title: A detailed study of the substrate specificity of a chimeric restriction enzyme.
abstract: 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.
authors.affiliation: Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine,725 North Wolfe Street, Baltimore, MD 21205-2179, USA.
authors.forename: J
authors.initials: J
authors.lastname: Smith
authors.forename: J M
authors.initials: JM
authors.lastname: Berg
authors.forename: S
authors.initials: S
authors.lastname: Chandrasegaran
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D004247:DNA; D004262:DNA Restriction Enzymes; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific