The E. coli strains used in the laboratory for cloning and DNA isolation contain three site-specific DNA methylation systems. Modification of DNA using these methylation systems protects the potential restriction enzyme recognition sites from cleavage. This genetic engineering technique sheds light on the sensitivity of restrictions endonuclease towards DNA methylation. Such DNA methylation systems are:
- Dam methylation: The Dam gene encodes for methylase enzyme that methylates the N-6 position of the adenine nucleotide of the sequence 5′-GATC-3′. Such methylation does not impair the ability of adenine to bind with thymine residue. This type of methylation happens in many type of cellular functions such as DNA replication. The newly synthesized DNA is hemimethylated during the DNA replication process. Dam methylase is a slow enzyme and eventually, both strands will be methylated. This methylation process is imporatnt because such methylation of origin of replication, oriC, will determine if replication will proceed or not. If the oriC is hemimethylated, replication process will not occur.
- Dcm methylation: Dcm methylase is another methylation enzyme that methylates C-5 postion of the internal cytosine residue of the sequence 5′-CCWGG-3′, where W is either adenine or thymine residue.
- Mcr system: Earlier, when DNA from various bacterial and eukaryotic sources could only be cloned at a lower efficiency in E. coli. This was because the incoming DNA was restricted by the host. This phenomenon was caused by methylcytosine in DNA and is called modified cytosine restriction (Mcr). Two genes in E. coli genome, McrB and McrC, that are located closer to the EcoRI restriction site. This is a naturally occuring modification-restriction system found in E. coli. Cleavage occurs at ultiple sites of the double stranded DNA between the methylated cytosines and requires GTP.