Module 3: Genome Deletion Overview

Dam methylation

Restriction enzymes often come together with methylation enzymes to form a restriction-modification system. This well-known combination prevents the genome of the cell from being cleaved by its own restriction enzyme.

In our system, to protect against DNA destruction due to basal levels of restriction enzyme production, we have made use of the native E. coli Dam methylase protection system. Methylation as a protection device against resriction enzymes is well documented, and has been proven to work using its endogenous promoter. The Dam system is chosen as both DpnII and TaqI enzyme activity can be blocked by Dam methylation.

Dam methylases recognise the sequence GATC and methylate the Adenine base. This prevents the restriction enzymes from recognising the sequence and cleaving it. Therefore, only high levels of restriction enzyme (ie. after thermal triggering) will cleave the DNA.

There is an asymmetry between the function of restriction enzymes and methylases. Restriction enzymes can cause just one cleavage, which if unrepaired, kills the cell. However, to effectively protect the cell, methylases need to methylate all the recognition sites.

The methylase protection system has been used with its native promoter to prevent basal levels of restriction enzyme expression from killing the cell. The use of a native promoter shows the highly sensitive balance that exist between restriction enzymes and methylases.