On-Chip Analysis of Intermittent Molecular Encounters in Nuclease Digestion of Specific DNA Sequence

Abstract

DNA-binding proteins such as transcription factors react with their targets at faster than estimation from diffusion-controlled rates. This is explained by the fact that the protein can slide along DNA molecule with appreciable affinity for nonspecific sequences. However, does this sliding function really play a key role in this type of reaction kinetics? Most of the previous methods are optimized for one-dimensional protein diffusion along DNA maintained in an extended configuration. Here we show that a site-specific nuclease digestion efficiently occurs in the course of an intermittent encounter by the same single DNA molecule to a number of nucleolytic enzyme molecules. We found in a limited protein's sliding-free condition that the intermittent molecular encounter and complex formation process gives an enhanced rate for the reaction. Our results demonstrate how the flowing DNA encounters the immobilized restriction enzyme ApaI by which the site-specific DNA-break occurs. Moving tags of both DNA ends transmitted the breakup of single DNA molecule into fragments. Quite a number of digestions occurred at the first encounter (62%), but the remaining 38% of digestion occurred in the course of the subsequent intermittent encounters. The complex formation was maintained after the DNA break. This is suggestive of an intrinsic behaviour in which the DNA and protein molecules are continuously held together by switching their binding positions with short-range interactions. It also provides a clue to understanding an efficient and effective reaction by the DNA-binding proteins in bacterial cells.