Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains.
This study concerns chimeric restriction enzymes that are hybrids between a zinc finger DNA-binding domain and the non-specific DNA-cleavage domain from the natural restriction enzyme FOK:I. Because of the flexibility of DNA recognition by zinc fingers, these enzymes are potential tools for cleaving DNA at arbitrarily selected sequences. Efficient double-strand cleavage by the chimeric nucleases requires two binding sites in close proximity. When cuts were mapped on the DNA strands, it was found that they occur in pairs separated by approximately 4 bp with a 5' overhang, as for native FOK:I. Furthermore, amino acid changes in the dimer interface of the cleavage domain abolished activity. These results reflect a requirement for dimerization of the cleavage domain. The dependence of cleavage efficiency on the distance between two inverted binding sites was determined and both upper and lower limits were defined. Two different zinc finger combinations binding to non-identical sites also supported specific cleavage. Molecular modeling was employed to gain insight into the precise location of the cut sites. These results define requirements for effective targets of chimeric nucleases and will guide the design of novel specificities for directed DNA cleavage in vitro and in vivo.
Smith
J
J
Department of Environmental Health Sciences, The Johns Hopkins University School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, MD 21205, USA.
Bibikova
M
M
Whitby
F G
FG
Reddy
A R
AR
Chandrasegaran
S
S
Carroll
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific