Most laboratory strains of E. coli contain three site-specific DNA methylases. The methylase encoded by the dam gene (Dam methylase) transfers a methyl group from S-adenosylmethionine (SAM) to the N6 position of the adenine residues in the sequence GATC (1,2). The Dcm methylase (encoded by the dcm gene; referred to as the Mec methylase in earlier references) methylates the internal cytosine residues in the sequences CCAGG and CCTGG (1,3) at the C5 position. The EcoKI methylase, M. EcoKI, modifies adenine residues in the sequences AAC(N6)GTGC and GCAC(N6)GTT. EcoKI sites (~1 site per 8 kb) are much less common than Dam sites (~1 site per 256 bp) or Dcm sites (~1 site per 512 bp) in DNA of random sequence (GC=AT).
These methylases are of interest here for two reasons. First, some or all of the sites for a restriction endonuclease may be resistant to cleavage when isolated from strains expressing the Dam or Dcm methylases. This occurs because DNA is protected from cleavage when a particular base in the recognition site of a restriction endonuclease is methylated. The relevant base may be methylated by one of the E. coli methylases if the methylase recognition site overlaps the endonuclease recognition site. For example, plasmid DNA isolated from Dam+ E. coli is completely resistant to cleavage by MboI (NEB #R0147), which cleaves at GATC sites. The table below summarizes the possible overlaps and what is known about cleavability of DNA modified by Dam or Dcm. Almost all cloning strains are Dam+Dcm+, and many are M+ EcoKI. For more detailed information about K methylation, consult REBASE, the restriction enzyme database.
The second reason these methylases are of interest is that the modification state of plasmid DNA can affect the frequency of transformation in special situations. Transformation efficiency will be reduced when 1) Dam-modified plasmid DNA is introduced into Dam- E. coli (one might do this in order to prepare DNA for digestion with BclI (NEB #R0160), for example); and 2) Dam or Dcm-modified DNA is introduced into certain other bacterial species. In the first instance, it has been shown that replication initiation is suppressed when plasmid DNA is hemimethylated at Dam sites (4). Dam-modified plasmids therefore replicate once in Dam- cells, and are unable to replicate again. The second phenomenon, poor transformation into other bacterial species, has not been comprehensively characterized, but may be due to modification-dependent restriction systems (5-8) analogous to the Mcr and Mrr systems found in E. coli. By using Dam- Dcm- strains to grow shuttle vectors, the modification-dependent restriction found in such bacteria can be avoided.
Not all DNA isolated from E. coli is methylated to the same extent. While pBR322 DNA is fully modified (and is therefore completely resistant to MboI digestion), only about 50% of λ DNA Dam sites are methylated, presumably because the methylase does not have the opportunity to methylate the DNA fully before it is packaged into the phage head. As a result, enzymes blocked by Dam or Dcm modification will yield partial digestion patterns with λ DNA.
- Marinus, M.G. and Morris, N.R. (1973) J. Bacteriol. 114, 1143-1150. PMID: 4576399
- Geier, G.E.and Modrich, P. (1979) J. Biol. Chem. 254, 1408-1413. PMID: 368070
- May, M.S. and Hattman, S.(1975) J. Bacteriol. 123, 768-770. PMID: 1097428
- Russell, D.W. and Zinder, N.D. (1987) Cell 50, 1071-1079. PMID: 3304662
- MacNeil, D.J. (1988) J. Bacteriol. 170, 5607-5612. PMID: 3068070
- Holmes, M.L. et al (1991) J. Bacteriol. 173, 3807-3813. PMID: 1711028
- Sladek, R.L. et al (1986) J. Bacteriol. 165, 219-225. PMID: 3001023
- Macaluso, A. and Mettus, A.M. (1991) J. Bacteriol. 173, 1353-1356. PMID: 1991728
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.
Table Notes: Recognition sites appear in bold type. Sequence, in addition to the recognition site, required to produce an overlapping methylation site appears as normal type (not bold). Bases constrained by the requirements of an overlapping methylation site that would otherwise be degenerate (N, R, or Y) are in red. For palindromic enzymes, both ends of the recognition sequence must be considered for possible overlapping methylation, e.g. ClaI is blocked by Dam methylation at GATCGAT and ATCGATC.
Table data from: REBASE
- Cleavage rate slowed significantly (impaired) by methylation.
- Multiple overlaps required to block cleavage.
Single Letter Code
|Dam Methylation: GmATC||Dcm Methylation: CmCWGG|
|Blocked by Overlapping Dam||Blocked by Overlapping Dcm|
|Not Blocked by Overlapping Dam||PspGI||CCWGG|
|Not Blocked by Overlapping Dcm|