An exonuclease protection assay reveals heat-shock element and TATA box DNA-binding proteins in crude nuclear extracts.
The ability to identify and purify trans-acting cellular factors that regulate eukaryotic genes is limited by the lack of a practical general assay. Current procedures using crude whole cell or nuclear extracts that restore transcriptional function in vitro or permit reconstruction of native chromatin at control sequences are effective only in select systems. I now present an exonuclease protection assay that is generally applicable for detecting sequence-specific DNA-binding proteins. The assay extends earlier work on the binding to the Drosophila heat-shock gene control element of a protein factor (HAP) present in crude nuclear extracts; the binding was shown by reconstitution of specific exonuclease resistance within a nuclease-hypersensitive site in chromatin. We show here that this same exonuclease resistance can be reconstituted on free linear DNA, despite many nonspecific binding activities present in unfractionated nuclear extracts. We have further applied this assay method to fractionate the protein factor that is bound constitutively to the heat-shock gene TATA box region in native chromatin. Exonuclease protection offers a sensitive, precise and rapid assay for any sequence-specific DNA-binding protein.
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D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics; D005092:Exonucleases / Q000378:metabolism*; D005796:Genes; D006360:Heat-Shock Proteins / Q000235:genetics; D014158:Transcription, Genetic
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D005092:Exonucleases