Orientation-Guided Immobilization of Probe DNA on swCNT-FET for Enhancing Sensitivity of EcoRV Detection.

We present a novel approach that integrates electrical measurements with molecular dynamics (MD) simulations to assess the activity of type-II restriction endonucleases, specifically EcoRV. Our approach employs a single-walled carbon nanotube field-effect transistor (swCNT-FET) functionalized with the EcoRV substrate DNA, enabling the detection of enzymatic cleavage events. Notably, we leveraged the methylene blue (MB) tag as an "orientation guide" to immobilize the EcoRV substrate DNA in a specific direction, thereby enhancing the proximity of the DNA cleavage reaction to the swCNT surface and consequently improving the sensitivity in EcoRV detection. We conducted computational modeling to compare the conformations and electrostatic potential (ESP) of MB-tagged DNA with its MB-free counterpart, providing strong support for our electrical measurements. Both conformational and ESP simulations exhibited robust agreement with our experimental data. The inhibitory efficacy of the EcoRV inhibitor aurintricarboxylic acid (ATA) was also evaluated, and the selectivity of the sensing device was examined.

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.

smFRET Detection of Cis and Trans DNA Interactions by the BfiI Restriction Endonuclease.

Protein-DNA interactions are fundamental to many biological processes. Proteins must find their target site on a DNA molecule to perform their function, and mechanisms for target search differ across proteins. Especially challenging phenomena to monitor and understand are transient binding events that occur across two DNA target sites, whether occurring in cis or trans. Type IIS restriction endonucleases rely on such interactions. They play a crucial role in safeguarding bacteria against foreign DNA, including viral genetic material. BfiI, a type IIS restriction endonuclease, acts upon a specific asymmetric sequence, 5-ACTGGG-3, and precisely cuts both upper and lower DNA strands at fixed locations downstream of this sequence. Here, we present two single-molecule Förster resonance energy-transfer-based assays to study such interactions in a BfiI-DNA system. The first assay focuses on DNA looping, detecting both "Phi"- and "U"-shaped DNA looping events. The second assay only allows in trans BfiI-target DNA interactions, improving the specificity and reducing the limits on observation time. With total internal reflection fluorescence microscopy, we directly observe on- and off-target binding events and characterize BfiI binding events. Our results show that BfiI binds longer to target sites and that BfiI rarely changes conformations during binding. This newly developed assay could be employed for other DNA-interacting proteins that bind two targets and for the dsDNA substrate BfiI-PAINT, a useful strategy for DNA stretch assays and other super-resolution fluorescence microscopy studies.

The type IIS restriction enzyme MmeI can cut across a double-strand break.

BACKGROUND: Type-IIS restriction enzymes cut outside their recognition sites, allowing them to remove their binding sites upon digestion. This feature has resulted in their wide application in molecular biology techniques, including seamless cloning methods, enzymatic CRISPR library generation, and others. We studied the ability of the Type-IIS restriction enzyme MmeI, which recognizes an asymmetric sequence TCCRAC and cuts 20 bp downstream, to cut across a double-strand break (DSB). METHODS AND RESULTS: We used synthetic double-stranded oligos with MmeI recognition sites close to 5' end and different overhang lengths to measure digestion after different periods of time and at different temperatures. We found that the MmeI binding and cutting sites can be situated on opposite sides of a DSB if the edges of the DNA molecules are held together by transient base-pairing interactions between compatible overhangs. CONCLUSION: We found that MmeI can cut across a DSB, and the efficiency of the cutting depends on both overhang length and temperature.

Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI.

Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.

Structure-Function Studies of Two Yeast Homing Endonucleases that Evolved to Cleave Identical Targets with Dissimilar Rates and Specificities.

The LAGLIDADG family of homing endonucleases (LHEs) bind to and cleave their DNA recognition sequences with high specificity. Much of our understanding for how these proteins evolve their specificities has come from studying LHE homologues. To gain insight into the molecular basis of LHE specificity, we characterized I-WcaI, the homologue of the Saccharomyces cerevisiae I-SceI LHE found in Wickerhamomyces canadensis. Although I-WcaI and I-SceI cleave the same recognition sequence, expression of I-WcaI, but not I-SceI, is toxic in bacteria. Toxicity suppressing mutations frequently occur at I-WcaI residues critical for activity and I-WcaI cleaves many more non-cognate sequences in the Escherichia coli genome than I-SceI, suggesting I-WcaI endonuclease activity is the basis of toxicity. In vitro, I-WcaI is a more active and a less specific endonuclease than I-SceI, again accounting for the observed toxicity in vivo. We determined the X-ray crystal structure of I-WcaI bound to its cognate target site and found that I-WcaI and I-SceI use residues at different positions to make similar base-specific contacts. Furthermore, in some regions of the DNA interface where I-WcaI specificity is lower, the protein makes fewer DNA contacts than I-SceI. Taken together, these findings demonstrate the plastic nature of LHE site recognition and suggest that I-WcaI and I-SceI are situated at different points in their evolutionary pathways towards acquiring target site specificity.

Quantum biology.π-πentanglement signatures in protein-DNA interactions.

The biological functions of DNA are carried out by individual proteins that interact with specific sequences along the DNA in order to prime the molecular processes required by the cellular metabolism. Protein-DNA interactions include DNA replication, gene expression and its regulation, DNA repair, DNA restriction and modification by endonucleases, generally classified as enzymatic functions, or transcription factors functions. To find specific binding target sequences and achieve their aims, in less than one second proteins operate in symbiosis with a crowded cellular environment, identifying extremely small cognate sequences along the DNA chain, which range from 15-20 bps for repressors to 4-6 bps for restriction enzymes. In a previous work, we proposed that the extraordinary ability of proteins to identify consensus sequences on DNA in a short time appears to be dependent on specific quantum signatures such as the entanglement ofπ-πelectrons between DNA nucleotides and protein amino acids, where the couple ofπelectrons function as a radical pair, oneπelectron is located on a specific site of sequence to be identified and the other one performs a quantum walk to identify possible sites of consensus sequence. In this paper, we use the restriction endonucleases enzymes, EcoRV and EcoRI as a case study. These enzymes are able to recognize 3'-GATACT-5' or 3'-GAATCT-5' sequences, respectively. We exploit the analogy of a coin operator with a Bloch sphere to demonstrate that the entanglement betweenπ-πelectrons generated at the contacts on specific GA dimers between proteins and DNA relies on the spin of the electrons that form an initial singlet state. The latter is a maximally entangled state so that the identification of specific nucleotides is associated with the formation of singlet states. On the other hand, during the identification of subsequent GA dimers, the spin-orbit interaction on walkingπelectron induces triplet transitions so that singlet-triplet transitions should manifest an experimentally measurable effect. We propose that the possible experimental evidence of entanglement betweenπ-πelectrons may be due to the phosphorescence signal correspondence to triplet decay processes.

Pretransition state and apo structures of the filament-forming enzyme SgrAI elucidate mechanisms of activation and substrate specificity.

Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both accelerates its DNA cleavage activity and expands its DNA sequence specificity, thus allowing for many additional DNA sequences to be rapidly cleaved. Both outcomes-the acceleration of DNA cleavage and the expansion of sequence specificity-are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca2+ resolved to ∼2.5 Å within the catalytic center, which represents the trapped enzyme-DNA complex prior to the DNA cleavage reaction. This structure reveals important conformational changes that contribute to the catalytic mechanism and the binding of a second divalent cation in the enzyme active site, which is expected to contribute to increased DNA cleavage activity of SgrAI in the filamentous state. Second, we present an X-ray crystal structure of DNA-free (apo) SgrAI resolved to 2.0 Å resolution, which reveals a disordered loop involved in DNA recognition. Collectively, these multiple new observations clarify the mechanism of expansion of DNA sequence specificity of SgrAI, including the indirect readout of sequence-dependent DNA structure, changes in protein-DNA interactions, and the disorder-to-order transition of a crucial DNA recognition element.

Defining genome-wide CRISPR-Cas genome-editing nuclease activity with GUIDE-seq.

Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.

The VDR gene confers a genetic predisposition to Graves' disease and Graves' ophthalmopathy in the Southwest Chinese Han population.

OBJECTIVE: Graves' disease (GD) is a common autoimmune disease manifesting with diffuse symmetric thyroid gland enlargement, pretibial myxedema, and Graves' ophthalmopathy (GO). Recently, the vitamin D receptor (VDR) gene has been linked to various autoimmune diseases. This study aimed to investigate the association of VDR gene polymorphisms with susceptibility to GD and GO in the Southwest Chinese Han population. METHODS: A two-stage association study was performed in 1,209 controls and 650 GD patients by PCR-RFLP assay. Real-time PCR and ELISA were carried out to quantify gene expression and cytokine production. RESULTS: The first-stage study showed that the frequency of VDR/Apa I AA genotype was significantly increased in GD (Pc = 1.67 × 10-2, OR = 1.98). The second-stage and combined studies confirmed the association of VDR/Apa I with GD (AA genotype: Pc = 3.45 × 10-4, OR = 1.87; A allele: Pc = 2.62 × 10-2, OR = 1.20). The stratification analysis showed that GO patients had a higher frequency of the VDR/Apa I AA genotype (Pc = 8.69 × 10-5, OR = 2.84). Functional experiments showed a decreased VDR expression and TGF-ß1 production as well as an increased IL-17 production in VDR/Apa I AA genotype carriers. CONCLUSION: The VDR/Apa I polymorphism is significantly associated with GD and GO, and it may be involved in the development of GD and GO by influencing VDR mRNA expression levels and the secretion levels of cytokines.

Restriction endonuclease T.Smu451I with new cleavage specificity-neoschizomer of T.AsuI.

A specific type II restriction endonuclease T.Smu451I has been purified to electrophoretic homogeneity from the frozen cells of soil bacterium Sphingobacterium multivorum 451 (formerly Flavobacterium multivorum 451), using ultrasonic grinding, nucleic acid removal by streptomycin sulfate, protein precipitation by ammonium sulfate and phosphocellulose P-11, DEAE-Cellulose DE-52, Hepharin-Sepharose CL-6B chromatography, and elucidated several characteristics of T.Smu451I. The molecular weight of the enzyme determined by gel filtration and SDS-polyacrylamide gel electrophoresis was calculated to be 45,000 ± 2000 D (dimer) and 23,000 ± 1000 D (monomer), respectively. The isoelectric point (pI) of T.Smu451I is 5.4. T.Smu451I recognizes pentanucleotide palindromic sequences 5'-GGNC↓C-3' and cleaves between C and C in position shown by arrow to produce 3'-cohesive terminus of trinucleotide. Therefore, T.Smu451I is a neoschizomer of T.AsuI.

Protein-DNA target search relies on quantum walk.

Protein-DNA interactions play a fundamental role in all life systems. A critical issue of such interactions is given by the strategy of protein search for specific targets on DNA. The mechanisms by which the protein are able to find relatively small cognate sequences, typically 15-20 base pairs (bps) for repressors, and 4-6 bps for restriction enzymes among the millions of bp of non-specific chromosomal DNA have hardly engaged researchers for decades. Recent experimental studies have generated new insights on the basic processes of protein-DNA interactions evidencing the underlying complex dynamic phenomena involved, which combine three-dimensional and one-dimensional motion along the DNA chain. It has been demonstrated that protein molecules have an extraordinary ability to find the target very quickly on the DNA chain, in some cases, with two orders of magnitude faster than the diffusion limit. This unique property of protein-DNA search mechanism is known as facilitated diffusion. Several theoretical mechanisms have been suggested to describe the origin of facilitated diffusion. However, none of such models currently has the ability to fully describe the protein search strategy. In this paper, we suggest that the ability of proteins to identify consensus sequences on DNA is based on the entanglement of π-π electrons between DNA nucleotides and protein amino acids. The π-π entanglement is based on Quantum Walk (QW), through Coin-position entanglement (CPE). First, the protein identifies a dimer belonging to the consensus sequence, and localize a π on such dimer, hence, the other π electron scans the DNA chain until the sequence is identified. Focusing on the example of recognition of consensus sequences of EcoRV or EcoRI, we will describe the quantum features of QW on protein-DNA complexes during the search strategy, such as walker quadratic spreading on a coherent superposition of different vertices and environment-supported long-time survival probability of the walker. We will employ both discrete- or continuous-time versions of QW. Biased and unbiased classical Random Walk (CRW) have been used for a long time to describe the Protein-DNA search strategy. QW, the quantum version of CRW, has been widely studied for its applications in quantum information applications. In our biological application, the walker (the protein) resides at a vertex in a graph (the DNA structural topology). Differently to CRW, where the walker moves randomly, the quantum walker can hop along the edges in the graph to reach other vertices entering coherently a superposition across different vertices spreading quadratically faster than CRW analogous evidencing the typical speed up features of the QW. When applied to a protein-DNA target search problem, QW gives the possibility to achieve the experimental diffusional motion of proteins over diffusion classical limits experienced along DNA chains exploiting quantum features such as CPE and long-time survival probability supported by the environment. In turn, we come to the conclusion that, under quantum picture, the protein search strategy does not distinguish between one-dimensional (1D) and three-dimensional (3D) cases.

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.

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV.

Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg2+ ions, EcoRV is able to cleave the DNA but not in presence of Ca2+ , although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg2+ (A)-EcoRV-DNA complex compared to Ca2+ (S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca2+ (S)-EcoRV-DNA compared to Mg2+ (A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca2+ (S) bound complex than in Mg2+ (A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.

ATP-powered molecular recognition to engineer transient multivalency and self-sorting 4D hierarchical systems.

Biological systems organize multiple hierarchical structures in parallel, and create dynamic assemblies and functions by energy dissipation. In contrast, emerging artificial non-equilibrium self-assembling systems have remained relatively simplistic concerning hierarchical design, and non-equilibrium multi-component systems are uncharted territory. Here we report a modular DNA toolbox allowing to program transient non-equilibrium multicomponent systems across hierarchical length scales by introducing chemically fueled molecular recognition orchestrated by reaction networks of concurrent ATP-powered ligation and cleavage of freely programmable DNA building blocks. Going across hierarchical levels, we demonstrate transient side-chain functionalized nucleic acid polymers, and further introduce the concept of transient cooperative multivalency as a key to bridge length scales to pioneer fuel-driven encapsulation, self-assembly of colloids, and non-equilibrium transient narcissistic colloidal self-sorting on a systems level. The fully programmable and functionalizable DNA components pave the way to design chemically fueled 4D (3 space, 1 time) molecular multicomponent systems and autonomous materials.

TALEN-Based Chemically Inducible, Dimerization-Dependent, Sequence-Specific Nucleases.

For precise genome editing, it is important to control the activity of sequence-specific nucleases. We have constructed a chemically inducible nuclease system based on the dimerization of FKBP and FRB domains in the presence of rapamycin and designated it as a chemically inducible dimerization (CID). The CID was designed to occur at the interlinker section between DNA binding domains and the FokI catalytic domain. Thus, induction of cleavage should occur quickly after addition of rapamycin because components of proteins are already in active form and located in the nucleus. This CID-dependent sequence-specific nuclease has potential to be applied for time-resolved analysis of the mutation induction mechanism in the genome.

Structure of HhaI endonuclease with cognate DNA at an atomic resolution of 1.0 Å.

HhaI, a Type II restriction endonuclease, recognizes the symmetric sequence 5'-GCG↓C-3' in duplex DNA and cleaves ('↓') to produce fragments with 2-base, 3'-overhangs. We determined the structure of HhaI in complex with cognate DNA at an ultra-high atomic resolution of 1.0 Å. Most restriction enzymes act as dimers with two catalytic sites, and cleave the two strands of duplex DNA simultaneously, in a single binding event. HhaI, in contrast, acts as a monomer with only one catalytic site, and cleaves the DNA strands sequentially, one after the other. HhaI comprises three domains, each consisting of a mixed five-stranded ß sheet with a defined function. The first domain contains the catalytic-site; the second contains residues for sequence recognition; and the third contributes to non-specific DNA binding. The active-site belongs to the 'PD-D/EXK' superfamily of nucleases and contains the motif SD-X11-EAK. The first two domains are similar in structure to two other monomeric restriction enzymes, HinP1I (G↓CGC) and MspI (C↓CGG), which produce fragments with 5'-overhangs. The third domain, present only in HhaI, shifts the positions of the recognition residues relative to the catalytic site enabling this enzyme to cleave the recognition sequence at a different position. The structure of M.HhaI, the biological methyltransferase partner of HhaI, was determined earlier. Together, these two structures represent the first natural pair of restriction-modification enzymes to be characterized in atomic detail.

Comparative analysis of IGFBP-3 gene sequence in Egyptian sheep, cattle, and buffalo.

OBJECTIVE: A total of 205 animals from four Egyptian livestock species; cattle (n = 18), buffaloes (n = 12), sheep (n = 150) and goats (n = 25) were used in this study to detect polymorphism and perform comparative analysis for IGFBP-3 gene using DNA sequencing and (PCR-RFLP). RESULTS: The amplified fragments were found to be of length 654 bp in sheep, 651 bp in cattle and 655 bp in buffalo. For Falahy goats, PCR was performed to amplify a 316 bp fragment from exon 2 of the IGFBP-3 gene. The digestion of 654 bp with HaeIII restriction enzyme yielded a single restriction pattern for goats, while for cattle, 3 genotypes were identified; (AA), (AB), and (BB). Moreover, for buffalo one genotype (AA) only was found with HaeIII and TaqI restriction enzymes, separately. Also, the digestion profile for goats with HaeIII revealed one pattern only. Nucleotide sequencing of the amplified fragments of IGFBP-3 gene in sheep, cattle, buffalo, and goat was submitted to the NCBI GenBank (Accession no. MG738671.1, MG738673.1, MG738674.1, and MG738672.1, respectively). The nucleotide sequencing analysis indicated similarity percentages in IGFBP-3 gene fragments of 88.54, 89.63 and 95.06% between "sheep and cattle", "sheep and buffalo", and "cattle and buffalo", respectively.

Enzyme-Mediated Endogenous and Bioorthogonal Control of a DNAzyme Fluorescent Sensor for Imaging Metal Ions in Living Cells.

Bioorthogonal control of metal-ion sensors for imaging metal ions in living cells is important for understanding the distribution and fluctuation of metal ions. Reported here is the endogenous and bioorthogonal activation of a DNAzyme fluorescent sensor containing an 18-base pair recognition site of a homing endonuclease (I-SceI), which is found by chance only once in 7×1010  bp of genomic sequences, and can thus form a near bioorthogonal pair with I-SceI for DNAzyme activation with minimal effect on living cells. Once I-SceI is expressed inside cells, it cleaves at the recognition site, allowing the DNAzyme to adopt its active conformation. The activated DNAzyme sensor is then able to specifically catalyze cleavage of a substrate strand in the presence of Mg2+ to release the fluorophore-labeled DNA fragment and produce a fluorescent turn-on signal for Mg2+ . Thus I-SceI bioorthogonally activates the 10-23 DNAzyme for imaging of Mg2+ in HeLa cells.

Mechanism of Filamentation-Induced Allosteric Activation of the SgrAI Endonuclease.

Filament formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the cryoelectron microscopy (cryo-EM) structure of the filament of SgrAI in its activated configuration. The structural data illuminate the mechanistic origin of hyperaccelerated DNA cleavage activity and suggests how indirect DNA sequence readout within filamentous SgrAI may enable recognition of substantially more nucleotide sequences than its low-activity form, thereby altering and partially relaxing its DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI's catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.