STABLE FLUORESCENT DYE-DNA COMPLEXES IN HIGH-SENSITIVITY DETECTION OFPROTEIN-DNA INTERACTIONS - APPLICATION TO HEAT-SHOCK TRANSCRIPTION FACTOR

The gel mobility-shift assay is an important tool for the study of pro tein-nucleic acid interactions. High detection sensitivity is typicall y attained by radioisotopic labeling of the target nucleic acid fragme nts. A novel fluorescence methology offers significant advantages over this conventional approach. Ethidium, thiazole orange, and oxazole ye llow homodimers form stable, highly fluorescent complexes with double- stranded DNA that can be detected in gels by a laser-excited, confocal , fluorescence scanning system with a sensitivity higher than that att ainable with radioisotopic labeling. We describe here the use of these dyes in a gel-mobility assay to detect complexes of a truncation of t he Kluyveromyces lactis heat shock transcription factor, containing th e trimerization and DNA-binding domains (HSF(DT)), with target DNA. At an appropriate molar DNA base pair to dye ratio, the labeling of a DN A fragment with dimeric dye did not affect the binding to HSF(DT). The detection of the fluorescent-dye labeled HSF(DT)-DNA complexes with t he laser scanner achieves a spatial resolution far superior to that of conventional autoradiography and permits analysis of multimer protein -DNA complexes that are not resolved by traditional detection methods. We have used this technique to demonstrate that HSF forms multimeric complexes on DNA by addition of trimeric units. The latter conclusion is based on an analysis of the mobilities of the multiple HSF(DT)-DNA complexes and on a two-color mobility-shift fluorescence assay that us es a mutant of HSF(DT) engineered for site-specific labeling with fluo rescein and target DNA labeled with an ''energy transfer'' dye, thiazo le orange-thiazole blue heterodimer.