Drybench Plasmid.
dam and dcm methylation and blocked sites
Plasmid DNA grown in most E. coli strains carries methyl groups at GATC and CCWGG, and some enzymes cannot cut a site where one falls.
What the methylases mark
The dam methylase adds a methyl group to the adenine of GATC on both strands (Geier and Modrich 1979), and E. coli strains lacking it can be isolated (Marinus and Morris 1973). The dcm methylase marks the second cytosine of CCWGG, where W is A or T.
A site blocks only when a methylated base falls on a position the enzyme is sensitive to. That is recorded enzyme by enzyme in a compilation by McClelland, Nelson and Raschke (1994). For example, XbaI's TCTAGA is blocked when a GATC overlaps its end.
Blocked, cuts or may be blocked
The map marks each site as cutting, blocked by dam or dcm, or may be blocked. A site reads "may be blocked" when a mark falls on it and the compilation records nothing that settles the question; it is never counted as a cut. DNA from a PCR or made synthetically carries no marks.
DpnI is the reverse case: it cuts GATC only when it is methylated, so it digests plasmid grown in a dam host and leaves PCR products alone.
See it on a real plasmid
Open pUC19, switch the host between dam⁺ dcm⁺ and PCR product, and watch which sites change.
Methyl marks sit on the DNA bases themselves; the tour's first stop shows the double helix and how it is packed.
References
- McClelland M, Nelson M, Raschke E. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res 1994;22:3640-3659. PubMed 7937074
- Geier GE, Modrich P. Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease. J Biol Chem 1979;254:1408-1413. PubMed 368070
- Marinus MG, Morris NR. Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J Bacteriol 1973;114:1143-1150. PubMed 4576399
Statements summarised by the site's builder from the references, for scientific review.