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Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA

By John S. Graham, Reid C. Johnson and John F. Marko

Abstract

The multistep kinetics through which DNA-binding proteins bind their targets are heavily studied, but relatively little attention has been paid to proteins leaving the double helix. Using single-DNA stretching and fluorescence detection, we find that sequence-neutral DNA-binding proteins Fis, HU and NHP6A readily exchange with themselves and with each other. In experiments focused on the Escherichia coli nucleoid-associated protein Fis, only a small fraction of protein bound to DNA spontaneously dissociates into protein-free solution. However, if Fis is present in solution, we find that a concentration-dependent exchange reaction occurs which turns over the bound protein, with a rate of kexch = 6 × 104 M−1s−1. The bacterial DNA-binding protein HU and the yeast HMGB protein NHP6A display the same phenomenon of protein in solution accelerating dissociation of previously bound labeled proteins as exchange occurs. Thus, solvated proteins can play a key role in facilitating removal and renewal of proteins bound to the double helix, an effect that likely plays a major role in promoting the turnover of proteins bound to DNA in vivo and, therefore, in controlling the dynamics of gene regulation

Topics: Molecular Biology
Publisher: Oxford University Press
OAI identifier: oai:pubmedcentral.nih.gov:3064784
Provided by: PubMed Central

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