4 publications
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Chemical Conversion of a DNA-Binding Protein into a Site-Specific Nuclease
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Science 1987, 237, 1197-1201, 10.1126/science.2820056
The tryptophan gene (trp) repressor of Escherichia coli has been converted into a site-specific nuclease by covalently attaching it to the 1,10-phenanthroline-copper complex. In its cuprous form, the coordination complex with hydrogen peroxide as a coreactant cleaves DNA by oxidatively attacking the deoxyribose moiety. The chemistry for the attachment of 1,10-phenanthroline to the trp repressor involves modification of lysyl residues with iminothiolane followed by alkylation of the resulting sulfhydryl groups with 5-iodoacetamido-1,10-phenanthroline. The modified trp repressor cleaves the operators of aroH and trpEDCBA upon the addition of cupric ion and thiol in a reaction dependent on the corepressor L-tryptophan. Scission was restricted to the binding site for the repressor, defined by deoxyribonuclease I footprinting. Since DNA-binding proteins have recognition sequences approximately 20 base pairs long, the nucleolytic activities derived from them could be used to isolate long DNA fragments for sequencing or chromosomal mapping.
Metal: CuLigand type: PhenanthrolineHost protein: Tryptophan gene repressor (trp)Anchoring strategy: CovalentOptimization: ---Notes: Engineered sequence specificity
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Flavohemoglobin: A Semisynthetic Hydroxylase Acting in the Absence of Reductase
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J. Am. Chem. Soc. 1987, 109, 606-607, 10.1021/ja00236a062
n/a
Notes: ---
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Generation of a Hybrid Sequence-Specific Single Stranded Deoxyribonuclease
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Science 1987, 238, 1401-1403, 10.1126/science.3685986
The relatively nonspecific single-stranded deoxyribonuclease, staphylococcal nuclease, was selectively fused to an oligonucleotide binding site of defined sequence to generate a hybrid enzyme. A cysteine was substituted for Lys116 in the enzyme by oligonucleotide-directed mutagenesis and coupled to an oligonucleotide that contained a 3'-thiol. The resulting hybrid enzyme cleaved single-stranded DNA at sites adjacent to the oligonucleotide binding site.
Metal: CaLigand type: UndefinedHost protein: Staphylococcal nucleaseAnchoring strategy: ---Optimization: ---Notes: Engineered sequence specificity
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Synthesis of a Sequence-Specific DNA-Cleaving Peptide
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Science 1987, 238, 1129-1132, 10.1126/science.3120311
A synthetic 52-residue peptide based on the sequence-specific DNA-binding domain of Hin recombinase (139-190) has been equipped with ethylenediaminetetraacetic acid (EDTA) at the amino terminus. In the presence of Fe(II), this synthetic EDTA-peptide cleaves DNA at Hin recombination sites. The cleavage data reveal that the amino terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin recombination sites. This work demonstrates the construction of a hybrid peptide combining two functional domains: sequence-specific DNA binding and DNA cleavage.
Metal: FeLigand type: EDTAHost protein: Domain of Hin recombinaseAnchoring strategy: CovalentOptimization: ---Notes: Engineered sequence specificity