Sharp DNA bends as landmarks of protein-binding sites on straightened DNA

We have developed a fluorescence-based method for mapping single or multipl e protein-binding sites on straightened, large-size DNA molecules (>5 kbp). In the described method, protein-DNA complexes were straightened and immob ilized on a flat surface using surface tension. A fraction of the immobiliz ed complexes displayed a sharp DNA bend with two DNA segments extending fro m the apex. The presence of DNA-binding proteins at the apex was verified b y 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 tem plate and to evaluate relative binding affinity. The proposed protein-bindi ng-site mapping technique is simple and easy to perform. Practical applicat ions include screening for DNA-binding proteins and the localization of pro tein-binding sites on large segments of DNA.