Specific DNA Cleavage Research Articles

A DNA sensor based on CbAgo effector protein and on a dual electrochemical signal amplification strategy for B19 parvovirus detection

Human parvovirus B19 is a prevalent childhood infectious virus that poses a great challenge to public health, so the detection of B19V is of great importance. In this study, a DNA sensor based on CbAgo, a Cas effector, and a dual electrochemical signal amplification strategy was developed by using a novel nanocomposite MnO2/CMK-3/g-C3N4/AgNPs for initial signal amplification, with CMK being an ordered mesoporous carbon nanomaterial. Single-walled carbon nanotubes (SWCNTs) were used as electrocatalytic probes for secondary signal amplification to detect B19 DNA. The detection process begins with polymerase chain reaction (PCR) amplification using the B19V infectious clone plasmid (pB19-M20) as a template and NS1-F/R as primers, followed by specific cleavage of B19 DNA based on the programmable cutting sites of CbAgo effector protein. This study enriches the application of Argonaute proteins in sensing and introduces a novel method to detect B19V. Under optimized conditions, the biosensor can detect B19 DNA in the range of 10−15–10−10 M, with a detection limit (LOD) of 0.2 fM. The results indicate that the developed DNA sensor holds promise for reliable and sensitive detection of B19 DNA in human serum.

Photocleavable Guide crRNAs for a Light-Controllable CRISPR/Cas9 System.

The design of controllable and precise RNA-targeted CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) systems is an important problem of modern molecular biology and genetic technology. Herein, we have designed a series of photocleavable guide CRISPR RNAs (crRNA) and their 2'-modified (2'-fluoro and locked nucleic acid) analogs containing one or two 1-(2-nitrophenyl)-1,2-ethanediol photolabile linkers (PL). We have demonstrated that these crRNAs can be destroyed by relatively mild UVA irradiation with the rate constants 0.24-0.77 min-1 and that the photocleavage markedly slows down the action of Cas9 nuclease in the model in vitro system. Two PLs provide more rapid crRNA destruction than a single linker. PLs in the crRNA structure improve the specificity of DNA cleavage by Cas9 nuclease for the fully complementary target. The application of photocleavable crRNA in CRISPR/Cas9 genome editing permits the system to be switched off in a spatiotemporally controlled manner, thus alleviating its off-target effects.

Development of an on-site real-time dual detection method for norovirus and rotavirus using RPA-CRISPR/Cas12,13

Norovirus (NoV) and rotavirus (RoV) are the two most prevalent foodborne viruses responsible for infectious gastroenteritis, with high contamination rates in fruits and vegetables. Monitoring and accurately identifying these pathogens are crucial for preventing human foodborne illnesses. Therefore, developing a rapid, highly specific, and sensitive detection method for NoV and RoV is essential. An on-site real time dual detection method for Noroviruses of genogroup I (GI-NoV) and Rotavirus of group A (A-RoV) was established by integrating Recombinase Polymerase Amplification (RPA) with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas) system. The DNA amplification products generated by multiplex RPA were simultaneously detected by Cas12a and Cas13a in a single tube. Utilizing the trans-cleavage preferences of Cas12a and Cas13a, the specific cleavage of DNA and RNA probes carrying different fluorescent groups within the reaction system was achieved. The results indicated that the established dual detection method had a detection limit of 102 copies/μL for GI-NoV and A-RoV. Specificity assay showed the established method only detected GI-NoV and A-RoV, with no cross-reactivity with other four foodborne viruses. Furthermore, the detection capability of this method was validated in strawberries and lettuce. The method enables the simultaneous detection of GI-NoV and A-RoV in fresh produce at 37 °C within 60 min. Detection results were interpreted by measuring fluorescence values or observing color changes under blue light at the end of the reaction. This technique required minimal instrumentation, provided excellent visual representation of results, and served as a technical reference for the rapid on-site detection of foodborne viruses in food matrices.

CRISPR-Cas9 target-strand nicking provides phage resistance by inhibiting replication.

Cas endonucleases, like Cas9 and Cas12a, are RNA-guided immune effectors that provide bacterial defense against bacteriophages. Cas endonucleases rely on divalent metal ions for their enzymatic activities and to facilitate conformational changes that are required for specific recognition and cleavage of target DNA. While Cas endonucleases typically produce double-strand breaks (DSBs) in DNA targets, reduced, physiologically relevant Mg2+ concentrations and target mismatches can result in incomplete second-strand cleavage, resulting in the production of a nicked DNA. It remains poorly understood whether nicking by Cas endonucleases is sufficient to provide protection against phage. To address this, we tested phage protection by Cas9 nickases, in which only one of two nuclease domains is catalytically active. By testing a large panel of guide RNAs, we find that target strand nicking can be sufficient to provide immunity, while non-target nicking does not provide any additional protection beyond Cas9 binding. Target-strand nicking inhibits phage replication and can reduce the susceptibility of Cas9 to viral escape when targeting non-essential regions of the genome. Cleavage of the non-target strand by the RuvC domain is strongly impaired at low Mg2+ concentrations. As a result, fluctuations in the concentration of other biomolecules that can compete for binding of free Mg2+ strongly influences the ability of Cas9 to form a DSB at targeted sites. Overall, our results suggest that Cas9 may only nick DNA during CRISPR-mediated immunity, especially under conditions of low Mg2+ availability in cells.

Mycobacterium smegmatis putative Holliday junction resolvases RuvC and RuvX play complementary roles in the processing of branched DNA structures

In eubacteria, Holliday junction (HJ) resolvases (HJRs) are crucial for faithful segregation of newly replicated chromosomes, homologous recombination and repair of stalled/collapsed DNA replication forks. However, compared with the Escherichia coli HJRs, little is known about their orthologs in mycobacterial species. A genome-wide analysis of Mycobacterium smegmatis identified two genes encoding putative HJRs, namely RuvC (MsRuvC) and RuvX (MsRuvX); but whether they play redundant, overlapping, or distinct roles remains unknown. Here, we reveal that MsRuvC exists as a homodimer while MsRuvX as a monomer in solution, and both showed high-binding affinity for branched DNAs compared with unbranched DNA species. Interestingly, the DNA cleavage specificities of MsRuvC and MsRuvX were found to be mutually exclusive: the former efficiently promotes HJ resolution, in a manner analogous to the E. coli RuvC, but does not cleave other branched DNA species; whereas the latter is a versatile DNase capable of cleaving a variety of branched DNA structures, including 3' and 5' flap DNA, splayed-arm DNA and double-stranded DNA with 3' and 5' overhangs but lacks the HJ resolution activity. Point mutations in the RNase H-like domains of MsRuvC and MsRuvX pinpointed critical residues required for their DNA cleavage activities, and also demonstrated uncoupling between DNA-binding and DNA cleavage activities. Unexpectedly, we found robust evidence that MsRuvX possesses a double-strand/single-strand junction-specific endonuclease and single-stranded DNA exonucleolytic activities. Combined, our findings highlight that the RuvC and RuvX DNases play distinct complementary, and not redundant, roles in the processing of branched DNA structures in M. smegmatis.

CRISPR Based Gene Knock Out Study in Western Clawed Frog

The advancement of biotechnology has contributed a revolutionary change in field of gene editing for the medical research and industrial biotechnology. There are different types of technology developed in recent era but the contributions of CRISPR/Cas 9(Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) has the major role for the advancement of gene editing[1]. Anciently it was known as acquired immunity that protects the bacteria from plasmid infection. It switches the bacterial immune system to eliminate the odd genetic material[2]. The technology has enormous potential to edit the genomes very accurate and efficient manner. It also provides various applications in the field of medical research. There are several elements involved in the CRISPR process involved in the process of gene editing. The developments of the process increased its potential for various model organisms. The major two components of the process are generally the sgRNA(Synthetic Single guide RNA) and the Cas 9[3]. The sgRNA is associated with genome specific side targeting and the Cas 9 has the ability to form the side specific DNA cleavage. There are several advantages of this technology over the disease like cystic fibrosis, sickle cell anemia, hemophilia and single nucleotide based genetic disorders[4]

ChEC-seq2: an improved chromatin endogenous cleavage sequencing method and bioinformatic analysis pipeline for mapping in vivo protein-DNA interactions.

Defining the in vivo DNA binding specificity of transcription factors (TFs) has relied nearly exclusively on chromatin immunoprecipitation (ChIP). While ChIP reveals TF binding patterns, its resolution is low. Higher resolution methods employing nucleases such as ChIP-exo, chromatin endogenous cleavage (ChEC-seq) and CUT&RUN resolve both TF occupancy and binding site protection. ChEC-seq, in which an endogenous TF is fused to micrococcal nuclease, requires neither fixation nor antibodies. However, the specificity of DNA cleavage during ChEC has been suggested to be lower than the specificity of the peaks identified by ChIP or ChIP-exo, perhaps reflecting non-specific binding of transcription factors to DNA. We have simplified the ChEC-seq protocol to minimize nuclease digestion while increasing the yield of cleaved DNA. ChEC-seq2 cleavage patterns were highly reproducible between replicates and with published ChEC-seq data. Combined with DoubleChEC, a new bioinformatic pipeline that removes non-specific cleavage sites, ChEC-seq2 identified high-confidence cleavage sites for three different yeast TFs that are strongly enriched for their known binding sites and adjacent to known target genes.

Development of the Cas12a-based microdeletion and microinsertion detection system

CRISPR/Cas-based systems are widely used as genome editing systems, nucleic acid detection systems and molecular visualization instruments [1]. In our laboratory using CRISPR/Cas9 technology we have obtained several KO mouse lines harboring deletions ranging from 2 to 20 base pairs. While 20 bp deletions are easily PCR-detected, when it comes to 2 bp deletions it is essential to genotype numerous mice using time-consuming Sanger sequencing. We propose a microdeletion/microinsertion detection system based on Cas12a nuclease fromLachnospiraceaebacterium (LbCas12a). Its active complex consists of the Cas12a enzyme and one crisprRNA [2]. A special sequence called protospacer adjacent motif (PAM) is required for target recognition by LbCas12a. In our laboratory we have discovered new PAM TTAA recognized by LbCas12a [3]. Via agarose electrophoresis and fluorescent analysis using FAM-labeled probes we have shown that new PAM allowed detection of 1 bp substitutions in target DNAin vitro. Also we have tested different FAM-labeled probes and have shown that AT-rich probes longer than 10 bp are cleaved most effectively. Finally we have used our system for detecting 2 bp deletions in Pde6b-KO mice and Grin3A-KO mice and successfully distinguished these mice from wild type mice.
 In conclusion, new PAM TTAA greatly increases specificity of DNA cleavage allowing to use this system as an instrument for rapid detection if microdeletions in mice.
 This work was supported by a Saint Petersburg State University grant for the development of scientific research (ID 94030690) and the Genome Research Centre development program “Kurchatov Genome Centre–PNPI” (agreement No. 075-15-2019-1663)

In-silicon studies on hydration in EcoRI-cognate DNA complex

Restriction endonucleases (REs) cleave DNA at specific site in presence of Mg2+ ion. Experiments further emphasize the role of hydration in metal ion specificity and sequence specificity of DNA cleavage. However, the relation between hydration and specificity has not been understood till date. This leads us to study via all-atom molecular dynamics (MD) simulations how the hydration around the scissile phosphate group changes in presence of Mg2+ and Ca2+ and depend on the DNA sequence. We observe the least number of hydrogen bonds around the scissile phosphate group in presence of Mg2+ ion. We further find that the hydrogen bonds decrease at the scissile phosphate on mutating one base pair in the cleavage region of the DNA in Mg2+ loaded EcoRI-DNA complex. We also perform steered MD simulations and observe that the rate of decrease of fraction of hydrogen bonds is slower in the mutated complex than the unmutated complex.

Targeting of CRISPR-Cas12a crRNAs into human mitochondria

Mitochondrial gene editing holds great promise as a therapeutic approach for mitochondrial diseases caused by mutations in the mitochondrial DNA (mtDNA). Current strategies focus on reducing mutant mtDNA heteroplasmy levels through targeted cleavage or base editing. However, the delivery of editing components into mitochondria remains a challenge. Here we investigate the import of CRISPR-Cas12a system guide RNAs (crRNAs) into human mitochondria and study the structural requirements for this process by northern blot analysis of RNA isolated from nucleases-treated mitoplasts. To investigate whether the fusion of crRNA with known RNA import determinants (MLS) improve its mitochondrial targeting, we added MLS hairpin structures at 3′-end of crRNA and demonstrated that this did not impact crRNA ability to program specific cleavage of DNA in lysate of human cells expressing AsCas12a nuclease. Surprisingly, mitochondrial localization of the fused crRNA molecules was not improved compared to non-modified version, indicating that structured scaffold domain of crRNA can probably function as MLS, assuring crRNA mitochondrial import. Then, we designed a series of crRNAs targeting different regions of mtDNA and demonstrated their ability to program specific cleavage of mtDNA fragments in cell lysate and their partial localization in mitochondrial matrix in human cells transfected with these RNA molecules. We hypothesize that mitochondrial import of crRNAs may depend on their secondary structure/sequence. We presume that imported crRNA allow reconstituting the active crRNA/Cas12a system in human mitochondria, which can contribute to the development of effective strategies for mitochondrial gene editing and potential future treatment of mitochondrial diseases.

Measuring site specific DNA cleavage at the single molecule level.

We study the DNA cleavage mechanisms of type II restriction endonucleases. In our single molecule technique micron sized beads tethered with single DNAs are imaged using video microscopy. Bead loss is used to determine the moment of cleavage of individual DNAs. In one experiment, we are using dCas9 as a programmable roadblock to 1D diffusion. Previously, we have shown the target search rate of NdeI depends strongly on the presence of roadblocks at low salt, but is insensitive to roadblocks at high salt. This is consistent with roadblocks blocking 1D diffusion at low salt. We are currently investigating the mechanism by which NdeI bypasses roadblocks at high salt. In other work, we are characterizing the cleavage kinetics of EcoRI under varying the conditions. Our data show that EcoRI activity exhibits complex dependencies on pH, protein, MgCl2 and NaCl. At low protein concentration and high salt the reaction appears to be limited by target search. At high protein and low salt, the rate limiting step is independent of protein concentration, pH and MgCl2 concentration. The nature of this rate limiting step is unknown, but presumably occurs after target site binding. Determining the nature of this step is a focus of current research.

Structure and function of DNA transposition assemblies involved in antibiotic resistance spreading

Transposons are mobile genetic elements that drive evolution and adaptation throughout the tree of life. In bacteria, they often transfer antibiotic resistance genes and contribute to the emergence of multidrug‐resistant pathogens.In this talk, I will present our recent discoveries on a group of transposons that efficiently propagate antibiotic resistance genes across diverse microbial communities. Using a dedicated computational pipeline, we mapped the most wide‐spread transposons in bacterial genomes and characterized their diversity, genetic cargo, and transfer dynamics. Selected elements were experimentally reconstituted to describe their molecular mechanisms and biochemical pathways. We determined numerous high‐resolution crystal and cryo‐EM structures of the protein‐DNA assemblies involved in transposon movement. The structures capture various stages of transposition and reveal an intricate interplay between the core transposition machinery and host‐encoded proteins. Protein interactions shape transposon and genomic DNA in unique ways to cut, exchange and re‐join strands in a tightly regulated manner. Importantly, our data also explain how specific DNA distortion and cleavage mechanisms enable the transposons to move between diverse genomic sites, expanding gene transfer across many species.These results shed new light onto the molecular strategies of antibiotic resistance carrying transposons and show how they evolved to effectively spread key genetic traits. Our insights also open new opportunities to develop strategies for blocking transposition and thereby help control resistance spreading.

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA.

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.

CRISPR-/Cas12a-Mediated Liposome-Amplified Strategy for the Surface-Enhanced Raman Scattering and Naked-Eye Detection of Nucleic Acid and Application to Food Authenticity Screening

Surface-enhanced Raman scattering (SERS) has been recognized as a powerful tool for biosensors due to the ultrahigh sensitivity and unique fingerprint information. However, there are some limitations in trace target nucleic acid detection for the restricted signal-transducing and amplification strategies. Inspired by CRISPR/Cas12a with specific target DNA-activated collateral single-strand DNA (ssDNA) cleavage activity and liposome with signal molecule-loading properties, we first proposed a sensitive SERS-based on-site nucleic acid detection strategy mediated by CRISPR/Cas12a with trans-cleavage activity on ssDNA linkers utilized to capture liposomes. Liposomes loading two kinds of signal molecules, 4-nitrothiophenol (4-NTP) and cysteine, could achieve the dual-mode detection of target DNA with SERS and naked eye, respectively. The promptly amplified signals were initiated by the triggered breakdown of signal molecule-loaded liposomes. Emancipated 4-NTP, a biological-silent Raman reporter, would achieve highly selective and sensitive SERS measurement. Released cysteine induced the aggregation of plasmonic gold nanoparticles, leading to an obvious red to blue colorimetric shift to realize portable naked-eye detection. With this strategy, target nucleic acid concentration was dexterously converted into SERS and visualization signals and could be detected as low as 100 aM and 10 pM, respectively. The approach was also successfully applied to determine meat adulteration, achieving the detection of a low adulteration ratio in the complicated food matrix. We anticipate that this strategy will not only be regarded as a universal platform for the on-site detection of food authenticity but also broaden SERS application for the accurate determination of diverse biomarkers.

Production of Universal Anti-Bcma CAR-T Cells with Reduced Alloreactivity, but Potent Effector Function for the Treatment of Multiple Myeloma

Chimeric antigen receptor (CAR)-T therapy has recently shown great promise against multiple myeloma (MM) by targeting the B cell maturation antigen (BCMA). However, the production of autologous CAR-T cells on an individualized basis has significant challenges such as manufacture time, reproducibility, consistency and cost. In addition, viral-vector-produced CAR-T cells tend to be comprised of differentiated T cell subsets, which correlate with poor in vivo durability. Using the high-fidelity NextGEN™ (NG) CRISPR gene editing system in combination with the non-viral piggyBac™ (PB) DNA transposition technology, which produces CAR-T cells predominantly in a highly desirable stem cell memory T cell subset, we have developed P-BCMA-ALLO1, an off-the-shelf allogeneic CAR-T cell product that overcomes these limitations and can be given to any MM patient.The NG system was used to disrupt both TCR and MHCI expression in P-BCMA-ALLO1 to reduce possible alloreactivities. The DNA cleavage specificity is guaranteed by a truly dimeric system requiring the simultaneous binding of two obligate homodimers. High-throughput DNA sequencing analysis was performed on human T cells with gene disruption of TCRα, β2M or PD-1. Indel frequencies of amplified genomic on-target and off-target sites were evaluated from T cells that had been edited with either single or dual guide RNAs (gRNA)s. Herein, an average of 100,000 sequences were analyzed per site and non-edited T cells were included as a background reference. For these targets, the NG system demonstrated high-efficiency gene editing (>84% for TCRα, >64% for β2M, and >50% for PD-1) that was performed in resting T cells. No indel events were detected at any off-target site, proving the exquisite specificity of this system.P-BCMA-ALLO1 was incapable of mediating allo-responses such as graft-vs-host disease (GvHD) and graft rejection. Using a clinically-scalable purification method, P-BCMA-ALLO1 cells were enriched to be >99.5% TCR/MHCI double-negative and alloreactivities were evaluated by standard mixed lymphocyte reaction for either cell proliferation or IFNγ production in two separate assays: a 7-12 day CFSE-based cell proliferation assay and a 20-hour IFNγ-ELISPOT assay. Compared to non-edited CAR-T cells, P-BCMA-ALLO1 did not exhibit any detectable response to allogeneic PBMC stimulation, nor did it trigger any allogeneic T cell proliferative response above background level of cell proliferation or IFNγ production.Pre-clinical evaluation of P-BCMA-ALLO1 demonstrated optimal T cell phenotype and potent in vitro and in vivo efficacy. More than 95% of the P-BCMA-ALLO1 cells were CAR+ and greater than 50% of the product were CD45RA+CD62L+ stem cell memory T cells (TSCM), a highly desirable early memory T cell subset that exhibits the greatest capacity for self-renewal and the multipotent capacity to the entire spectrum of memory and effector T cell subsets. In addition, the CAR-T cells did not express the exhaustion markers PD-1, TIM-3 and LAG-3, nor did they exhibit any evidence of tonic signaling as IFNγ production in the absence of target antigen. In a short-term 24-hour in vitro co-culture assay with BCMA+ MM cell lines, P-BCMA-ALLO1 secreted high levels of IFNγ and mediated robust (70-90%) killing of cell targets. In addition, more than 80% of the cells proliferated in a longer 72-hour CFSE-based proliferation assay. P-BCMA-ALLO1 also showed strong potency in a NSG mouse model engrafted with MM.1S cells, an aggressive human MM-derived tumor model. While all control animals showed rapid tumor growth by bioluminescent imaging (BLI) and succumbed to disease within five weeks, all treated mice survived for the duration of the study. After P-BCMA-ALLO1 administration, tumor burden was rapidly reduced to the limit of detection by BLI within 7-10 days. Lastly, several occurrences of tumor relapse were observed in some of these animals, but were subsequently controlled without re-administration of the product. Altogether, these data demonstrate that P-BCMA-ALLO1 exhibited significantly reduced alloreactivity yet retained potent effector function, suggesting its safe and effective use for treating any MM patient. DisclosuresWang:Poseida Therapuetics: Employment. Barnett:Poseida Therapeutics, Inc.: Employment. Martin:Poseida Therapeutics, Inc.: Employment. Hermanson:Poseida Therapeutics: Employment. Li:Poseida Therapuetics: Employment. Smith:Poseida Therapeutics, Inc.: Employment. Wu:Poseida Therapuetics: Employment. Cranert:Poseida Therapuetics: Employment. Rengarajan:Poseida Therapeutics, Inc.: Employment. Tan:Poseida Therapeutics, Inc.: Employment. Tong:Poseida Therapuetics: Employment. Codde:Poseida Therapeutics, Inc.: Employment. Down:Poseida Therapeutics, Inc.: Employment. Ostertag:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Shedlock:Poseida Therapeutics, Inc.: Employment.

An extensive review to facilitate understanding of CRISPR technology as a gene editing possibility for enhanced therapeutic applications

CRISPR are the sequences in bacterial and archaeal genome which provide resistance against viral infections. They might be the natural part of bacterial genomes for providing protection against viruses like bacteriophages but science has successfully achieved their use in the benefit of man-kind by using them for the treatment of deadly diseases like cancer, AIDS or genetic disorders like sickle cell disease and Leber congenital amaurosis. CRISPR system is majorly divided into two classes i.e class I and class II, of which the class II CRISPR/Cas9 system performs site specific cleavage of DNA with a guide RNA Cas12 (Cpf1). With the new emerging discoveries it is being found that CRISPR not only works on double stranded DNA but can also be useful to induce any sort of site specific cleavage in RNA too by Cas13 earlier known as C2c2, which is a protein found in CRISPR system and has ability to cure viral infections in plants. CRISPR is being used in the field of gene manipulation and various animals models are available to serve this purpose with short lifespan, rapid reproducibility and lower maintenance cost. Many successful studies and experiments performed using CRISPR, reveals their potency and utility to bring revolution in the areas which were previously believed to be out of scope of science and medicine.

DNA-cleavage activity of the iron(II) complex with optically active ligands, meta- and para-xylyl-linked N’,N’-dipyridylmethyl-cyclohexane-1,2-diamine

DNA-cleavage agents such as bleomycin have potential anticancer applications. The development of a DNA-cleavage reagent that recognizes specific sequences allows the development of cancer therapy with reduced side effects. In this study, to develop novel compounds with specific DNA-cleavage activities, we synthesized optically active binuclear ligands, (1R,1′R,2R,2′R)-N1,N1′-(meta/para-phenylenebis(methylene))bis(N2,N2-bis(pyridin-2-ylmethyl)cyclohexane-1,2-diamine) and their enantiomers. The DNA-cleavage activities of these compounds were investigated in the presence of Fe(II)SO4 and sodium ascorbate. The obtained results indicated that the Fe(II) complexes of those compounds efficiently cleave DNA and that their cleavage was subtle sequence-selective. Therefore, we succeeded in developing compounds that can be used as small-molecule drugs for cancer chemotherapy.

Biochemical and molecular characterization of a restriction endonuclease Tvu2HI from Thermoactinomyces vulgaris 2H and study of its R-M system

A bacterial strain 2H isolated from soil and identified as Thermoactinomyces vulgaris produce a potent Type II restriction endonuclease activity that has been extracted by a PEG/dextran aqueous two-phase system. Optimal temperature for the restriction endonuclease activity was 55–65°C. Specific DNA cleavage was obtained at pH range 7–10 and 10–20mM MgCl2. Restriction cleavage analysis followed by sequencing confirms GG^CC as the recognition sequence. This enzyme, named Tvu2HI, is a thermostable isoschizomer of the mesophilic prototype restriction endonuclease HaeIII. Sequencing of the complete Thermoactinomyces vulgaris 2H genome revealed the presence of two adjacent ORFs coding for the restriction endonuclease Tvu2HI and the corresponding methyltransferase; an ORF coding for a putative Vsr nicking enzyme was found close to those coding for the Tvu2HI restriction-modification system. Phylogenetic analysis based on sequence alignment suggests a common origin of Tvu2HI R-M system with HaeIII-like R-M systems. This is the first investigation dealing with a Type II restriction endonuclease identified in a natural isolate of the genus Thermoactinomyces.

Contacts and Confirmations that Regulate Hexameric Helicases at the Replication Fork

Duplication of the genome requires efficient, rapid, and coordinated activities of many enzymes that interact within a larger replisome complex. The hexameric helicase is at the leading front of the replisome and is a nexus of control for both the initiation and elongation phases of DNA replication. These proteins are comprised of a N‐terminal DNA binding domain and a C‐terminal motor domain that assemble into a toroidal hexameric structure. Two main superfamilies of hexameric helicases exist: the RecA family, which contain helicases from phage and bacteria, and the AAA+ family, which contain helicases from archaea and eukaryotes. Interestingly, the measured translocation polarity of 5′‐3′ or 3′‐5′, respectively, dictates the orientation of these helicases at the replication fork.Using site specific DNA cleavage experiments and presteady‐state FRET, we have unequivocally shown the then N‐terminal domain of the archaeal MCM helicase leads the way for unwinding (N‐first). The MCM hexamer itself can bind model DNA substrates in multiple conformations; however, the correct ‘N‐first’ conformation is dictated by 1) the presence of an excluded strand, 2) the ssDNA polarity (3′‐5′), and 3) the C‐terminal winged‐helix (WH). This orientation is opposite to that of the bacterial DnaB helicase which proceeds C‐first in the 5′‐3′ direction.To determine the cellular impact of the excluded strand interaction in the steric exclusion and unwinding model (SEW), we created and tested several CRISPR/Cas9 genetic mutations of E. coli dnaB that correlate with mutations on the exterior surface. These DnaB mutations alter the conformation of the hexamer creating a spectrum of constricted states that all result in faster DNA unwinding. Genetic dnaB mutations lead to decoupling within the replisome, buildup of ssDNA, activation of the SOS response, and loss of genomic integrity. Therefore, the SEW interaction and the central channel diameter is adaptive and dynamic to properly maintain the DNA unwinding within the confines of the replisome to regulate speed of DNA replication and prevent chromosomal instability.Support or Funding InformationNational Science Foundation Division of Molecular and Cellular Biosciences (NSF1613534)

Using Single Molecule Methods to Study Mechanisms of Site Specific DNA Cleavage

Using Single Molecule Methods to Study Mechanisms of Site Specific DNA Cleavage