Splase: a new class IIS zinc-finger restriction endonuclease with specificity for Sp1 binding sites.
A new restriction endonuclease, named Splase, was constructed by genetically fusing the DNA-cleavage domain of the restriction endonuclease Fok1 with the zinc-finger DNA-binding domain of the transcription factor Sp1. The resulting protein was expressed in Escherichia coli., partially purified, and shown to selectively digest plasmid DNA harboring consensus Sp1 sites. Splase was also shown to selectively digest the long terminal repeat of the HIV-1 DNA at Sp1 sites. Splase recognizes a 10-bp DNA sequence and hydrolyzes phosphodiester bonds upstream of the binding sequence. The binding specificity of Splase makes this a "rare cutter" restriction enzyme which could be valuable in creating large DNA fragments for genome sequencing projects. The result also presents the opportunity to create other restriction enzymes by altering the binding specificity of the zinc-finger recognition helix.
Ohio State Biochemistry Program, Ohio State University, Columbus 43210, USA.
B
B
Huang
C J
CJ
Schaeffer
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Q
Li
M D
MD
Tsai
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D016335:Zinc Fingers
D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D004247:DNA; C104571:splase; D015252:Deoxyribonucleases, Type II Site-Specific