Mapping a protein-binding site on straightened DNA by atomic force microscopy

We have developed an Atomic Force Microscopy (AFM)-based method for mapping protein-binding sites on individual, long DNA molecules (>5 kb) at nanomet er resolution. The protein is clearly detected at the apex of the bent DNA molecules. Randomly coiled DNA molecules or protein:DNA complexes were exte nded by a motor-controlled moving meniscus on an atomically hat surface. Th e immobilized molecules were detected by AFM. The straightened DNA displaye d a sharp bend at the site of bound protein with the two DNA segments linea rly extending from the protein-binding site. Using GAL4, a yeast transcript ion factor, we demonstrate good agreement of the position of the observed b inding site on straightened DNA templates to the predicted binding site. Th e technique is expected to have significant implications in elucidating DNA and protein interactions in general, and specifically, for the measurement of promoter occupancy with unlabeled regulatory proteins at the single-mol ecule level. (C) 1998 Academic Press.