Site-specific Cleavage Research Articles

The Construction of Heterothallic Strains of Komagataella kurtzmanii Using the I-SceI Meganuclease.

The methylotrophic yeast Komagataella kurtzmanii belongs to the group of homothallic fungi that are able to spontaneously change their mating type by inversion of chromosomal DNA in the MAT locus region. As a result, natural and genetically engineered cultures of these yeasts typically contain a mixture of sexually dimorphic cells that are prone to self-diploidisation and spore formation accompanied by genetic rearrangements. These characteristics pose a significant challenge to the development of genetically stable producers for industrial use. In the present study, we constructed heterothallic strains of K. kurtzmanii, ensuring a constant mating type by unifying the genetic sequences in the active and silent MAT loci. To obtain such strains, we performed site-directed inactivation of one of the two yeast MAT loci, replacing its sequence with a selective HIS4 gene surrounded by I-SceI meganuclease recognition sites. We then used transient expression of the SCE1 gene, encoding a recombinant I-SceI meganuclease, to induce site-specific cleavage of HIS4, followed by damage repair by homologous recombination in mutant cells. As a result, heterothallic strains designated 'Y-727-2(alpha)' and 'Y-727-9(a)', which correspond to the α and a mating type, respectively, were obtained. The strains demonstrated a loss of the ability to self-diploidize. The results of PCR and whole genome analysis confirmed the identity of the contents of the MAT loci. Analysis of the genomes of the final strains, however, revealed a fusion of chromosome 3 and chromosome 4 in strain Y-727-2(alpha)-1. This finding was subsequently confirmed by pulsed-field gel electrophoresis of yeast chromosomes. However, the ability of the Y-727-2(alpha)-derived producers to efficiently secrete recombinant β-galactosidase was unaffected by this genomic rearrangement.

How to shift the equilibrium of dna break repair in favor of homology recombination

With the practical implementation of the CRISPR/Cas technology for targeted genome editing, it has become possible to carry out genetic engineering manipulations with eukaryotic genomes with high efficiency. One of the key stages of this technology is the targeted induction of site-specific DNA cleavages (breaks). The cell repairs these breaks via one of two pathways: nonhomologous end joining or homologous recombination. The choice of DNA repair pathway is determined by the architecture of the sites at the DNA break area formed as a result of terminal resection and depends on the phases of the cell cycle. Nonhomologous end joining is the main pathway for repair of double-stranded DNA breaks in mammalian cells. It involves a nonspecific ligation reaction, the accuracy of which depends on the structure of the ends of the break, and can result in various insertions or deletions in the target region of the genome. Integration of the desired sequence into the genome occurs along the path of homologous recombination, the implementation of which requires a matrix with homology regions on both sides of the double-strand break. The introduction of a genetic construct into a given location in the genome is an important, but currently complex and labor-intensive task. At the same time, for fundamental studies of gene function and the creation of animal models of human diseases, the choice of the repair pathway can be of fundamental importance. This review is an attempt to combine and structure all known information on approaches to increasing the efficiency of DNA repair involving homologous recombination. The article lists the most effective strategies to shift the balance towards homologous repair, such as the use of inhibitors of the non-homologous end joining mechanism, regulation of key factors of homologous recombination, control of the cell cycle, chromatin status, construction of templates for homologous recombination.

Self-Adaptive Activation of DNAzyme Nanoassembly for Synergistically Combined Gene Therapy.

DNAzyme represents a promising gene silencing toolbox yet is obstructed by the poor substrate accessibility in specific cells. Herein, a compact DNA nanoassembly, incorporating multimeric therapeutic DNAzyme, was prepared for selective delivery of gene-silencing DNAzyme with requisite cofactors and auxiliary chemo-drugs. By virtue of the sequence-conservative duplex-specific nuclease, the endogenous miRNA catalyzes the successive and site-specific cleavage of DNA nanoassembly substrate (nominated as the localized RNA walking machine) and thus ensures the liberation/activation of therapeutic agents with high accuracy and efficacy. The miR-10b-stimulated DNAzyme was designed to downregulate the TWIST transcription factor, an upstream promotor of miR-10b, thus acquiring the self-sufficient downregulation of TWIST/miR-10b signaling nodes (self-adaptive negative feedback loop) for abrogating tumor metastasis and chemo-resistance issues.

Elucidating Evolutionary Mechanisms and Variants of the Hammerhead Ribozyme Using In Vitro Selection.

The Hammerhead Ribozyme (HHR) is a ubiquitous RNA enzyme that catalyzes site-specific intramolecular cleavage. While mutations to its catalytic core have traditionally been viewed as detrimental to its activity, several discoveries of naturally occurring variants of the full-length ribozyme challenge this notion, suggesting a deeper understanding of HHR evolution and functionality. By systematically introducing mutations at key nucleotide positions within the catalytic core, we generated single-, double-, and triple-mutation libraries to explore the sequence requirements and evolution of a full-length HHR. In vitro selection revealed many novel hammerhead variants, some of which possess mutations at nucleotides previously considered to be essential. We also demonstrate that the evolutionary trajectory of each nucleotide in the catalytic core directly correlates with their functional importance, potentially giving researchers a novel method to assess the sequence requirements of functional nucleic acids.

Preventing Site-Specific Calpain Proteolysis of Junctophilin-2 Protects Against Stress-Induced Excitation-Contraction Uncoupling and Heart Failure Development.

Excitation-contraction (E-C) coupling processes become disrupted in heart failure (HF), resulting in abnormal Ca2+ homeostasis, maladaptive structural and transcriptional remodeling, and cardiac dysfunction. Junctophilin-2 (JP2) is an essential component of the E-C coupling apparatus but becomes site-specifically cleaved by calpain, leading to disruption of E-C coupling, plasmalemmal transverse tubule degeneration, abnormal Ca2+ homeostasis, and HF. However, it is not clear whether preventing site-specific calpain cleavage of JP2 is sufficient to protect the heart against stress-induced pathological cardiac remodeling in vivo. Calpain-resistant JP2 knock-in mice (JP2CR) were generated by deleting the primary JP2 calpain cleavage site. Stress-dependent JP2 cleavage was assessed through in vitro cleavage assays and in isolated cardiomyocytes treated with 1 μmol/L isoproterenol by immunofluorescence. Cardiac outcomes were assessed in wild-type and JP2CR mice 5 weeks after transverse aortic constriction compared with sham surgery using echocardiography, histology, and RNA-sequencing methods. E-C coupling efficiency was measured by in situ confocal microscopy. E-C coupling proteins were evaluated by calpain assays and Western blotting. The effectiveness of adeno-associated virus gene therapy with JP2CR, JP2, or green fluorescent protein to slow HF progression was evaluated in mice with established cardiac dysfunction. JP2 proteolysis by calpain and in response to transverse aortic constriction and isoproterenol was blocked in JP2CR cardiomyocytes. JP2CR hearts are more resistant to pressure-overload stress, having significantly improved Ca2+ homeostasis and transverse tubule organization with significantly attenuated cardiac dysfunction, hypertrophy, lung edema, fibrosis, and gene expression changes relative to wild-type mice. JP2CR preserves the integrity of calpain-sensitive E-C coupling-related proteins, including ryanodine receptor 2, CaV1.2, and sarcoplasmic reticulum calcium ATPase 2a, by attenuating transverse aortic constriction-induced increases in calpain activity. Furthermore, JP2CR gene therapy after the onset of cardiac dysfunction was found to be effective at slowing the progression of HF and superior to wild-type JP2. The data presented here demonstrate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain cleavage of JP2 offers multifaceted beneficial effects, conferring cardiac protection against stress-induced proteolysis, hypertrophy, and HF. Our data also indicate that specifically targeting the primary calpain cleavage site of JP2 by gene therapy approaches holds great therapeutic potential as a novel precision medicine for treating HF.

Fusion of FokI and catalytically inactive prokaryotic Argonautes enables site-specific programmable DNA cleavage

Site-specific nucleases are crucial for genome engineering applications in medicine and agriculture. The ideal site-specific nucleases are easily reprogrammable, highly specific in target site recognition, robust in nuclease activities. Prokaryotic Argonaute (pAgo) proteins have received much attention as biotechnological tools due to their ability to recognize specific target sequences without a protospacer adjacent motif (PAM), but their lack of intrinsic double-stranded DNA (dsDNA) unwinding activity limits their utility in key applications such as gene editing. Recently, we developed a pAgo-based system for site-specific DNA cleavage at physiological temperatures independently of the DNA form, using peptide nucleic acids (PNAs) to facilitate unwinding dsDNA targets. Here, we fused catalytically dead pAgos with the nuclease domain of the restriction endonuclease FokI and named this modified platform PNA-assisted FokI-(d)pAgo (PNFP) editors. In the PNFP system, catalytically inactive pAgo recognizes and binds to a specific target DNA sequence based on a programmable guide DNA sequence; upon binding to the target site, the FokI domains dimerize and introduce precise dsDNA breaks. We explored key parameters of the PNFP system including the requirements of PNA and guide DNAs, the specificity of PNA and guide DNA on target cleavage, the optimal concentration of different components, reaction time for invasion and cleavage and ideal temperature and reaction buffer, to ensure efficient DNA editing in vitro. The results demonstrated robust site-specific target cleavage by PNFP system at optimal conditions in vitro. We envision that the PNFP system will provide higher editing efficiency and specificity with fewer off-target effects in vivo.

Protein-Protein Interface Identification by Site-Specific Photo-Cross-linking/Cleavage in Mammalian Cells.

Identification of protein-protein interfaces is necessary for understanding and regulating biological events. Genetic code expansion enables site-specific photo-cross-linking by introducing photo-reactive non-canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein-protein interactions and is applicable in mammalian cells. However, the identification of the cross-linked region still remains challenging. Our new protocol enables its identification by pre-installing a site-specific cleavage site, an α-hydroxy acid (Nε-allyloxycarbonyl-α-hydroxyl-L-lysine acid, AllocLys-OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α-hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N-terminus or C-terminus, the crosslinked site is located on within the target protein. A series of AllocLys-OH introductions narrows down the crosslinked region. This combination of site-specific crosslinking and cleavage promises to be useful for revealing binding interfaces and protein complex geometries. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Search for crosslinkable sites Basic Protocol 2: Site-specific photo-cross-linking/cleavage.

Mechanisms for HNH-mediated target DNA cleavage in type I CRISPR-Cas systems

The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-FHNH and I-EHNH Cascade complexes at different states are reported. In type I-FHNH Cascade, Cas8fHNH loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8fHNH to detach and rotate ∼150° to accommodate target ssDNA for cleavage. In type I-EHNH Cascade, Cas5eHNH domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.

Sample-in-answer-out centrifugal microfluidic chip reaction biosensor powered by Thermus thermophilus Argonaute (TtAgo) for rapid, highly sensitive and multiplexed molecular diagnostics of foodborne bacterial pathogens

Foodborne pathogens endanger public health and rapid, sensitive and accurate detection of them is vital. Argonaute stands in the frontline as the next generation nucleic acid detection tool, bearing a few comparable advantages. Centrifugal microfluidic chips (CMCs) use centrifugal force to achieve liquid flowing, mixing and reaction, eliminating complicated designs of valves and pumps. In this study, for the first time, we devised a Thermus thermophilusArgonaute (TtAgo)-powered centrifugal microfluidic chip reaction biosensor for Sample-in-Answer-out detection of Pathogen S. aureus (termed as ASAP) with ultrahigh rapidity and sensitivity in a one-pot fashion. The samples subjected to testing were mixed with a home-made nucleic acid fast extraction reagent and then the mixture was injected into the sample cell of CMC, which was centrifuged down into the reaction cell. The reaction cell was preloaded with both LAMP (Loop-mediated isothermal amplification) and TtAgo systems. As such, the species-specific nuc genes were amplified by LAMP, while the TtAgo performed site-specific cleavage to output fluorescent (FL) signals. The sample-to-answer time was 17 min, and the limit of detection (LOD) reached 1 CFU/mL. ASAP was able to perform simultaneous multiplexed detection and up to 16 samples can be detected at the same time. ASAP reduced reagent consumption and minimized the influence of carry-over and cross-contamination. ASAP was capable of detecting S. aureus in foods, but also detecting physiological samples from infect, holding great promise for practical applications. Overall, our work has enriched the Argonaute-powered biosensing and CMC biosensor technology by providing a conceptually novel bacterial detection platform.

Tumour-specific activation of a tumour-blood transport improves the diagnostic accuracy of blood tumour markers in mice

The accuracy of blood-based early tumour recognition is compromised by signal production at non-tumoral sites, low amount of signal produced by small tumours, and variable tumour production. Here we examined whether tumour-specific enhancement of vascular permeability by the particular tumour homing peptide, iRGD, which carries dual function of binding to integrin receptors overexpressed in the tumour vasculature and is known to promote extravasation via neuropilin-1 receptor upon site-specific cleavage, might be useful to improve blood-based tumour detection by inducing a yet unrecognised vice versa tumour-to-blood transport. To detect an iRGD-induced tumour-to-blood transport, we examined the effect of intravenously injected iRGD on blood levels of α-fetoprotein (AFP) and autotaxin in several mouse models of hepatocellular carcinoma (HCC) or in mice with chronic liver injury without HCC, and on prostate-specific antigen (PSA) levels in mice with prostate cancer. Intravenously injected iRGD rapidly and robustly elevated the blood levels of AFP in several mouse models of HCC, but not in mice with chronic liver injury. The effect was primarily seen in mice with small tumours and normal basal blood AFP levels, was attenuated by an anti-neuropilin-1 antibody, and depended on the concentration gradient between tumour and blood. iRGD treatment was also able to increase blood levels of autotaxin in HCC mice, and of PSA in mice with prostate cancer. We conclude that iRGD induces a tumour-to-blood transport in a tumour-specific fashion that has potential of improving diagnosis of early stage cancer. Deutsche Krebshilfe, DKTK, LOEWE-Frankfurt Cancer Institute.

Force-Induced Site-Specific Enzymatic Cleavage Probes Reveal That Serial Mechanical Engagement Boosts T Cell Activation.

The T cell membrane is studded with >104 T cell receptors (TCRs) that are used to scan target cells to identify short peptide fragments associated with viral infection or cancerous mutation. These peptides are presented as peptide-major-histocompatibility complexes (pMHCs) on the surface of virtually all nucleated cells. The TCR-pMHC complex forms at cell-cell junctions, is highly transient, and experiences mechanical forces. An important question in this area pertains to the role of the force duration in immune activation. Herein, we report the development of force probes that autonomously terminate tension within a time window following mechanical triggering. Force-induced site-specific enzymatic cleavage (FUSE) probes tune the tension duration by controlling the rate of a force-triggered endonuclease hydrolysis reaction. This new capability provides a method to study how the accumulated force duration contributes to T cell activation. We screened DNA sequences and identified FUSE probes that disrupt mechanical interactions with F > 7.1 piconewtons (pN) between TCRs and pMHCs. This rate of disruption, or force lifetime (τF), is tunable from tens of minutes down to 1.9 min. T cells challenged with FUSE probes with F > 7.1 pN presenting cognate antigens showed up to a 23% decrease in markers of early activation. FUSE probes with F > 17.0 pN showed weaker influence on T cell triggering further showing that TCR-pMHC with F > 17.0 pN are less frequent compared to F > 7.1 pN. Taken together, FUSE probes allow a new strategy to investigate the role of force dynamics in mechanotransduction broadly and specifically suggest a model of serial mechanical engagement boosting TCR activation.

Cleaving DNA with DNA: Cooperative Tuning of Structure and Reactivity Driven by Copper Ions.

A copper-dependent self-cleaving DNA (DNAzyme or deoyxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics (MD) simulations and advanced Electron Paramagnetic Resonance (Electron Spin Resonance) EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction. The modeled 46-nucleotide deoxyribozyme in MD simulations forms duplex and triplex sub-structures that flank a highly conserved catalytic core. The DNA self-cleaving construct can also form a bimolecular complex that has a distinct substrate and enzyme domains. The highly dynamic structure combined with an oxidative site-specific cleavage of the substrate are two key-aspects to elucidate. By combining EPR/ESR spectroscopy with selectively isotopically labeled nucleotides it has been possible to overcome the major drawback related to the "metal-soup" scenario, also known as "super-stoichiometric" ratios of cofactors versus substrate, conventionally required for the DNA cleavage reaction within those nucleic acids-based enzymes. The focus on the endogenous paramagnetic center (Cu2+ ) here described paves the way for analysis on mixtures where several different cofactors are involved. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic perspective towards the applicability of EPR/ESR spectroscopic studies.

T790M mutation upconversion fluorescence biosensor via mild ATRP strategy and site-specific DNA cleavage of restriction endonuclease.

A unique combination of a specific nucleic acid restriction endonuclease (REase) and atom transfer radical polymerization (ATRP) signal amplification strategy was employed for the detection of T790M mutations prevalent in the adjuvant diagnosis of lung cancer. REase selectively recognizes and cleaves T790M mutation sites on double-stranded DNA formed by hybridization of a capture sequence and a target sequence. At the same time, the ATRP strategy resulted in the massive aggregation of upconverted nanoparticles (UCNPs), which significantly improved the sensitivity of the biosensor. In addition, the UCNPs have excellent optical properties and can eliminate the interference of autofluorescence in the samples, thus further improving the detection sensitivity. The proposed upconversion fluorescent biosensor is characterized by high specificity, high sensitivity, mild reaction conditions, fast response time, and a detection limit as low as 0.14 fM. Theperformance of the proposed biosensor is comparable to that of clinical PCR methods when applied to clinical samples. This work presents a new perspective for assisted diagnosis in the pre-intervention stage of tumor diagnostics in the early stage of precision oncology treatments.

Measuring site-specific DNA cleavage at the single-molecule level

Measuring site-specific DNA cleavage at the single-molecule level

A high-fidelity DNAzyme-assisted CRISPR/Cas13a system with single-nucleotide resolved specificity.

A CRISPR/Cas system represents an innovative tool for developing a new-generation biosensing and diagnostic strategy. However, the off-target issue (i.e., mistaken cleavage of nucleic acid targets and reporters) remains a great challenge for its practical applications. We hypothesize that this issue can be overcome by taking advantage of the site-specific cleavage ability of RNA-cleaving DNAzymes. To test this idea, we propose a DNAzyme Operation Enhances the Specificity of CRISPR/Cas13a strategy (termed DOES-CRISPR) to overcome the problem of relatively poor specificity that is typical of the traditional CRISPR/Cas13a system. The key to the design is that the partial hybridization of the CRISPR RNA (crRNA) with the cleavage fragment of off-target RNA was not able to activate the collateral cleavage activity of Cas13a. We showed that DOES-CRISPR can significantly improve the specificity of traditional CRISPR/Cas13a-based molecular detection by up to ∼43-fold. The broad utility of the strategy is illustrated through engineering three different systems for the detection of microRNAs (miR-17 and let-7e), CYP2C19*17 gene, SARS-Cov-2 variants (Gamma, Delta, and Omicron) and Omicron subtypes (BQ.1 and XBB.1) with single-nucleotide resolved specificity. Finally, clinical evaluation of this assay using 10 patient blood samples demonstrated a clinical sensitivity of 100% and specificity of 100% for genotyping CYP2C19*17, and analyzing 20 throat swab samples provided a diagnostic sensitivity of 95% and specificity of 100% for Omicron detection, and a clinical sensitivity of 92% and specificity of 100% for XBB.1 detection.

SCARPET: site-specific quantification of methylated and nonmethylated adenosines reveals m6A stoichiometry.

m6A has different stoichiometry at different positions in different mRNAs. However, the exact stoichiometry of m6A is difficult to measure. Here, we describe SCARPET (site-specific cleavage and radioactive-labeling followed by purification, exonuclease digestion, and thin-layer chromatography), a simple and streamlined biochemical assay for quantifying m6A at any specific site in any mRNA. SCARPET involves a site-specific cleavage of mRNA immediately 5' of an adenosine site in an mRNA. This site is radiolabeled with 32P, and after a series of steps to purify the RNA and to remove nonspecific signals, the nucleotide is resolved by TLC to visualize A and m6A at this site. Quantification of these spots reveals the m6A stoichiometry at the site of interest. SCARPET can be applied to poly(A)-enriched RNA, or preferably purified mRNA, which produces more accurate m6A stoichiometry measurements. We show that sample processing steps of SCARPET can be performed in a single day, and results in a specific and accurate measurement of m6A stoichiometry at specific sites in mRNA. Using SCARPET, we measure exact m6A stoichiometries in specific mRNAs and show that Zika genomic RNA lacks m6A at previously mapped sites. SCARPET will be useful for testing specific sites for their m6A stoichiometry and to assess how m6A stoichiometry changes in different conditions and cellular contexts.

Engineering of a Chimeric Template Triggers RNase H-Based Isothermal Amplification Approach for Sensitive Detection of Pathogen RNA.

RNA-based detection of pathogenic organisms is an emerging field of research that is crucial for disease diagnosis and environmental and food safety. By rationally engineering an RNA-DNA tandem (RDT) structural template, we proposed a novel RNase H-based isothermal exponential amplification (RH-IEA) reaction to rapidly identify long-stranded RNA. In this strategy, the rigid and compact RDT template selectively recognized the target RNA and formed a stable hybrid with it. Upon site-specific cleavage of RNase H, the 3' overhang of the target RNA was cut off, and a free hydroxyl end at the hydrolysis site was generated to trigger an exponential amplification reaction (EXPAR). This method maintained the high efficiency and rapid amplification kinetics of EXPAR. As a result, the RH-IEA strategy was able to sensitively and specifically detect the characteristic sequence of Escherichia coli O157:H7 RNA, with a detection sensitivity of 1 fg/μL. Besides, the RDT template can be used as an RNA protector to prevent specific segments of the target RNA from being degraded by RNase enzymes, allowing the sample to be stored at room temperature for a long time. With this advantage, the practicality of RH-IEA will be more flexible than the reverse transcription polymerase chain reaction. It was successfully applied in the identification of E. coli O157:H7 in milk with a minimum detection concentration of 1.0 × 102 CFU/mL. Therefore, the RH-IEA method will serve as a powerful tool for detecting long-stranded RNA and will also shed light on the pathogen detection in food safety and molecular diagnosis.

Argonaute-DNAzyme tandem biosensing for highly sensitive and simultaneous dual-gene detection of methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA), a common zoonotic multidrug-resistant bacterium, puts a great threat to public health and food safety. Rapid and reliable detection of MRSA is crucial to guide effective patient treatment at early stages of infection and control the spread of MRSA infections. Herein, we developed a Simultaneous dual-gene and ulTra-sensitive detection for methicillin-resistant Staphylococcus aureus using Argonaute-DNAzyme tandem Detection (STAND). Simply, loop-mediated isothermal amplification (LAMP) was used for the amplification of the species-specific mecA and nuc gene, followed by STAND enabled by the site-specific cleavage of programable Argonaute. The Argonaute-DNAzyme tandem reaction rendered a conceptually novel signal amplification and transduction module that was more sensitive (1 or 2 order of magnitude higher) than the original Argonaute-based biosensing. With the strategy, the target nucleic acid signals gene were dexterously converted into fluorescent signals. STAND could detect the nuc gene and mecA gene simultaneously in a single reaction with 1 CFU/mL MRSA and a dynamic range from 1 to 108 CFU/mL. This method was confirmed by clinical samples and challenged by identifying contaminated foods and MRSA-infected animals. This work enriches the arsenal of Argonaute-mediated biosensing and presents a novel biosensing strategy to detect pathogenic bacteria with ultra-sensitivity, specificity and on-site capability.

Bi-directional nucleosome sliding by the Chd1 chromatin remodeler integrates intrinsic sequence-dependent and ATP-dependent nucleosome positioning.

Chromatin remodelers use a helicase-type ATPase motor to shift DNA around the histone core. Although not directly reading out the DNA sequence, some chromatin remodelers exhibit a sequence-dependent bias in nucleosome positioning, which presumably reflects properties of the DNA duplex. Here, we show how nucleosome positioning by the Chd1 remodeler is influenced by local DNA perturbations throughout the nucleosome footprint. Using site-specific DNA cleavage coupled with next-generation sequencing, we show that nucleosomes shifted by Chd1 can preferentially localize DNA perturbations - poly(dA:dT) tracts, DNA mismatches, and single-nucleotide insertions - about a helical turn outside the Chd1 motor domain binding site, super helix location 2 (SHL2). This phenomenon occurs with both the Widom 601 positioning sequence and the natural+1 nucleosome sequence from the Saccharomycescerevisiae SWH1 gene. Our modeling indicates that localization of DNA perturbations about a helical turn outward from SHL2 results from back-and-forth sliding due to remodeler action on both sides of the nucleosome. Our results also show that barrier effects from DNA perturbations can be extended by the strong phasing of nucleosome positioning sequences.

Successful full-length genomic cloning and characterization of site-specific nick structures of Phytophthora endornaviruses 2 and 3 in yeast, Saccharomyces cerevisiae.

Two endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3), have been discovered in pathogens targeting asparagus. In this study, we analyzed the nick structure in the RNA genomes of PEV2 and PEV3 in the host oomycetes. Northern blot hybridization using positive and negative strand-specific RNA probes targeting the 5' and 3' regions of PEV2 and PEV3 RNA genomes revealed approximately 1.0 kilobase (kb) RNA fragments located in the 5' regions of the two genomes. 3' RACE analysis determined that the size of the RNA fragments were 958 nucleotides (nt) for PEV2 and 968 nt for PEV3. We have successfully constructed full-length cDNA clones of the entire RNA genomes of PEV2 and PEV3 using a homologous recombination system in the yeast, Saccharomyces cerevisiae. These full-length cDNA sequences were ligated downstream of a constitutive expression promoter (TDH3) or a galactose-inducing promoter (GAL1) in the shuttle vector to enable the production of the full-length RNA transcripts of PEV2 and PEV3 in yeast cells. Interestingly, a 1.0 kb RNA fragment from the PEV3 positive-strand transcript was also detected with a 5'-region RNA probe, indicating that site-specific cleavage also occurred in yeast cells. Further, when PEV2 or PEV3 mRNA was overexpressed under the GAL1 promoter, yeast cell growth was suppressed. A fusion protein combining EGFP to the N-terminus of the full-length PEV2 ORF or C-terminus of the full-length PEV3 ORF was expressed, and allowed PEV2 and PEV3 ORFs to be successfully visualized in yeast cells. Expression of the fusion protein also revealed presence of heterogeneous bodies in the cells.