Mechanism Of DNA Cleavage Research Articles

Structural insights into the diversity and DNA cleavage mechanism of Fanzor

Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts.

An Insight into the Mechanism of DNA Cleavage by DNA Endonuclease from the Hyperthermophilic Archaeon Pyrococcus furiosus.

Hyperthermophilic archaea such as Pyrococcus furiosus survive under very aggressive environmental conditions by occupying niches inaccessible to representatives of other domains of life. The ability to survive such severe living conditions must be ensured by extraordinarily efficient mechanisms of DNA processing, including repair. Therefore, in this study, we compared kinetics of conformational changes of DNA Endonuclease Q from P. furiosus during its interaction with various DNA substrates containing an analog of an apurinic/apyrimidinic site (F-site), hypoxanthine, uracil, 5,6-dihydrouracil, the α-anomer of adenosine, or 1,N6-ethenoadenosine. Our examination of DNA cleavage activity and fluorescence time courses characterizing conformational changes of the dye-labeled DNA substrates during the interaction with EndoQ revealed that the enzyme induces multiple conformational changes of DNA in the course of binding. Moreover, the obtained data suggested that the formation of the enzyme-substrate complex can proceed through dissimilar kinetic pathways, resulting in different types of DNA conformational changes, which probably allow the enzyme to perform its biological function at an extreme temperature.

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.

Two-metal ion mechanism of DNA cleavage by activated, filamentous SgrAI

Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-electron microscopy (cryo-EM). The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of filamentation induced conformational changes.

A Data Mining Study of Zinc Oxide Nanostructures Utilization in Drug Delivery Nanosystems

AbstractThe present investigation offers a thorough bibliometric exploration spotlighting the pivotal utilization of zinc oxide nanostructures, encompassing both zero‐dimensional (0D) nanoparticles and one‐dimensional (1D) nanostructures, within the realm of nanotechnology‐driven drug delivery systems. This analysis particularly illuminates the distinctive potential of these nanostructures in the cancer therapy context, not merely as carriers and deliverers of pharmaceutical agents, but as entities capable of selectively inducing apoptosis in cancer cells through the orchestrated generation of reactive oxygen species (ROS). Recent research endeavors notably underscore the application prospects of ZnO nanostructures in domains like DNA cleavage, bioimaging, and agricultural defense mechanisms. Moreover, the study elucidates the pronounced enhancement in solubility and biocompatibility that these nanostructures bring about when harmoniously integrated into polymeric nanocomposites. Concomitantly, the involvement of polymers in this symbiotic system is unveiled, wherein they play a multifaceted role in dictating release selectivity, thereby facilitating targeted and efficacious interactions with tissues. Functionally engineered polymeric nanocomposites emerge as promising candidates for targeted delivery modalities in cancer treatment, demonstrating an affinity for specific cell receptors and exhibiting enhanced cellular uptake within the size range of 100–200 nm. The study unearths robust associations among pivotal research terminologies through an exhaustive analysis of Link Strength Between Items (LSBI), culminating in the presentation of a cogent map delineating the evolving trends and forthcoming trajectories in this dynamic domain. In summation, this all‐encompassing bibliometric inquiry serves as a poignant testament to the prodigious potential of zinc oxide nanostructures in the realm of forthcoming nanotechnology‐driven drug delivery paradigms.

DNA Interaction and Cleavage Modes of Anthraquinone-Fused Enediynes: A Study on Tiancimycins, Yangpumicins, and Their Semisynthetic Analogues.

Dynemicin A has been the sole prototypical anthraquinone-fused enediyne (AFE) explored since its discovery in 1989. This study investigates the distinct DNA binding and cleavage mechanisms of emerging AFEs, represented by tiancimycins and yangpumicins, along with semisynthetic analogues. Our findings reveal their potent cytotoxicity against various tumor cell lines, while 18-methoxy tiancimycin A treatment could significantly suppress breast tumor growth with minimal toxicity. One of the most potent AFEs, i.e., tiancimycin A, preferentially targets DNA sequences 5'-ATT, 5'-CTT, 5'-GAA, 5'-GAT, and 5'-TTA. Molecular dynamics simulations suggest that emerging AFEs intercalate deeper into AT-rich DNA base pairs compared to dynemicin A. Importantly, tiancimycin A may equilibrate between insertional and intercalative modes without deintercalation, enabling selective cleavage of T and A bases. This study underscores how subtle structural variations among AFEs significantly influence their DNA recognition and cleavage, facilitating future design of novel AFEs as potent and selective payloads for antibody-drug conjugates.

Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications

Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering.

ÜÇ YENİ BİS(AÇİLHİDRAZON) MONONÜKLEER GEÇIŞ METALI KOMPLEKSİ: SENTEZ, KARAKTERİZASYON VE DNA ETKİLEŞİM ÇALIŞMALARI

In recent years, studies on the design and synthesis of artificial nucleases with biological, structural and coordination properties have been increasing. With this study, mononuclear complex compounds were obtained with Cu(II), Ni(II) and Zn(II) transition metal ion salts of bis(acylhydrazone) ligand, which has the potential to show biological activity and has revolving atoms such as N and O in its structure. The characteristic structures of the obtained complex compounds were elucidated using various techniques such as FTIR, UV-Visible, 1H-NMR, TGA, elemental analysis and magnetic susceptibility. The DNA-binding activities of potential artificial metallonucleases, whose structures were elucidated, were investigated using the UV-visible absorption titration method. Additionally, their DNA-cleavge activities were analyzed using the agarose gel electrophoresis method. The DNA binding mode and cleavage mechanisms were determined as part of this study.. In line with the results obtained, it was determined that the binding mode between DNA and the complexes is a noncovalent interaction. It has been demonstrated that the compounds cleavage DNA both oxidatively and hydrolytically. It has been revealed that the radicals involved in DNA cleavage activity are H2O2, superoxide and hydroxyl radicals. It was revealed that the compound with the highest cleavage and bonding interaction was the Cu(II) complex, followed by Ni(II) and Zn(II) complexes.

Fluorescence Polarization Assay Based on a New Recognition Motif QepA for the One-Step Detection of Fluoroquinolones in Eggs.

A recognition motif is vital in determining the specificity and sensitivity of the fluorescence polarization assay (FPA) for detecting chemical contaminants in food. Four candidates (Gyrase, GyrBA, TopIV, and QepA) were prepared for this study. The applicability of QepA was confirmed through DNA cleavage assay, inhibition effects, and mechanism investigations using molecular docking, compared to other counterparts. Finally, a novel FPA based on QepA and a CIP-FITC tracer for the detection of fluoroquinolones (FQs) in eggs was developed. The limits of detection (LODs) for eight fluoroquinolones ranged from 2.2 to 5.1 ng g-1, with enrofloxacin, danofloxacin, and difloxacin meeting the maximum residue limits (MRLs). The spiked recoveries ranged from 65.8 to 103.6% with coefficients of variation (CVs) of 5.4-12.8%. Therefore, a new recognition motif for FQs that did not belong to conventional antibodies was identified, and QepA-based FPA could be a potential tool for rapid, homogeneous, and sensitive monitoring of the residue of FQs in eggs.

The crystal structures of Sau3AI with and without bound DNA suggest a self-activation-based DNA cleavage mechanism

The type II restriction endonuclease Sau3AI cleaves the sequence 5'-GATC-3' in double-strand DNA producing two sticky ends. Sau3AI cuts both DNA strands regardless of methylation status. Here, we report the crystal structures of the active site mutant Sau3AI-E64A and the C-terminal domain Sau3AI-C with a bound GATC substrate. Interestingly, the catalytic site of the N-terminal domain (Sau3AI-N) is spatially blocked by the C-terminal domain, suggesting a potential self-inhibition of the enzyme. Interruption of Sau3AI-C binding to substrate DNA disrupts Sau3AI function, suggesting a functional linkage between the N- and C-terminal domains. We propose that Sau3AI-C behaves as an allosteric effector binding one GATC substrate, which triggers a conformational change to open the N-terminal catalytic site, resulting in the subsequent GATC recognition by Sau3AI-N and cleavage of the second GATC site. Our data indicate that Sau3AI and UbaLAI might represent a new subclass of type IIE restriction enzymes.

Functional copper complexes with benzofurans tridentate ligand: Synthesis, crystal structure, DNA binding and anticancer studies

Metal complexes, particularly copper(II) complexes, are often used as anticancer drugs due to their ability to generate reactive oxygen species (ROS) in cells. Four copper(II) complexes have been designed based on ligands for triplet pyridine derivatives (complexes 1–4), and their structures have been determined using X-ray single crystal analysis. The interactions of these complexes with calf thymus DNA (CT-DNA) have been investigated using various techniques, including UV–vis absorption, viscosity measurements, and circular dichroism spectroscopy. The results indicate that complexes 1–4 strongly interact with DNA through partial intercalations. Further investigation using agarose gel electrophoresis shows that all four complexes can cleave pBR322 DNA in the presence of ascorbic acid as a reducing agent, and the DNA cleavage mechanism is through the generation of singlet oxygen (1O2). In vitro anticancer activities of these complexes have been evaluated using A549, MDA-MB-231, HeLa, and HepG2 cells. The calculated IC50 values indicate significant efficacy against cancer cells. Additionally, AO/EB staining assays reveal that these complexes induce cell apoptosis in HeLa cell line.

Synthesis and DNA Cleavage Activity of Cationic Methylene-Blue-Backboned Polymers.

Methods of DNA cleavage have broad bioapplications in gene editing, disease treatment, and biosensor design. The traditional method for DNA cleavage is mainly through oxidation or hydrolysis mediated by small molecules or transition metal complexes. However, DNA cleavage by artificial nucleases using organic polymers has been rarely reported. Methylene blue has been extensively studied in the fields of biomedicine and biosensing due to its excellent singlet oxygen yield, redox properties, and good DNA affinity. Methylene blue mainly relies on light and oxygen for DNA cleavage, and the cutting rate is slow. Here, we synthesize cationic methylene-blue-backboned polymers (MBPs) that can bind DNA efficiently and induce DNA cleavage through free radical mechanisms in the absence of light and exogenous reagents, showing high-efficiency nuclease activity. In addition, MBPs with different structures showed selectivity for DNA cleavage, and the cleavage efficiency of the flexible structure was significantly higher than that of the rigid structure. Studies on the DNA cleavage mechanism have shown that the cleavage mechanism of MBPs is not through the common ROS-mediated oxidative cleavage pathway, but through the radical of MBP• inducing DNA cleavage. Meanwhile, MBPs can simulate topoisomerase I (Topo I)-mediated topological rearrangement of superhelical DNA. This work paved a way for the application of MBPs in the field of artificial nucleases.

TnpB structure reveals minimal functional core of Cas12 nuclease family.

The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells1,2. Bioinformatic analysis identified TnpB proteins as the likely predecessors of Cas12 nucleases3-5, which along with Cas9 are widely used for targeted genome manipulation. Whereas Cas12 family nucleases are well characterized both biochemically and structurally6, the molecular mechanism of TnpB remains unknown. Here we present the cryogenic-electron microscopy structures of the Deinococcus radiodurans TnpB-reRNA (right-end transposon element-derived RNA) complex in DNA-bound and -free forms. The structures reveal the basic architecture of TnpB nuclease and the molecular mechanism for DNA target recognition and cleavage that is supported by biochemical experiments. Collectively, these results demonstrate that TnpB represents the minimal structural and functional core of the Cas12 protein family and provide a framework for developing TnpB-based genome editing tools.

Cryo-EM structure of the transposon-associated TnpB enzyme

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR–Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA–target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR–Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR–Cas12 effectors.

Single-molecule fluorescence assays for studying the mechanism of DNA cleavage by CRISPR-Cas12a

Single-molecule fluorescence assays for studying the mechanism of DNA cleavage by CRISPR-Cas12a

Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.

Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.

What's on the Other Side of the Gate: A Structural Perspective on DNA Gate Opening of Type IA and IIA DNA Topoisomerases.

DNA topoisomerases have an essential role in resolving topological problems that arise due to the double-helical structure of DNA. They can recognise DNA topology and catalyse diverse topological reactions by cutting and re-joining DNA ends. Type IA and IIA topoisomerases, which work by strand passage mechanisms, share catalytic domains for DNA binding and cleavage. Structural information has accumulated over the past decades, shedding light on the mechanisms of DNA cleavage and re-ligation. However, the structural rearrangements required for DNA-gate opening and strand transfer remain elusive, in particular for the type IA topoisomerases. In this review, we compare the structural similarities between the type IIA and type IA topoisomerases. The conformational changes that lead to the opening of the DNA-gate and strand passage, as well as allosteric regulation, are discussed, with a focus on the remaining questions about the mechanism of type IA topoisomerases.

The electronic structure of genome editors from the first principles

Ab-initio molecular dynamics enables following the dynamics of biological systems from the first principles, describing the electronic structure and offering the opportunity to “watch” the evolution of biochemical processes with unique resolution, beyond the capabilities of state-of-the-art experimental techniques. This article reports the role of first-principles (ab-initio) molecular dynamics (MD) in the CRISPR-Cas9 genome editing revolution, achieving a profound understanding of the enzymatic function and offering valuable insights for enzyme engineering. We introduce the methodologies and explain the use of ab-initio MD simulations to establish the two-metal dependent mechanism of DNA cleavage in the RuvC domain of the Cas9 enzyme, and how a second catalytic domain, HNH, cleaves the target DNA with the aid of a single metal ion. A detailed description of how ab-initio MD is combined with free-energy methods—i.e., thermodynamic integration and metadynamics—to break and form chemical bonds is given, explaining the use of these methods to determine the chemical landscape and establish the catalytic mechanism in CRISPR-Cas9. The critical role of classical methods is also discussed, explaining theory and application of constant pH MD simulations, used to accurately predict the catalytic residues’ protonation states. Overall, first-principles methods are shown to unravel the electronic structure and reveal the catalytic mechanism of the Cas9 enzyme, providing valuable insights that can serve for the design of genome editing tools with improved catalytic efficiency or controllable activity.

Theory and computation early career award talk: unraveling the CRISPR-Cas revolution through the lens of computational biophysics.

The clustered regularly interspaced short palindromic repeat (CRISPR) genome-editing revolution established the beginning of a new era in life sciences. I will report the role of state-of-the-art computations in the CRISPR-Cas9 revolution, from the early refinement of cryo-EM data to enhanced simulations of large-scale conformational transitions. Molecular simulations reported a mechanism for RNA binding and the formation of a catalytically competent Cas9 enzyme, corroborated by recent cryo-EM data of the activated state. Inspired by single-molecule experiments, molecular dynamics offered a rationale for the onset of off-target effects, while graph theory unveiled the allosteric regulation. Finally, the use of a mixed quantum-classical approach established the catalytic mechanism of DNA cleavage. Overall, molecular simulations have been instrumental in understanding the dynamics and mechanism of CRISPR-Cas9, contributing to understanding function, catalysis, allostery, and specificity. Future computational studies will bridge the scales from quantum mechanical approaches to enhanced simulations and cryo-EM processing methods to push the boundaries of molecular simulations and tackle the most exciting problems in the biophysics of genome editing.

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.