An Engineered SS Bridge Blocks the Conformational Change Required for the Nuclease Activity of BfiI

The type IIS restriction endonuclease BfiI is a homodimer, and each monomer is composed of the N-terminal catalytic and C-terminal DNA recognition domains connected by a 28-residue linker segment. In the crystal in the absence of cognate DNA, BfiI exists in a “closed” conformation, in which an interdomain linker occludes a putative DNA binding surface at the catalytic domain and sterically hinders access to the active site. Cognate DNA binding presumably triggers a conformational change from the inactive “closed” state to the catalytically competent “open” state. Here we show that the disulfide SS bridge engineered at the domain interface locks the enzyme in the “closed” state. In the “closed” SS-linked state, BfiI binds cognate DNA with the same affinity as the wild-type enzyme but does not cut it, indicating that cross-linking introduces a restraint on the conformational transition, which couples DNA recognition and cleavage. Disruption of the interdomain SS bridge by the reducing agent restores the DNA cleavage ability of BfiI

Catalytic Activity Control of Restriction EndonucleaseTriplex Forming Oligonucleotide Conjugates

Targeting of individual genes in complex genomes requires endonucleases of extremely high specificity. To direct cleavage at the unique site­(s) in the genome, both naturally occurring and artificial enzymes have been developed. These include homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and restriction or chemical nucleases coupled to a triple-helix forming oligonucleotide (TFO). The desired cleavage has been demonstrated both <i>in vivo</i> and <i>in vitro</i> for several model systems. However, to limit cleavage strictly to unique sites and avoid undesired reactions, endonucleases with controlled activity are highly desirable. In this study we present a proof-of-concept demonstration of two strategies to generate restriction endonuclease–TFO conjugates with controllable activity. First, we combined the restriction endonuclease caging and TFO coupling procedures to produce a caged MunI–TFO conjugate, which can be activated by UV-light upon formation of a triple helix. Second, we coupled TFO to a subunit interface mutant of restriction endonuclease Bse634I which shows no activity due to impaired dimerization but is assembled into an active dimer when two Bse634I monomers are brought into close proximity by triple helix formation at the targeted site. Our results push the restriction endonuclease–TFO conjugate technology one step closer to potential <i>in vivo</i> applications

Domain architectures of () protein families sharing homology with the BpuJI nuclease domain identified in this study and () previously characterized Type III Res subunits and the McrBC system

<p><b>Copyright information:</b></p><p>Taken from "Restriction endonuclease BpuJI specific for the 5′-CCCGT sequence is related to the archaeal Holliday junction resolvase family"</p><p></p><p>Nucleic Acids Research 2007;35(7):2377-2389.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874659.</p><p>© 2007 The Author(s)</p> AHJR, the archaeal Holliday junction resolvase domain similar to the C-terminal catalytic domain of BpuJI; for domains of superfamily II helicases, SF-II, the closest homolog of known structure is indicated; HSP90N, the N-terminal domain of HSP90; McrB-N and McrB-C, respectively, the N-terminal DNA binding and the C-terminal GTPase domains of the McrB subunit of 5′-methylcytosine-specific restriction enzyme; DBD?, unclassified putative DNA binding domain; UPF0102, uncharacterized protein family with the predicted single-domain nuclease fold related to AHJRs; Region X and PD-(D/E)XK, endonuclease domains of Type III and McrC restriction enzymes, respectively. Each architecture in (A) is illustrated with a single representative and corresponds to the grouping in the alignment (Supplementary Figure 5B)

The 3′-end directed endonucleolytic activity of the isolated C-terminal domain of BpuJI

<p><b>Copyright information:</b></p><p>Taken from "Restriction endonuclease BpuJI specific for the 5′-CCCGT sequence is related to the archaeal Holliday junction resolvase family"</p><p></p><p>Nucleic Acids Research 2007;35(7):2377-2389.</p><p>Published online 28 Mar 2007</p><p>PMCID:PMC1874659.</p><p>© 2007 The Author(s)</p> () Cleavage of the PCR fragment by the C-terminal domain. Total of 1 nM of the PCR fragment, 5′-labelled in bottom strand, was incubated with 3 μM of the C-terminal domain at 4°C. Aliquots were removed at timed intervals (indicated above the relevant lanes) and analysed by electrophoresis through the standard sequencing gel. Lanes G, A, T, C are sequence ladders. () Cleavage of oligonucleotide duplexes by the C-terminal domain. Total of 2 nM of oligoduplex, 3′-labelled (upper gels) or 5′-labelled (lower gels) in top strand, was incubated with 100 nM of the C-terminal domain at 25°C. Aliquots were removed at timed intervals (indicated above the relevant lanes) and analysed by electrophoresis through denaturing 20% polyacrylamide. In cartoons below the gels the position of the label is indicated by a star, the arrows show the position of initial cleavage

Restriction Enzyme Ecl18kI-Induced DNA Looping Dynamics by Single-Molecule FRET

Many type II restriction endonucleases require binding of two copies of a recognition site for efficient DNA cleavage. Simultaneous interaction of the enzyme with two DNA sites results in DNA loop formation. It was demonstrated with the tethered particle motion technique that such looping is a dynamic process where a DNA loop is repeatedly formed and disrupted. Here we use a better and in the context of protein-induced DNA looping virtually unexploited strategy of single-molecule Förster resonance energy transfer of surface immobilized biomolecules to quantitatively study the dynamics of Ecl18kI endonuclease-induced DNA looping and determine the rate constants of loop formation and disruption. We show that two DNA-bound Ecl18kI dimers efficiently form a bridging tetramer looping out intervening DNA with a rate that is only a few orders of magnitude lower than the diffusion limited rate. On the other hand, the existence of Ecl18kI tetramer is only transient, and the loop is rapidly disrupted within about 1 s

Additional file 1: of Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements

Figures S1–S14 and Tables S1 and S2. (PDF 993 kb

Analytical gel filtration experiments to determine the MvaI oligomeric state and the stoichiometry of DNA binding for the oligoduplex 1 shown in ()

<p><b>Copyright information:</b></p><p>Taken from "Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetrically"</p><p></p><p>Nucleic Acids Research 2007;35(6):2035-2046.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874612.</p><p>© 2007 The Author(s)</p> Elution profiles were recorded simultaneously at 260 and 280 nm and deconvoluted to obtain separate curves for the MvaI (blue) and DNA (red) concentration. () MvaI alone, () DNA alone, () mixture with a 2:1 molar excess of MvaI over DNA, () stoichiometric mixture, () mixture with a 2:1 molar excess of DNA over MvaI, () calibration curve for Superose ™ 12 HR 10/30 column (Amersham Biosciences) with standards from Biorad (vitamin B-12, 1.35 kDa; myoglobin, 17 kDa; ovalbumin, 44kDa; IgG, 150 kDa and thyroglobin, 670 kDa)

Targeted gene editing by transfection of <i>in vitro</i> reconstituted <i>Streptococcus thermophilus</i> Cas9 nuclease complex

Targeted gene editing by transfection of <i>in vitro</i> reconstituted <i>Streptococcus thermophilus</i> Cas9 nuclease comple