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DNA methyltransferases (MTases) that transfer a methyl group from S-adenosylmethionine to either adenine or
cytosine residues, are found in a wide variety of prokaryotes and eukaryotes. Methylation should be considered
when digesting DNA with restriction endonucleases because cleavage can be blocked or impaired when a particular
base in the recognition site is methylated.
Prokaryotic Methylation
In prokaryotes, MTases have most often been identified as elements of restriction/modification systems that
act to protect host DNA from cleavage by the corresponding restriction endonuclease. Most laboratory strains
of E. coli contain three site-specific DNA methylases.
- Dam methylase–methylation at the N6 position of the adenine in the sequence GATC (1,2).
- Dcm methylase–methylation at the C5 position of cytosine in the sequences CCAGG and CCTGG
(1,3).
- EcoKI methylase–methylation of adenine in the sequences AAC(N6A)GTGC and GCAC(N6A)GTT.
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 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, which cleaves at GATC sites.
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.
Restriction sites that are blocked by Dam or Dcm methylation can be un-methylated by cloning your DNA into
a dam–, dcm– strain of E.
coli, such as dam–/dcm– Competent E.
coli (NEB #C2925).
Restriction sites can also be blocked if an overlapping site is present. In this case, part of the Dam or
Dcm sequence is generated by the restriction enzyme sequence, followed by the flanking sequence. This situation
should also be considered when designing restriction enzyme digests.
Eukaryotic Methylation
CpG MTases, found in higher eukaryotes (e.g., Dnmt1), transfer a methyl group to the C5 position of cytosine
residues. 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 (4).
Note: The effects of CpG methylation are mainly a concern when digesting
eukaryotic genomic DNA. CpG methylation patterns are not retained once the DNA is cloned into a bacterial
host.
Methylation Sensitivity
The table below summarizes methylation sensitivity for NEB restriction enzymes, indicating whether or not
cleavage is blocked or impaired by Dam, Dcm or CpG methylation if or when it overlaps each recognition site.
This table 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.
References
- Marinus, M.G. and Morris, N.R. (1973) J. Bacteriol. 114, 1143–1150.
- Geier, G.E.and Modrich, P. (1979) J. Biol. Chem. 254, 1408–1413.
- May, M.S. and Hattman, S.(1975) J. Bacteriol. 123, 768–770.
- Siegfried, Z. and Cedar, H. (1997) Curr. Biol. 7, r305–307.
| Legend |
 |
Not Sensitive |
|
|
 |
Blocked |
 |
Impaired |
 |
Blocked by Overlapping |
 |
Impaired by Overlapping |
 |
Blocked by Some Combinations of Overlapping |
 |
Impaired by Some Combinations of Overlapping |
A | B | C | D | E | F | H | I | K | M | N | P | R | S | T | X | Z | single letter code
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