blog

A paper in the Indian Journal of Pathology and Microbiology describes a simple screening tool (evaluated by Samuel Kariuki) for detecting a mutation in Salmonella enterica that confers resistance to standard treatment options. The point mutation Ser 83–>Tyr or Ser 83–>Phe (TCC–>TAC or TTC, respectively) can be identified using the restriction enzyme SSiI, which recognizes the sequence C^CGC.

A little while ago, another restriction enzyme found its way into F1000. Petra Imhof’s lab in Heidelberg published an analysis of EcoRV binding to its recognition/cleavage sequence GATATC. Mechanisms of restriction cleavage usually concentrate on the protein, but this paper shows that the intrinsic flexibility of the DNA sequence is critical. I asked Petra a few questions about the work, and here’s what she said:

rpg: What’s so important about restriction enzymes in general, and what you’ve found specifically?

Petra: Restriction enzymes are crucial for the survival of the cell. They cleave invading foreign DNA at a specific DNA sequence which is recognised among an enormous molar excess of structurally similar non-specific DNA. DNA is not just a stiff double helix, but can be strongly bent.

EcoRV bends DNA before cleaving it and our computer simulation research provides a clear picture of the mechanism by which this bending, clamping and cleavage is achieved. Also, we have been able to show how the intrinsic propensity of specific DNA sequences to bend helps the enzyme be highly selective, and how the protein amplifies this propensity to clamp onto it.

rpg: What’s the wider impact of this research?

Petra: People might not know that DNA is intrinsically bendy, depending on the sequence, and that this is used by enzymes that cleave DNA to bind to it and distort it, hence looking at the recognition process (also) from a DNA perspective. This view can be transferred to DNA processing enzymes in general: the intrinsic DNA dynamics can help those enzymes find their target sites, such as specific sequences or mispaired or damaged DNA .

rpg: How did you get interested?

Petra: The University of Heidelberg has a special research area in catalysis, and research on metalloenzyme function forms part of that. We decided that these restriction enzymes, or DNA scissors, that are so commonly used in molecular biology labs, needed to be understood in detail. We felt that besides investigating the catalytic cleavage mechanism itself, we should learn more about how the enzyme achieves its specificity and elucidate the mechanism of sequence recognition.

The paper, evaluated by B Montgomery Pettitt , is Mechanism of DNA recognition by the restriction enzyme EcoRV and you can read it in full at J Mol Biol.