Transforming the Escherichia coli Trp repressor into a site-specific nuclease.
The Escherichia coli Trp repressor has been converted into an operator-specific nuclease by alkylating cysteine-49, inserted by site-directed mutagenesis, with 5-(iodoacetamido)-1,10-phenanthroline. In the presence of copper ion and thiol, high yields (> 50%) of double-stranded breaks of DNA are observed after a 20-min reaction. The high cleavage efficiency of this derivatized protein (Trp repressor E49C-OP) can be attributed to the proximity of cysteine-49 to the minor groove, the site of the C-1H of the deoxyribose and the target of the oxidative nuclease activity of (1,10-phenanthroline)copper. Since sequence position 49 is close to the protein's C2 dyad axis and adjacent to the minor groove, Trp repressor E49C-OP reacts with the operator DNA near the binding site of this symmetry locus of the protein. The patterns of scission of the trpR, aroH, and trpEDCBA operators (a) confirm the orientation of the repressor to the operator predicted from the X-ray study of a cocrystal (Otwinowski et al., 1988) and (b) support the model for tandem binding of the repressor to the trpR, aroH, and trpEDCBA operators based on DNase I footprinting and methylation interference (Kumamoto et al., 1987). There are one, two, and three binding sites for the repressor on the trpR, aroH, and trpEDCBA operators, respectively. In addition to providing a novel approach to studying the interactions of DNA binding proteins, 1,10-phenanthroline-derivatized proteins such as Trp repressor E49C-OP may be useful as rare cutters in the analysis of high molecular weight DNAs, especially if their binding specificities can be altered.
Sutton
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Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024-1570.
Mazumder
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A
Chen
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Sigman
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D013698:Templates, Genetic; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D003545:Cysteine; D004269:DNA, Bacterial; D003851:Deoxyribonucleases; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins