Type III CRISPR-Cas systems in prokaryotes provide immunity against invading nucleic acids through the coordinated degradation of transcriptionally active DNA and its transcripts by Csm effector complex. The Cas10 subunit complex contains an HD nuclease domain that is responsible for two Palm domains with elusive functions. In addition, Csm6, a ribonuclease not part complex, also required to full immunity. We show here target RNA binding Streptococcus thermophilus triggers synthesize cyclic...

The study of interactions protein with DNA is important for gaining a fundamental understanding how numerous biological processes occur, including recombination, transcription, repair, etc. In this study, we use the EcoRII restriction enzyme, which employs three-site binding mechanism to catalyze cleavage single recognition site. Using high-speed atomic force microscopy (HS-AFM) image single-molecule in real time, were able observe binding, translocation, and dissociation mechanisms protein....

DNA cytosine methylation is a widespread epigenetic mark. Biological effects of are mediated by the proteins that preferentially bind to 5-methylcytosine (5mC) in different sequence contexts. Until now two structural mechanisms have been established for 5mC recognition eukaryotes; however, it still unknown how discrimination modification achieved prokaryotes. Here we report crystal structure N-terminal DNA-binding domain (McrB-N) methyl-specific endonuclease McrBC from Escherichia coli. The...

Abstract The type III CRISPR–Cas systems provide immunity against invading nucleic acids through the coordinated transcription-dependent DNA targeting and cyclic adenylate (cAn)-activated RNA degradation. Here, we show that both these pathways contribute to Streptococcus thermophilus (St) III-A immunity. HPLC-MS analysis revealed in heterologous Escherichia coli host StCsm effector complex predominantly produces cA5 cA6. cA6 acts as a signaling molecule binds CARF domain of StCsm6 activate...

Prokaryotic type III CRISPR-Cas antiviral systems employ cyclic oligoadenylate (cAn) signaling to activate a diverse range of auxiliary proteins that reinforce the defense. Here we characterize class cAn-dependent effector named CRISPR-Cas-associated messenger RNA (mRNA) interferase 1 (Cami1) consisting CRISPR-associated Rossmann fold sensor domain fused winged helix-turn-helix and RelE-family mRNA domain. Upon activation by tetra-adenylate (cA4), Cami1 cleaves exposed at ribosomal A-site...

The type III-A Csm complex of Streptococcus thermophilus (StCsm) provides immunity against invading nucleic acids through the coordinated action three catalytic domains: RNase (Csm3), ssDNase (Cas10-HD), and cyclic oligoadenylates synthase (Cas10-Palm). matured StCsm is composed Cas10:Csm2:Csm3:Csm4:Csm5 subunits 40-nt CRISPR RNA (crRNA). We have carried out gene disruptions for each subunit isolated deletion complexes to reveal role individual in assembly function. show that Cas10-Csm4...

RNA interference is a powerful experimental tool for knockdown, but not all organisms are amenable. Here, we provide proof of principle demonstration that type III Csm effector complex can be used programmable mRNA transcript degradation in eukaryotes. In zebrafish, Streptococcus thermophilus (StCsm) proved effective knockdown maternally expressed EGFP germ cells Tg(ddx4:ddx4-EGFP) fish. It also led to significant, albeit less drastic, fluorescence reduction at one day postfertilization...

Interactions between distantly separated DNA regions mediated by specialized proteins lead to the formation of synaptic protein−DNA complexes. This is a ubiquitous phenomenon which critical in various genetic processes. Although such interactions typically occur two sites, among three specific have been identified, and corresponding model has proposed. Atomic force microscopy was used test this for EcoRII restriction enzyme provide direct visualization characterization complexes involving...

Restriction endonuclease MvaI recognizes the sequence CC/WGG (W stands for A or T, ‘/’ designates cleavage site) and generates products with single nucleotide 5′-overhangs. The enzyme has been noted its tolerance towards DNA modifications. Here, we report a biochemical characterization crystal structures of in an apo-form complex target at 1.5 Å resolution. Our results show that is monomer pseudosymmetric asymmetrically. consists two lobes. catalytic lobe anchors active site residues Glu36,...

SUMMARY To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cA n ) signaling to activate various auxiliary effectors, including the CRISPR-associated SAVED-Lon protease CalpL, which forms a tripartite effector system together with an anti-σ factor, CalpT, and ECF-like σ CalpS. Here we report characterization of Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA 4 binding triggers CalpL filament formation activates it cleave...

A catalytic sequence motif PDX 10–30 (E/D)XK is found in many restriction enzymes. On the basis of similarities and mapping conserved residues to crystal structure Ngo MIV we suggest that D160, K182, R186, R188 E195 contribute catalytic/DNA binding site Ecl 18kI endonuclease. Mutational analysis confirms functional significance 18kI. Therefore, conclude active 159 VDX 21 KX 12 E differs from canonical characteristic for most Moreover, propose two subfamilies endonucleases 18kI/ Psp GI/ Eco...

PspGI is a representative of group restriction endonucleases that recognize pentameric sequence related to CCNGG. Unlike the previously investigated Ecl18kI, which does not have any specificity for central base pair, prefers A/T over G/C in its target site. Here, we present structure with DNA at 1.7 Å resolution. In this structure, bases center recognition are extruded from and flipped into pockets PspGI. The thymine usual anti conformation, but adenine takes normally unfavorable syn...

To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to copy. However, free solution dimer. find out whether dimer or represents functionally important assembly, we generated mutants aimed disrupting putative dimer–dimer interface and analysed functional properties mutant variants. We show by atomic force microscopy that on two-site DNA, loops an...

Many DNA modification and repair enzymes require access to bases therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in vicinity of target recognition site do not base extrusion for sequence readout catalysis. Therefore, observation extrahelical nucleotides a co-crystal REase Ecl18kI with cognate sequence, CCNGG, was unexpected. It turned out that reads directly only CCGG skips unspecified N nucleotides, flipping them from helix....

Endonucleases that generate double-strand breaks in DNA often possess two identical subunits related by rotational symmetry, arranged so the active sites from each subunit act on opposite strands. In contrast to many endonucleases, Type IIP restriction enzyme BcnI, which recognizes pseudopalindromic sequence 5'-CCSGG-3' (where S stands for C or G) and cuts both strands after second C, is a monomer possesses single catalytic center. We show here break BcnI nicks one strand, switches its...

The archetypal Type IIE restriction endonuclease Eco RII is a dimer that has modular structure. DNA binding studies indicate the isolated C‐terminal domain an interface binds single cognate molecule whereas N‐terminal monomer also copy of DNA. Hence, full‐length contains three putative interfaces: one at and two each domains. Mutational analysis indicates shares conserved active site architecture elements with tetrameric enzyme Ngo MIV. Data provided here suggest possible evolutionary...

Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition adjust DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy 2-aminopurine-labelled in complex with each solution explore nucleotide flipping mechanism obtain a detailed picture molecular environment extrahelical bases. also report first study 7-bp cutter, PfoI, whose...

Although all Type II restriction endonucleases catalyze phosphodiester bond hydrolysis within or close to their DNA target sites, they form different oligomeric assemblies ranging from monomers, dimers, tetramers higher order oligomers generate a double strand break in DNA. IIP endonuclease AgeI recognizes palindromic sequence 5΄-A/CCGGT-3΄ and cuts it ('/' denotes the cleavage site) producing staggered ends. Here, we present crystal structures of apo DNA-bound forms. The structure is...

Abstract The type III CRISPR-Cas effector complex Csm functions as a molecular Swiss army knife that provides multilevel defense against foreign nucleic acids. coordinated action of three catalytic activities the enables simultaneous degradation invader's RNA transcripts, destruction template DNA and synthesis signaling molecules (cyclic oligoadenylates cAn) activate auxiliary proteins to reinforce defense. Here, we employed single-molecule techniques connect kinetics binding, dissociation,...