Module 3: Genome Deletion Overview

Dam methylation

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.

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.

Module 3 - Genome Deletion