Sharp DNA bends as landmarks of protein-binding sites on straightened DNA.
We have developed a fluorescence-based method for mapping single or multiple protein-binding sites on straightened, large-size DNA molecules (> 5 kbp). In the described method, protein-DNA complexes were straightened and immobilized on a flat surface using surface tension. A fraction of the immobilized complexes displayed a sharp DNA bend with two DNA segments extending from the apex. The presence of DNA-binding proteins at the apex was verified by atomic force microscopy. The position of protein binding relative to the ends of the DNA molecule was determined by measuring the length of two DNA segments using fluorescence microscopy. We demonstrate the potential of the fluorescence-based method to localize protein-binding sites on the DNA template and to evaluate relative binding affinity. The proposed protein-binding-site mapping technique is simple and easy to perform. Practical applications include screening for DNA-binding proteins and the localization of protein-binding sites on large segments of DNA.
D010582:Bacteriophage lambda / Q000235:genetics; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism