Inhibition of MspI cleavage activity by hydroxymethylation of the CpG site: a concern for DNA modification studies using restriction endonucleases.

In mammalian genomic DNA, cytosine methylation predominantly occurs at CpG dinucleotides and provides epigenetic information. In some cells, 5-methyl-cytosine (5-mC) can be further converted to 5-hydroxymethyl-cytosine (5-hmC) by the ten-eleven translocation family of proteins. MspI restriction endonuclease has been used to analyze these modified cytosines. However, the kinetic analysis in this study revealed that MspI activity is dramatically decreased by symmetrical hydroxymethylation of its recognition sequence and partly inhibited by hemi-hydroxymethylation, whereas TaqI and HaeIII are relatively resistant to hydroxymethylation. Therefore, DNA modification studies that use MspI, for example, reduced representation bisulfite shotgun sequencing, quantitative analysis of 5-hmC, and cleavage-sensitivity analysis, should be carefully interpreted.