Chemical conversion of a DNA-binding protein into a site-specific nuclease.
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.
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D001426:Bacterial Proteins; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D007202:Indicators and Reagents; D010618:Phenanthrolines / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004262:DNA Restriction Enzymes; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D007202:Indicators and Reagents; D010618:Phenanthrolines; D012097:Repressor Proteins; D014157:Transcription Factors; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins; C014741:bis(1,10-phenanthroline)copper(2+) ion