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 enzymes.

There is a strong 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. There is a fine balance that exists naturally between rstriction enzymes and methylases which can be easily disrupted. This is why endogenous promoters are often preferred.

Methylation as a protection device against resriction enzymes is well documented, and has been proven to work. 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. 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 transfer a methyl group to 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.

References

[1] Alfred M. Pingoud, 2004, Restriction Endonucleases (Nucleic Acids and Molecular Biology), Springer pp10-40

[2] Luke, McCallum and Halford, 1987, The EcoRV restriction endonuclease|Gene Amplification and Analysis pp183-205

Module 3 - Genome Deletion