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

摘要

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