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Chemical Conversion of a DNA-Binding Protein into a Site-Specific Nuclease
Chi-Hong B. Chen and David S. Sigman
New Series, Vol. 237, No. 4819 (Sep. 4, 1987), pp. 1197-1201
Published by: American Association for the Advancement of Science
Stable URL: http://www.jstor.org/stable/1699519
Page Count: 5
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The tryptophan gene (trp) repressor of Escherichia coli has been converted into a sitespecific 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.
Science © 1987 American Association for the Advancement of Science