A detailed study of the substrate specificity of a chimeric restriction enzyme

Recently, the crystal structure of the designed zinc finger protein, Delta QNK, bound to a preferred DNA sequence was reported. We have converted Delta QNK into a novel site-specific endonuclease by linking it to the Fokl cleavage domain (F-N) The substrate specificity and DNA cleavage properties of the resulting chimeric restriction enzyme (Delta QNK-F-N) were investigated, and the binding affinities of Delta QNK and Delta QNK-F-N for various DNA substrates were determined. Substrates that are bound by Delta QNK with high affinity are the same as those that are cleaved efficiently by Delta QNK-F-N. Substrates bound by Delta QNK with lower affinity are cleaved with very low efficiency or not at all by Delta QNK-F-N. The binding of Delta QNK-F-N to each substrate was similar to 2-fold weaker than that for Delta QNK. Thus, the fusion of the Fokl cleavage domain to the zinc finger motif does not change the DNA sequence specificity of the zinc finger protein and does not change its binding affinity significantly.