Nucleic Acids Research, Vol 27, Issue 2 674-681, Copyright © 1999 by Oxford University Press
J Smith, JM Berg and S Chandrasegaran
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.
ARTICLES
A detailed study of the substrate specificity of a chimeric restriction enzyme
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine,725 North Wolfe Street, Baltimore, MD 21205-2179, USA.
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