6 research outputs found

    Chemical Display of Pyrimidine Bases Flipped Out by Modification-Dependent Restriction Endonucleases of MspJI and PvuRts1I Families

    No full text
    <div><p>The epigenetic DNA modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in eukaryotes are recognized either in the context of double-stranded DNA (e.g., by the methyl-CpG binding domain of MeCP2), or in the flipped-out state (e.g., by the SRA domain of UHRF1). The SRA-like domains and the base-flipping mechanism for 5(h)mC recognition are also shared by the recently discovered prokaryotic modification-dependent endonucleases of the MspJI and PvuRts1I families. Since the mechanism of modified cytosine recognition by many potential eukaryotic and prokaryotic 5(h)mC “readers” is still unknown, a fast solution based method for the detection of extrahelical 5(h)mC would be very useful. In the present study we tested base-flipping by MspJI- and PvuRts1I-like restriction enzymes using several solution-based methods, including fluorescence measurements of the cytosine analog pyrrolocytosine and chemical modification of extrahelical pyrimidines with chloroacetaldehyde and KMnO<sub>4</sub>. We find that only KMnO<sub>4</sub> proved an efficient probe for the positive display of flipped out pyrimidines, albeit the method required either non-physiological pH (4.3) or a substitution of the target cytosine with thymine. Our results imply that DNA recognition mechanism of 5(h)mC binding proteins should be tested using a combination of all available methods, as the lack of a positive signal in some assays does not exclude the base flipping mechanism.</p></div

    DNA cleavage and binding by YkrI and BmeDI.

    No full text
    <p>The sequences at the top of the figure schematically depict the 39-H/H (optimal substrate with two 5hmC bases), 39-M/H, 39-C/H, 39-H, 39-P/H, 39-T/H, and 39-T (one or both 5hmC-G base pairs replaced with a 5mC-G, C-G, T-A, pyrrolocytosine-G, and thymine-G base pairs, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114580#pone-0114580-t001" target="_blank">Table 1</a>) oligoduplexes. (A) The observed first-order DNA cleavage rate constants. Cleavage reactions were performed with 1 nM substrate and 100 nM enzyme (monomer) at 15°C. In our experimental setup, BmeDI cleavage of the 39-T oligoduplex was not detectable. Denaturing PAGE analysis of cleavage products formed with various DNA substrates is shown on the right-hand side. Gel lane ‘M’ contained a synthetic single-stranded oligonucleotide that corresponds to cleavage of the bottom strand 11 nt downstream of the 5hmC nucleotide. (B) Electrophoretic mobility shift assay with YkrI. DNA binding experiments were performed in a pH 8.3 buffer in the presence of 5 mM Ca<sup>2+</sup> ions. The final DNA concentration was 1 nM, and YkrI concentrations are indicated above the gel lanes. Red arrows mark the location of the specific YkrI-DNA complexes. The upper band corresponds to the low-mobility non-specific YkrI-DNA complex formed due to binding/aggregation of multiple protein molecules. (C) Electrophoretic mobility shift experiments with BmeDI. Reaction conditions were as in panel (B).</p

    The modified cytosine binding pockets.

    No full text
    <p>(A-B) 5-methylcytosine recognition by the UHRF1 SRA domain (PDB ID 3fde) and the DNA recognition domain of MspJI endonuclease (PDB ID 4r28). The indicated protein pocket residues make base-specific contacts to the extrahelical base <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114580#pone.0114580-Horton2" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114580#pone.0114580-Hashimoto2" target="_blank">[45]</a>. (C-D) The models for the modified cytosine recognition by the DNA binding domains of AspBHI (PDB ID 4oc8) and PvuRts1I (PDB ID 4oq2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114580#s2" target="_blank">Materials and Methods</a> for details). The indicated amino acid residues could form base-specific contacts to the extruded base. In the case of AspBHI, this would require protonation of the D71 residue. In each panel the dark line marks the boundaries of the protein pocket cut at the plane of the cytosine ring.</p

    DNA cleavage and binding by LpnPI.

    No full text
    <p>The sequences in all panels depict recognition sites in the oligoduplex substrates. (A) DNA cleavage experiments. The reactions were performed with 500 nM enzyme (monomer) and 400 nM substrate at 25°C. Time courses of the reactions are shown. The reaction rate constant for the 30-M substrate equals 0.20±0.05 min<sup>−1</sup>. Reaction rate constants for other substrates were lower than 1×10<sup>−5</sup> min<sup>−1</sup>. (B) Electrophoretic mobility shift assay with LpnPI. DNA binding experiments were performed in a pH 6.3 buffer in the presence of 5 mM Ca<sup>2+</sup> ions. The final substrate concentration was 10 nM, LpnPI concentrations (in terms of monomer) are indicated above the gel lanes. Red arrows mark the location of the protein-DNA complexes. (C) Electrophoretic mobility shift assay with LpnPI DNA binding domain (LpnPI-N). Experiments with the cognate (16-M) and non-cognate (16-C) substrates were performed in a pH 6.3 buffer in the absence of Ca<sup>2+</sup> ions. (D) Electrophoretic mobility shift assay with LpnPI in a pH 8.3 buffer in the presence of Ca<sup>2+</sup> ions.</p

    Catalytic Activity Control of Restriction EndonucleaseTriplex Forming Oligonucleotide Conjugates

    No full text
    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

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

    No full text
    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
    corecore