Chemical Conversion of a DNA-Binding Protein into a Site-Specific Nuclease

Abstract

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.

Gal4-VP16 and Gal4-AH Increase the Orientational and Axial Specificity of TATA Box Recognition by TATA Box Binding Protein

“…All five Gal4 binding sites contain the same 17 bp sequence. (72). Addition of cupric ion, hydrogen peroxide, and a reducing agent to K97C-OP generated a reactive yet nondiffusible OP-Cu complex (73,74) able to abstract hydrogen atoms from the deoxyribose backbone and effect DNA cleavage.…”

Metal–nucleic acid interactions

“…Through this scheme, again the conversion of a DNA-binding moiety into a cleaving moiety by incorporation of a redox-active metal, the specific binding sites of repressor proteins can be readily identified (far more quickly on large DNA than through footprinting). 78 Another scheme, which perhaps takes advantage more directly of bioinorganic chemistry, involves engineering redox metal-binding sites into DNA-binding proteins and peptides. The DNA-binding domain of the protein Hin recombinase was synthesized chemically, and first, to examine the folding of the peptide on the DNA helix, EDTA was tethered onto the peptide for Fe(II) cleavage experiments.…”

DNA‐binding proteins as site‐specific nucleases

“…This protein is composed of two 108 amino aoid polypeptide chains and requires tryptophan for high-affinity site-specific binding. It is stable and can be expressed in high yields by using vectors kindly provided by UCLA colleague Robert Gunsalus (Gunsalus and Yanofsky, 1980), We had previously converted it into a site-specific scission reagent by modifying its four lysyl residues successively with iminothiolane and 5-iodoacetamido-1,10-phenanthroline (Chen and Sigman, 1987). Although chemically heterogeneous, this derivatized protein cleaved the trpEDCBA and aroH operators in a tryptophan-dependent reaction.…”