Metilintą DNR atpažįstančių restrikcijos endonukleazių sąveikos su modifikuotu citozinu mechanizmas

The ability of proteins to discriminate between modified and unmodified cytosine is of key importance for such processes as epigenetic regulation in eukaryotes and activity of methyl-directed restriction endonucleases (REases) in bacteria. In eukaryotes modified cytosine is recognized either in the context of dsDNA (by the MBD domains), or in the flipped-out state (by the SRA domains). Structurally related SRA-like domains are also found in methyl-directed REases of MspJI and PvuRts1I families. Another methyl-directed endonuclease McrBC, despite an unrelated structure, also employs the base flipping mechanism. However, the mechanism of modified cytosine recognition by many uncharacterized eukaryotic and prokaryotic modified cytosine “readers” remains unknown, as is the DNA recognition and reaction mechanism of the classical methyl-directed enzyme McrA. In this study using variuos chemical methods we demonstrate that MspJI and PvuRts1I enzymes flip out modified base from dsDNA. We also identify protein loops in MspJI family enzymes that act as DNA binding/recognition modules, replacement of which results in altered sequence specificity, and present the first detailed study of McrBC specificity for various cytosine modifications. Finally, this is the first study that demonstrates McrA nuclease activity in vitro and confirms functions of its N-terminal (DNA binding) and C-terminal (catalysis) domains. Our findings pave the way for engineering of new tools for epigenome studies

Recognition of modified cytosine variants by the DNA‐binding domain of methyl‐directed endonuclease McrBC

Cytosine modifications expand the information content of genomic DNA in both eukaryotes and prokaryotes, providing means for epigenetic regulation and self versus nonself discrimination. For example, the methyl‐directed restriction endonuclease, McrBC, recognizes and cuts invading bacteriophage DNA containing 5‐methylcytosine (5mC), 5‐hydroxymethylcytosine (5hmC), and N4‐methylcytosine (4mC), leaving the unmodified host DNA intact. Here, we present cocrystal structures of McrB‐N bound to DNA oligoduplexes containing 5hmC, 5‐formylcytosine (5fC), and 4mC, and characterize the relative affinity of McrB‐N to various cytosine variants. We find that McrB‐N flips out modified bases into a protein pocket and binds cytosine derivatives in the order of descending affinity: 4mC > 5mC > 5hmC ≫ 5fC. We also show that pocket mutations alter the relative preference of McrB‐N to 5mC, 5hmC, and 4mC

Recognition of modified cytosine variants by the DNA-binding domain of methyl-directed endonuclease McrBC

Cytosine modifications expand the information content of genomic DNA in both eukaryotes and prokaryotes, providing means for epigenetic regulation and self versus nonself discrimination. For example, the methyl‐directed restriction endonuclease, McrBC, recognizes and cuts invading bacteriophage DNA containing 5‐methylcytosine (5mC), 5‐hydroxymethylcytosine (5hmC), and N4‐methylcytosine (4mC), leaving the unmodified host DNA intact. Here, we present cocrystal structures of McrB‐N bound to DNA oligoduplexes containing 5hmC, 5‐formylcytosine (5fC), and 4mC, and characterize the relative affinity of McrB‐N to various cytosine variants. We find that McrB‐N flips out modified bases into a protein pocket and binds cytosine derivatives in the order of descending affinity: 4mC > 5mC > 5hmC ≫ 5fC. We also show that pocket mutations alter the relative preference of McrB‐N to 5mC, 5hmC, and 4mC

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

<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 KMnO₄. We find that only KMnO₄ 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

The modified cytosine binding pockets.

<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.

<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⁻¹. Reaction rate constants for other substrates were lower than 1×10⁻⁵ min⁻¹. (B) Electrophoretic mobility shift assay with LpnPI. DNA binding experiments were performed in a pH 6.3 buffer in the presence of 5 mM Ca²⁺ 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 Ca²⁺ ions. (D) Electrophoretic mobility shift assay with LpnPI in a pH 8.3 buffer in the presence of Ca²⁺ ions.</p

DNA cleavage and binding by YkrI and BmeDI.

<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 Ca²⁺ 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

Crystal structure of the EcoKMcrA N-terminal domain (NEco): recognition of modified cytosine bases without flipping

EcoKMcrA from Escherichia coli restricts CpG methylated or hydroxymethylated DNA, and may act as a barrier against host DNA. The enzyme consists of a novel N-terminal specificity domain that we term NEco, and a C-terminal catalytic HNH domain. Here, we report that NEco and full-length EcoKMcrA specificities are consistent. NEco affinity to DNA increases more from hemi- to full-methylation than from non- to hemi-methylation, indicating cooperative binding of the methyl groups. We determined the crystal structures of NEco in complex with fully modified DNA containing three variants of the Y5mCGR EcoKMcrA target sequence: C5mCGG, T5mCGA and T5hmCGA. The structures explain the specificity for the two central base pairs and one of the flanking pairs. As predicted based on earlier biochemical experiments, NEco does not flip any DNA bases. The proximal and distal methyl groups are accommodated in separate pockets. Changes to either pocket reduce DNA binding by NEco and restriction by EcoKMcrA, confirming the relevance of the crystallographically observed binding mode in solution