Restriction endonucleases are enzymes which cut DNA at specific target sequences. The proteins of this family recognize approximately 300 different specific sequences, which has made them irreplaceable tools for the manipulation of DNA, and a necessary part of the modern molecular biology toolbox.

The specificity of a restriction endonuclease for its target DNA can be achieved through mechanisms such as direct readout (direct contact between protein and DNA), indirect readout (utilizing molecules such as water to bridge between the protein and DNA), and DNA distortion (altering the DNA structure upon protein binding). These mechanisms of recognition are intimately connected to the catalytic mechanisms, making it difficult to engineer one aspect without affecting the other.

We are particularly interested in the latter two mechanisms, and how they contribute to the function of the restriction endonuclease HincII, which utilizes both indirect readout and DNA distortion to increase its specificity. This enzyme possesses an intercalating strand, which seems to be the key to specific DNA distortion; bulky side chains behind this strand stabilize its shift into the major groove of the DNA helix.

In order to better understand the role of these mechanisms in DNA recognition, we are re-designing the protein-DNA interface of HincII in order to change its DNA sequence specificity. Our approach is to utilize directed evolution, using random priming to alter this region. Desired HincII mutants with novel specificities will then be selected for through phage challenge.

Engineering of novel binding specificities, while useful in its own right, will also allow us to elucidate the mechanisms of DNA recognition, and allow for further rational design of novel DNA-binding proteins.