Nuclease Domain Research Articles

Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families

ABSTRACTA study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Northern blotting and interference assays in Synechocystis confirmed that one particular non-CARF accessory protein family was involved in crRNA maturation. Non-CARF accessory genes were generally diverse, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains with some encoding no known domains. We infer that additional families of non-CARF accessory proteins remain to be found. The method employed is scalable for potential application to metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk.

Single-site DNA cleavage by Type III restriction endonuclease requires a site-bound enzyme and a trans-acting enzyme that are ATPase-activated

Endonucleolytic cleavage of DNA by Type III restriction-modification (RM) enzymes requires long-range communication between at least two recognition sites in inverted orientation. This results in convergence of two nuclease domains, one each from the enzymes loaded at the recognition sites with one still bound to the site. The nucleases catalyze scission of the single-strands leading to double-strand DNA break. An obscure feature of the Type III RM enzymes EcoP1I and EcoP15I is their ability to cleave DNA having a single recognition site under certain conditions. Here we demonstrate that single-site cleavage is the result of cooperation between an enzyme bound to the recognition site in cis and one in trans. DNA cleavage is catalyzed by converging nucleases that are activated by hydrolysis-competent ATPase in presence of their respective DNA substrates. Furthermore, a single activated nuclease cannot nick a strand on its own, and requires the partner. Based on the commonalities in the features of single-site and two-site cleavage derived from this study, we propose that their mechanism is similar. Furthermore, the products of two-site cleavage can act as substrates and activators of single-site cleavage. The difference in the two modes lies in how the two cooperating enzymes converge, which in case of single-site cleavage appears to be via 3D diffusion.

MCPIP3 as a Potential Metastasis Suppressor Gene in Human Colorectal Cancer.

Monocyte chemotactic protein induced protein 3 (MCPIP3) belongs to the Cys–Cys–Cys–His (CCCH)-zinc finger protein family and contains a highly conserved CCCH-zinc finger domain and a Nedd4-BP1 YacP nuclease (NYN) domain. Previous studies showed that MCPIP3 inhibits the expression of proinflammatory genes, such as vascular cell adhesion molecule (VCAM)-1, in human endothelial cells, but the roles and functions of MCPIP3 in cancer cells are still unknown. In human colorectal cancer specimens, we found that the messenger RNA expression of MCPIP3 was significantly downregulated in cancer tissues compared to adjacent normal tissues (18/25; average fold change of 8.18). Two cell models were used to demonstrate the anti-migration activity of MCPIP3. First, Tet-on T-REx-293/HA-MCPIP3 cells were used to examine whether MCPIP3 can change epithelial–mesenchymal transition (EMT)-related gene expressions. Second, we used two human colorectal cancer cell lines, SW620 and HCT116, to prove the role of MCPIP3 in regulating EMT-related gene expressions. We found that overexpression of MCPIP3 inhibited cell migration according to a wound-healing assay and Transwell invasion assay and vimentin expression, and increased E-cadherin expression in these two cell lines. These results suggest that MCPIP3 might play a negative role in cell migration of human colorectal cancer cells.

Computational Characterization of Small Molecules Binding to the Human XPF Active Site and Virtual Screening to Identify Potential New DNA Repair Inhibitors Targeting the ERCC1-XPF Endonuclease.

The DNA excision repair protein ERCC-1-DNA repair endonuclease XPF (ERCC1-XPF) is a heterodimeric endonuclease essential for the nucleotide excision repair (NER) DNA repair pathway. Although its activity is required to maintain genome integrity in healthy cells, ERCC1-XPF can counteract the effect of DNA-damaging therapies such as platinum-based chemotherapy in cancer cells. Therefore, a promising approach to enhance the effect of these therapies is to combine their use with small molecules, which can inhibit the repair mechanisms in cancer cells. Currently, there are no structures available for the catalytic site of the human ERCC1-XPF, which performs the metal-mediated cleavage of a DNA damaged strand at 5′. We adopted a homology modeling strategy to build a structural model of the human XPF nuclease domain which contained the active site and to extract dominant conformations of the domain using molecular dynamics simulations followed by clustering of the trajectory. We investigated the binding modes of known small molecule inhibitors targeting the active site to build a pharmacophore model. We then performed a virtual screening of the ZINC Is Not Commercial 15 (ZINC15) database to identify new ERCC1-XPF endonuclease inhibitors. Our work provides structural insights regarding the binding mode of small molecules targeting the ERCC1-XPF active site that can be used to rationally optimize such compounds. We also propose a set of new potential DNA repair inhibitors to be considered for combination cancer therapy strategies.

Repetitive DNA Reeling by the Cascade-Cas3 Complex in Nucleotide Unwinding Steps

CRISPR-Cas provides RNA-guided adaptive immunity against invading genetic elements. Interference in type I systems relies on the RNA-guided Cascade complex for target DNA recognition and the Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here, we employed single-molecule Förster resonance energy transfer (FRET) to probe the helicase activity with high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling the DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of 3bp, which underlie three successive 1-nt unwinding events. Reeling is highly repetitive, allowing Cas3 to repeatedly present its inefficient nuclease domain with single-strand DNA (ssDNA) substrate. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure controlled degradation of target DNA only.

Real‐time observation of flexible domain movements in CRISPR–Cas9

The CRISPR‐associated protein Cas9 is widely used for genome editing because it cleaves target DNA through the assistance of a single‐guide RNA (sgRNA). Structural studies have revealed the multi‐domain architecture of Cas9 and suggested sequential domain movements of Cas9 upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains; however, it remains unclear whether these flexible movements occur in solution. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single‐molecule FRET. We found that the flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo‐Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9‐catalyzed DNA cleavage.

Pervasive Chimerism in the Replication-Associated Proteins of Uncultured Single-Stranded DNA Viruses.

Numerous metagenomic studies have uncovered a remarkable diversity of circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses, the majority of which are uncultured and unclassified. Unlike capsid proteins, the Reps show significant similarity across different groups of CRESS DNA viruses and have conserved domain organization with the N-terminal nuclease and the C-terminal helicase domain. Consequently, Rep is widely used as a marker for identification, classification and assessment of the diversity of CRESS DNA viruses. However, it has been shown that in certain viruses the Rep nuclease and helicase domains display incongruent evolutionary histories. Here, we systematically evaluated the co-evolutionary patterns of the two Rep domains across classified and unclassified CRESS DNA viruses. Our analysis indicates that the Reps encoded by members of the families Bacilladnaviridae, Circoviridae, Geminiviridae, Genomoviridae, Nanoviridae and Smacoviridae display largely congruent evolutionary patterns in the two domains. By contrast, among the unclassified CRESS DNA viruses, 71% appear to have chimeric Reps. Such massive chimerism suggests that unclassified CRESS DNA viruses represent a dynamic population in which exchange of gene fragments encoding the nuclease and helicase domains is extremely common. Furthermore, purging of the chimeric sequences uncovered six monophyletic Rep groups that may represent new families of CRESS DNA viruses.

The Structure and Mechanism of a Viral Genome Packaging Motor

Many dsDNA viruses use a multi-component ATPase motor to package their genomes into procapsids during viral maturation. These motors are among the most powerful known molecular machines, and how they couple ATP hydrolysis to DNA translocation is still poorly understood. Using a multi-faceted approach combining x-ray crystallography, small angle x-ray scattering, protein docking, and biochemical assays, we elucidate the mechanism of ATP hydrolysis-mediated DNA translocation. We present a novel genome packaging system from a thermophilic virus that provides new insight into motor structure and mechanism. We show that interfacial contacts mediate ATP hydrolysis, identify essential DNA binding and cleavage motifs, and reveal ATP-dependent conformational changes of the packaging motor. Additionally, we show that the motor's ATPase domain is both necessary and sufficient for DNA binding, an attribute previously ascribed to the nuclease domain. Our findings lead to a mechanism of translocation with a long lever-arm to generate high force, and a steric block mechanism for nuclease regulation. Support or Funding Information Pew Charitable Trusts, Hudson Hoagland Society, NIGMS Ruth L. Kirchstein National Research Service Award 1F31GM121019-01A1 Model for viral motor DNA translocation and nuclease regulation. During translocation, the ATPase domain of the motor grips and packages DNA into the capsid using an ATP hydrolysis-mediated lever-arm mechanism. The nuclease domain is hindered from cleaving DNA through contacts with portal and capsid. During packaging termination, the motor enters cleavage mode, and dissociates from the portal and capsid. The nuclease domains rearrange to cleave each of the DNA strands. Hilbert et al, NAR, 2017. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Preventing Autoimmunity in Transcription‐Dependent CRISPR‐Cas Systems

The CRISPR‐Cas systems are a collection of RNA‐based adaptive immune systems that protect many prokaryotes from invasion by viruses and plasmids. CRISPR‐Cas systems are very diverse and can be organized into six types and more than 15 subtypes. Type III systems are unique as their targeting mechanism requires transcription of the target DNA. Type III effector complexes use small CRISPR RNAs (crRNAs) to identify complementary transcripts. Transcript binding simultaneously activates two nuclease domains, leading to the degradation of both the bound transcript and adjacent DNA. Like all other immune systems, CRISPR‐Cas systems must differentiate between self and non‐self in order to prevent autoimmunity, which could arise from targeting of the host CRISPR‐array. Type III systems use two mechanisms to identify self‐transcripts. Studies of the Type III‐A system from Staphylococcus epidermidis revealed that base pairing between the crRNA and the sequence flanking the 3′ end of RNA target site prevents autoimmunity. However, a study of the Type III‐B system from Pyrococcus furiosus revealed that a short sequence motif flanking the RNA target sequence is required for immunity and the absence of this motif in CRISPR arrays prevents autoimmunity. Neither of these mechanisms is well understood biochemically and it is not clear if these mechanisms are conserved within the subtypes. To address these points, we monitored RNA‐activated DNA cleavage by the Type III‐B effector complex from Thermotoga maritima with purified components. A series of RNA transcripts containing various target 3′‐flanking sequences were tested for their ability to activate DNA cleavage. We find that two factors are important in preventing autoimmunity in the T. maritima Type III‐B system: (1) base pairing between the crRNA and the target 3′‐flanking sequence of the transcript and (2) the presence of a G, which does not base pair with the crRNA, immediately adjacent to the 3′‐end of the RNA target sequence. These data suggest that the mechanisms used to avoid autoimmunity by Type III systems are not subtype‐specific and that identification of self transcripts is complex, involving base pairing and specific sequence recognition.Support or Funding InformationThis work was supported by National Institutes of Health grant GM097330 to S.B.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rational Design of Mini-Cas9 for Transcriptional Activation.

Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

The H-subunit of the restriction endonuclease CglI contains a prototype DEAD-Z1 helicase-like motor.

CglI is a restriction endonuclease from Corynebacterium glutamicum that forms a complex between: two R-subunits that have site specific-recognition and nuclease domains; and two H-subunits, with Superfamily 2 helicase-like DEAD domains, and uncharacterized Z1 and C-terminal domains. ATP hydrolysis by the H-subunits catalyses dsDNA translocation that is necessary for long-range movement along DNA that activates nuclease activity. Here, we provide biochemical and molecular modelling evidence that shows that Z1 has a fold distantly-related to RecA, and that the DEAD-Z1 domains together form an ATP binding interface and are the prototype of a previously undescribed monomeric helicase-like motor. The DEAD-Z1 motor has unusual Walker A and Motif VI sequences those nonetheless have their expected functions. Additionally, it contains DEAD-Z1-specific features: an H/H motif and a loop (aa 163–aa 172), that both play a role in the coupling of ATP hydrolysis to DNA cleavage. We also solved the crystal structure of the C-terminal domain which has a unique fold, and demonstrate that the Z1-C domains are the principal DNA binding interface of the H-subunit. Finally, we use small angle X-ray scattering to provide a model for how the H-subunit domains are arranged in a dimeric complex.

The 5′ Nuclease Domain of DNA Polymerase I Mediates a Novel DNA Transfer Pathway during Proofreading

The high fidelity of many DNA polymerases is due, in part, to their ability to remove erroneously incorporated nucleotides at the terminus of the growing primer strand. This “proofreading” activity is catalyzed by the 3′-5′ exonuclease (exo) domain in E. coli DNA polymerase I (Pol I). Structural studies have shown that this domain is spatially separated from the 5′-3′ polymerase domain (pol), which is responsible for incorporation of dNTPs by template-directed polymerization. Pol I also contains another spatially distinct domain, the 5′ nuclease (nuc) domain, which is connected to the enzyme core by a flexible amino acid linker. This domain is involved in cleavage of downstream flaps formed during Okazaki fragment maturation and base excision repair. Using a single-molecule FRET system, we resolved three distinct conformations of Pol I-DNA complexes. Experiments with Pol I mutants established that these states correspond to DNA bound at the pol, exo, or nuc domains. Notably, the DNA substrate can transfer among all three domains without dissociation from the enzyme. We determined rate constants for site switching of model DNA substrates containing a correctly paired primer terminus, a terminal mismatch or an internal mismatch. The presence of mismatches accelerated transfer of DNA from the pol domain to the exo domain, as expected during proofreading. Surprisingly, the mismatches also accelerated transfer of DNA along the pol to nuc to exo domain pathway. These observations reveal the existence of a second intramolecular proofreading pathway, in which mismatched DNA first transfers from the pol domain to the nuc domain and then to the exo domain. Our results suggest an unexpected role for the 5′ nuclease domain in the proofreading activity of Pol I.

CRISPR Cas9 Mediated DNA Unwinding Detected using Site-Directed Spin Labeling

The RNA-guided CRISPR–Cas9 nuclease has revolutionized genome engineering, yet its mechanism for DNA target selection is not fully understood. A crucial step in Cas9 target recognition involves unwinding of the DNA duplex to form a three-stranded R-loop structure. Work reported here demonstrates direct detection of Cas9-mediated DNA unwinding by a combination of site-directed spin labeling and molecular dynamics simulations. The results support a model in which the unwound nontarget strand is stabilized by a positively charged patch located between the two nuclease domains of Cas9 and reveal uneven increases in flexibility along the unwound nontarget strand upon scissions of the DNA backbone. This work establishes the synergistic combination of spin-labeling and molecular dynamics to directly monitor Cas9-mediated DNA conformational changes and yields information on the target DNA in different stages of Cas9 function, thus advancing mechanistic understanding of CRISPR–Cas9 and aiding future technological ...

Simultaneous Real-Time Observation of DNA Unwinding and Nuclease Domain Activation in Cas9-RNA-DNA Complex via Three-Color Single Molecule FRET

Simultaneous Real-Time Observation of DNA Unwinding and Nuclease Domain Activation in Cas9-RNA-DNA Complex via Three-Color Single Molecule FRET

Enhanced Proofreading Governs CRISPR-Cas9 Targeting Accuracy

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer (smFRET), we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. We showed that the high-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) Cas9 variants are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 Activity

Bacterial adaptive immunity utilizes RNA-guided surveillance complexes comprising Cas proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single-guide RNA that base pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and the RuvC nuclease domains cleave opposing strands. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge. Our detailed kinetic characterization of the enzyme reveals that DNA binding is reversible, and R-loop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains. The specificity constant for cleavage is determined through an induced-fit mechanism as the product of the equilibrium binding affinity for DNA and the rate of R-loop formation.

DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 Activity

Bacterial adaptive immunity utilizes RNA-guided surveillance complexes composed of CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programmed with a single guide RNA that base-pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and RuvC nuclease domains can cleave opposing strands. Cas9 has been repurposed for a variety of important genome engineering applications. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge. Our detailed kinetic characterization of the enzyme reveals that DNA binding is reversible, R-loop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains. Although the HNH nuclease activity is stimulated by Mg2 with a single measureable Kd, the RuvC activity requires two distinct Mg2 binding events. The specificity constant for cleavage is determined through an induced-fit mechanism as the product of the equilibrium binding affinity for DNA and the rate of R-loop formation.

The multifaceted oncogene SND1 in cancer: focus on hepatocellular carcinoma.

Staphylococcal nuclease and tudor domain containing 1 (SND1) is a protein that regulates a complex array of functions. It controls gene expression through transcriptional activation, mRNA degradation, mRNA stabilization, ubiquitination and alternative splicing. More than two decades of research has accumulated evidence of the role of SND1 as an oncogene in various cancers. It is a promoter of cancer hallmarks like proliferation, invasion, migration, angiogenesis and metastasis. In addition to these functions, it has a role in lipid metabolism, inflammation and stress response. The participation of SND1 in such varied functions makes it distinct from most oncogenes that are relatively more focused in their role. This becomes important in the case of hepatocellular carcinoma (HCC) since in addition to typical cancer drivers, factors like lipid metabolism deregulation and chronic inflammation can predispose hepatocytes to HCC. The objective of this review is to provide a summary of the current knowledge available on SND1, specifically in relation to HCC and to shed light on its prospect as a therapeutic target.

Selection and Characterization of DNA Aptamers Against FokI Nuclease Domain.

Genome editing with site-specific nucleases (SSNs) may be effective for gene therapy, as SSNs can modify target genes. However, the main limitation of genome editing for clinical use is off-target effects by excess amounts of SSNs within cells. Therefore, a controlled delivery system for SSNs is necessary. Previously we have reported on a zinc finger nuclease (ZFN) delivery system, which combined DNA aptamers against FokI nuclease domain (FokI) and nanoneedles. Here, we describe how DNA aptamers against FokI were selected and characterized for genome editing applications.

Establishment of Cell Lines Stably Expressing dCas9-Fusions to Address Kinetics of Epigenetic Editing.

Epigenetic editing is a promising approach to modulate the local chromatin environment of target genes with the ultimate goal of stable gene expression reprogramming. Epigenetic editing tools minimally consist of a DNA-binding domain and an effector domain. The CRISPR/dCas9 platform, where mutations in the nuclease domains render the Cas9 protein inactive, is widely used to guide epigenetic effectors to their intended genomic loci. Its flexible nature, simple use, and relatively low cost have revolutionized the research field of epigenetic editing. Although effective expression modulation is readily achieved, only a few studies have addressed the maintenance of the induced effects on endogenous loci. Here, we describe a detailed protocol to engineer cells that stably express the CRISPR/dCas9-effectors. The protocol involves modification of published dCas9-based plasmid vectors for easy transfer of the effector domain between the vector designed for transient transfection and the vector used for establishing cell lines stably expressing the dCas9-effector fusion protein. Transient transfection of the dCas9-effector-producing cells with sgRNA-expressing plasmids allows studying of the maintenance of epigenetic editing. Targeting various genes in different chromatin contexts and/or co-targeting multiple CRISPR/dCas9-effectors can be used to unravel rules underlying maintained gene expression reprogramming.