PAUSING OF THE RESTRICTION-ENDONUCLEASE ECORI DURING LINEAR DIFFUSIONON DNA

Linear diffusion is a mechanism to accelerate association rates beyond their three-dimensional diffusional limit. It is employed by the rest riction endonuclease EcoRI as well as many other proteins interacting with specific DNA sequences to locate their target sites on the macrom olecular substrate. In order to investigate biochemical and biophysica l details of the linear diffusion process, we have developed a competi tive cleavage assay which allows us to assess with great accuracy the influence of sequence, sequence context, and other structural features on the linear diffusion of EcoRI on DNA. We show here that linear dif fusion is not a hopping but a sliding movement in which EcoRI follows the helical pitch of the DNA, because it does not ''overlook'' any cle avage site. Linear diffusion is slowed when EcoRI encounters sites on the DNA which resemble its recognition site (''star'' sites). Pauses o f up to 20 s are induced, depending on sequence and orientation of the star site. These data suggest that EcoRI can bind to DNA in two bindi ng modes: one tight, specific, and immobile, leading to DNA cleavage, and another one loose and nonspecific, allowing for linear diffusion. Depending on the similarity between the recognition sequence and the D NA sequence being encountered by EcoRI, there will be a continuous tra nsition between these binding modes. Other proteins bound to the DNA a nd irregular DNA structures such as bent DNA or a triple helix constit ute a barrier that cannot easily be passed by EcoRI.