Abstract
The multistep kinetics through which DNA-binding proteins bind their targets is heavily studied, but relatively little attention has been paid to mechanisms of how proteins leave the double helix. We used a single-DNA stretching and fluorescence detection approach to study the kinetics of unbinding of the E. coli nucleoid-associated protein Fis, which is known to have site-specific regulatory and nonspecific chromosome-compacting functions. We find that a fraction of Fis bound to DNA spontaneously dissociates into protein-free solution leaving some Fis tightly bound to the double helix on cell-cycle-long timescales. However, if Fis is present in solution, we find that a concentration-dependent exchange reaction occurs which turns over all the bound protein, with a rate of kexch = 4x105 M−1sec−1. Thus, solvated proteins can play a key role in facilitating removal and renewal of proteins bound to the double helix. In addition, we compare the pattern of Fis binding along the DNA to the base-pair sequence and find a greater amount of Fis bound to GC-rich regions than to AT-rich regions.
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