Abstract
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.
Highlights
Discrimination between methylated and unmethylated DNA was observed in electrophoretic mobility shift experiments (EMSA): both LpnPI and the N-terminal LpnPI DNA binding domain (LpnPI-N) formed protein-DNA complexes with methylated DNA at much lower protein concentrations than with unmethylated DNA (Fig. 2B,C)
The replacement of the target base with the fluorescent base pyrrolocytosine is a convenient method to detect base flipping by measuring the changes in pyrrolocytosine fluorescence intensity upon protein binding
For the method to work, the protein in question must have an adequately sized protein pocket to accommodate the bulky pyrrolocytosine base. While this seems to be the case with the DNA binding domain of McrBC, modeling, DNA binding and cleavage studies indicate that the SRA-like domains of MspJI- and PvuRts1I-like enzymes used in our study do not tolerate such a replacement (Figs. 2, 3 and S2 Figure)
Summary
5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are important epigenetic modifications of mammalian and plant DNA. Mutational analysis and close structural resemblance of DNA binding domains of AspBHI, PvuRts1I and AbaSI to the SRA domains of MspJI and eukaryotic proteins (Fig. 1C-D) suggest that these enzymes flip-out the modified cytosine [18, 20,21,22,23]. For many eukaryotic 5(h)mC-binding proteins identified in the recent pull-down and mass-spectrometry studies [24, 25], the mechanism of modified cytosine recognition remains unknown. Mechanistic studies of these proteins would benefit from a fast solution-based method for the detection of extrahelical 5(h)mC. Fluorescence measurements of the cytosine analog pyrrolocytosine and chemical display of extrahelical bases [26]
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