Identification of new Fis binding sites by DNA scission with Fis-1,10-phenanthroline-copper(I) chimeras.
The chimeric nuclease Fis-OP has been used to identify novel Fis binding sites. Tethering the chemical nuclease OP-Cu+ to position 73 of the protein with a newly developed longer acetyl-beta-alanylamino spacer has facilitated the localization of two high-affinity Fis binding sequences in a 3 kb pUC19 plasmid. The shorter acetamido linker has allowed the chimeric nuclease to locate two strong Fis binding sites in the 50 kb phage lambda genome. All four sites reside in biologically interesting loci and have been confirmed by gel-retardation and DNase I footprint analyses. A newly discovered site resides in the lac operon of Escherichia coli. The binding of Fis to this site may antagonize repression by the LacI repressor. These studies demonstrate the feasibility of applying chimeric chemical nucleases to the task of identifying functional protein binding sites of biological interest within genomes without any assumption about their sequence preference.
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Molecular Biology Institute, University of California, Los Angeles 90095-1570, USA.
Johnson
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Sigman
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D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002614:Chelating Agents; D002874:Chromosome Mapping; D003300:Copper / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D016679:Genome, Viral; D034982:Integration Host Factors; D007763:Lac Operon; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010618:Phenanthrolines / Q000737:chemistry; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D002614:Chelating Agents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; D010618:Phenanthrolines; D011993:Recombinant Fusion Proteins; C440502:integration host factor, E coli; D003300:Copper; C025205:1,10-phenanthroline