DNA methyltransferases (MTases) are found in a wide variety of prokaryotes and eukaryotes. In prokaryotes, MTases have most often been identified as elements of restriction/modification systems in which they act to protect host DNA from cleavage by the corresponding restriction endonuclease. CpG MTases found in higher eukaryotes(e.g., Dnmt1) are not involved in restriction and modification. Nonetheless, patterns of CpG methylation are heritable, tissue specific, and correlate with gene expression. Consequently CpG methylation has been postulated to play a role in differentiation and gene expression (1). CpG methylase patterns can be approximated in vitro using the prokaryotic MTase, M.Sss I (NEB #M0226).
We have screened a wide variety of restriction endonucleases whose recognition sites can be modified by M.Sss I in order to determine if cleavage is blocked by this modification. Three broad classes of enzymes were identified: (i) Those whose cleavage was completely blocked by modification, (ii) those whose cleavage was not blocked by modification, and (iii) those whose cleavage was blocked or impaired when the canonical restriction site overlapped the CG dinucleotide. The results are summarized below.
DNA modified by M.Sss I is restricted by McrA, McrBC, and Mrr systems in E. coli (2,3). Accordingly, DNAs which have been treated with M.Sss I should be transformed into Mcr- Mrr- strains, such as those available without charge from New England Biolabs.
In vivo methylation by M.Sss I has proven useful in overcoming methylation-based restriction during transformation (4) and in mapping chromatin structure in vivo (5).
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Note: Restriction enzyme cleavage is blocked when the recognition sequence is methylated by the cognate methylase. Methylation at other bases can block cleavage, leave cleavage unaffected or slow the rate or extent of cleavage. The rate of cleavage may also be affected by the DNA sequence flanking the recognition site. As a result, cleavage may depend on reaction conditions and on the site being studied, and cleavage of a substrate with multiple sites may yield a mixture of complete and partial digestion products. The accompanying tables should be viewed as a guide to the behavior of the enzymes listed rather than an absolute indicator. Consult REBASE, the restriction enzyme database, for more detailed information and specific examples upon which these guidelines are based.
- Cleavage rate slowed significantly (impaired) by methylation.
- Multiple overlaps required to block cleavage