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Science 4 September 1987:
Vol. 237. no. 4819, pp. 1197 - 1201
DOI: 10.1126/science.2820056
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Articles

Science, Vol 237, Issue 4819, 1197-1201
Copyright © 1987 by American Association for the Advancement of Science

articles

Chemical conversion of a DNA-binding protein into a site-specific nuclease

CH Chen and DS Sigman

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.


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Mechanistic studies on DNA damage by minor groove binding copper-phenanthroline conjugates.
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Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA.
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Nucleic Acids Res. 28, e52
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Bidirectional binding of the TATA box binding protein to the TATA box.
J. M. Cox, M. M. Hayward, J. F. Sanchez, L. D. Gegnas, S. van der Zee, J. H. Dennis, P. B. Sigler, and A. Schepartz (1997)
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High-specificity DNA cleavage agent: design and application to kilobase and megabase DNA substrates.
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The interplay between chemistry and biology in the design of enzymatic catalysts.
P. Schultz (1988)
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