Effects of CpG Methylation on Restriction Enzyme Cleavage
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).
NEB has 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. The information is located on the NEBuffer™ Activity/Performance Chart with Restriction Enzymes along with methylation sensitivities for Dam and Dcm methylases.
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).
- Siegfried, Z. and Cedar, H. (1997) Curr. Biol. 7, r305-307. PMID: 9115385
- Kelleher, J.E. and Raleigh, E. (1991) J. Bacteriol. 173, 5220-5223. PMID: 1830580
- Rees, P. et al. (1991) J. Bacteriol. 173, 5207-5219. PMID: 1650347
- Butler, C.A. and Gotschlich, E.C. (1991) J. Bacteriol. 173, 5793-5799. PMID: 1653220
- Kladde, M.P. and Simpson, R.T. (1996) Methods Enzymol. 274, 214-233. PMID: 8902807