Facilitated Dissociation of Protein from a Single DNA Binding Site

Protein-DNA interactions are central to chromosome compaction, defining the topology of chromatin, DNA repair, and gene regulation, and therefore control all aspects of cellular function. It is therefore important to understand the factors that regulate the dynamics of protein-DNA interactions since these factors will influence the dynamics of cellular processes. Previous single DNA studies involving the major nucleoid-associated protein Fis have shown that it stably binds 48.5 kb DNA in Fis-free buffer. However, protein in solution was found to accelerate the off-rate of Fis in a concentration dependent manner via an unknown mechanism. This effect has also recently been observed in a number of other cases suggesting its generality. It is unknown whether facilitated dissociation is an effect at the single binding site level or whether it involves protein clustering and/or cooperativity. Using single molecule fluorescence microscopy, we have measured the off-rate of Fis from 27bp dsDNAs, constituting individual Fis binding sites, and have observed facilitated exchange demonstrating an effect at the single binding site level. We have also found that the salt dependence of the off-rate is dramatically reduced when protein is in solution. This observation provides strong support for a simple microscopic theory that thermally excited, partial dissociation events lead to facilitated dissociation. In the absence of cooperativity, it is commonly assumed that finding the ratio of the off-rate to the on-rate constant will produce the same dissociation constant as finding the concentration of ligand for which half of the substrates are bound by ligand. However, if the off-rate is a function of protein concentration, this equivalence no longer holds. If facilitated exchange is found to be a general phenomenon, it will force a change in how we think about protein binding kinetics in biology.