of 15% and 50%, respectively (1). Annually 150,000 to 200,000 cases of hantavirus infections are reported worldwide, for which there is no treatment at present. Cap snatching is an early event in the initiation of virus replication in infected hosts. Interruption in cap snatching will inhibit virus replication and will likely improve the prognosis of the hantavirus disease. Our studies provide mechanistic insight into the cap-snatching mechanism and demonstrate the requirement of a host cell factor for successful cap snatching. Identification of this host cell factor will reveal a novel therapeutic target for combating this viral illness.

Computational and mutational analysis of TatD DNase of Bacillus anthracis.

The role of TatD DNases as DNA repair enzymes or cell death (apoptotic) nucleases is well established in prokaryotes as well as eukaryotes. The current study aims to characterize the TatD nuclease from Bacillus anthracis (Ba TatD) and to explore its key histidine catalytic residues. Ba TatD was found to be a metal-dependent, nonspecific endonuclease which could efficiently cleave double-stranded DNA substrates. Moreover, Ba TatD nuclease was observed to be thermostable up to 55°C and act in a wide pH range indicating its industrial applicability. Diethyl pyrocarbonate-based histidine-selective alkylation of the Ba TatD resulted in a loss of its nuclease activity suggesting a crucial role of the histidine residues in its activity. The key residues of Ba TatD were predicted using sequence analysis and structure-based approaches, and then the predicted residues were further tested by mutational analysis. Upon mutational analysis, H128 and H153 have been found to be crucial for Ba TatD activity, though H153 seems to bear an important but a dispensable role for the Ba TatD nuclease. Ba TatD had a uniform expression in the cytosol of B. anthracis, which indicates a significant role of the protein in the pathogen's life cycle. This is the first study to identify and characterize the TatD DNase from B. anthracis and will be helpful in gaining more insights on the role of TatD proteins in Gram-positive bacteria where it remains unexplored.

Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis.

RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.

Crystal structure of NucB, a biofilm-degrading endonuclease.

Bacterial biofilms are a complex architecture of cells that grow on moist interfaces, and are held together by a molecular glue of extracellular proteins, sugars and nucleic acids. Biofilms are particularly problematic in human healthcare as they can coat medical implants and are thus a potential source of disease. The enzymatic dispersal of biofilms is increasingly being developed as a new strategy to treat this problem. Here, we have characterized NucB, a biofilm-dispersing nuclease from a marine strain of Bacillus licheniformis, and present its crystal structure together with the biochemistry and a mutational analysis required to confirm its active site. Taken together, these data support the categorization of NucB into a unique subfamily of the ββα metal-dependent non-specific endonucleases. Understanding the structure and function of NucB will facilitate its future development into an anti-biofilm therapeutic agent.

Expression and Purification Procedure of Nonspecific Endonuclease Sma and Its Performance Study

Expression and Purification Procedure of Nonspecific Endonuclease Sma and Its Performance Study

Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer.

ABSTRACTBacteria use a variety of DNA-mobilizing enzymes to facilitate environmental niche adaptation via horizontal gene transfer. This has led to real-world problems, like the spread of antibiotic resistance, yet many mobilization proteins remain undefined. In the study described here, we investigated the uncharacterized family of YhgA-like transposase_31 (Pfam PF04754) proteins. Our primary focus was the genetic and biochemical properties of the five Escherichia coli K-12 members of this family, which we designate RpnA to RpnE, where Rpn represents recombination-promoting nuclease. We employed a conjugal system developed by our lab that demanded RecA-independent recombination following transfer of chromosomal DNA. Overexpression of RpnA (YhgA), RpnB (YfcI), RpnC (YadD), and RpnD (YjiP) increased RecA-independent recombination, reduced cell viability, and induced the expression of reporter of DNA damage. For the exemplar of the family, RpnA, mutational changes in proposed catalytic residues reduced or abolished all three phenotypes in concert. In vitro, RpnA displayed magnesium-dependent, calcium-stimulated DNA endonuclease activity with little, if any, sequence specificity and a preference for double-strand cleavage. We propose that Rpn/YhgA-like family nucleases can participate in gene acquisition processes.IMPORTANCE Bacteria adapt to new environments by obtaining new genes from other bacteria. Here, we characterize a set of genes that can promote the acquisition process by a novel mechanism. Genome comparisons had suggested the horizontal spread of the genes for the YhgA-like family of proteins through bacteria. Although annotated as transposase_31, no member of the family has previously been characterized experimentally. We show that four Escherichia coli K-12 paralogs contribute to a novel RecA-independent recombination mechanism in vivo. For RpnA, we demonstrate in vitro action as a magnesium-dependent, calcium-stimulated nonspecific DNA endonuclease. The cleavage products are capable of providing priming sites for DNA polymerase, which can enable DNA joining by primer-template switching.

RNA-directed off/on switch of RNase H activity using boronic ester formation.

RNase H is a non-specific endonuclease which degrades selectively the RNA strand in DNA/RNA duplexes. We demonstrate in the present study that 5'-boronic acid modified oligonucleotides hybridized to a RNA target sequence converts RNase H to an inactivated enzyme complex. The dynamic formation of a boronate ester upon addition of a diol moiety disrupts the enzyme-inhibitor complex and reactivates RNase H. Moreover, we show that reactivation of RNase H function can also be engineered through short RNA trimers inputs that fashion RNase H from a non-specific DNA-guided enzyme into an informational and programmable RNA-guided one. Examples of programmable RNA recognition and cleavage illustrate the potential of this new stimuli-responsive system.

DNase I SIM: A Simplified In-Nucleus Method for DNase I Hypersensitive Site Sequencing.

Identifying cis-regulatory elements is critical in understanding the direct and indirect interactions that occur within gene regulatory networks. Current approaches include DNase-seq, a technique that combines sensitivity to the nonspecific endonuclease DNase I with high-throughput sequencing to identify regions of regulatory DNA on a genome-wide scale. Yet, challenges still remain in processing recalcitrant tissues that have low DNA content. Here, we describe DNase I SIM (for Simplified In-nucleus Method), a protocol that simplifies and facilitates generation of DNase-seq libraries from plant tissues for high-resolution mapping of DNase I hypersensitive sites. By removing steps requiring the use of gel agarose plugs in DNase-seq, DNase I SIM reduces the time required to perform the protocol by at least 2 days, while also making possible the processing of difficult plant tissues including plant roots.