Question

You have been asked to clone the first enzyme of the leucine biosynthesis pathway from E. coli. To accomplish this objective, you have decided to purify the genomic DNA and treat with EcoRI (5’-G?AATTC-3’) restriction endonuclease. The genome size for E. coli is 6x106 bp (base pairs). After treatment with EcoRI with proper reaction conditions, you have found that the DNA was not digested by the restriction endonuclease EcoR1. However, when the same purified genomic DNA was treated with another restriction endonuclease BalI (5’-TGG?CCA-3’), the DNA was completely digested. The commercially purchased control E. coli DNA showed complete digestion under similar reaction conditions confirming that the EcoRI enzyme is functional and that the E. coli chromosome has multiple EcoRI restriction sites. What is the most probable cause for the E. coli DNA was not be digested by the EcoRI enzyme?

Answer 1

Restriction endonuclease can cleave DNA into fragments. Different RE has different restriction sites on DNA and different restriction endonucleases are isolated from different bacteria. Possibly, bacterial own DNA can also be digested with the same endonucleases but Bacteria have their own mechanism for protection against restriction endonuclease activity. Methylation protects bacterial DNA from digestion. Specific RE has specific cutting site present on DNA. Several bases present on this particular site is methylated, hence RE can’t recognize their cutting site and the activity of RE is nullify.

In this case isolated E.coli DNA can’t digest with EcoR1 due to this possible reason. However it is not protected against foreign restriction endonucleases. So it was completely digested with Ball (because Ball restriction endonuclease are not isolted from E.coli). Commercially available control Ecoli DNA is unmethylated. Hence EcoR1 can digest it. Mehtylation is a natural mechanism to protect bacteria from their own restriction endonucleases.