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|Author(s):||Ariel Michelman-Ribeiro; Davide Mazza; Tilman Rosales; Timothy J. Stasevich; Boukari Hacene; Vikas Rishi; Charles Vinson; Jay R. Knutson; James G. NcNally;|
|Title:||Direct Measurement of Association and Dissociation Rates of DNA Binding in Live Cells by Fluorescence Correlation Spectroscopy|
|Published:||July 01, 2009|
|Abstract:||Measurement of live-cell binding interactions is vital for understanding the biochemical reactions that drive cellular processes. Here we develop, characterize and apply a new procedure to extract information about binding to an immobile substrate from FCS autocorrelation data. We show that existing methods for analyzing such data by two- component diffusion fits can produce inaccurate estimates of diffusion constants and bound fractions, or even fail altogether to fit FCS binding data. By analyzing live-cell FCS measurements, we show that our new model can satisfactorily account for the binding interactions introduced by attaching a DNA binding domain to the dimerization domain of a site-specific transcription factor (VBP - the vitellogenin binding protein). Reassuringly, our FCS estimates are quantitatively consistent with our FRAP measurements on the same VBP peptides. However, due to the fast binding interactions introduced by the DNA binding domain, FCS generates independent estimates for the diffusion constant and the association and dissociation rates, while FRAP produces only a single estimate, the effective diffusion constant, which depends on all three parameters. We apply this new method to evaluate the efficacy of a potential anti-cancer drug that inhibits DNA binding of VBP in vitro, and find that in vivo the drug inhibits DNA binding in only a subset of cells.|
|Pages:||pp. 337 - 346|
|Keywords:||fluorescence correlation spectroscopy, FCS, FRAP, transcription, protein binding, binding model|
|Research Areas:||Biological Physics|
|PDF version:||Click here to retrieve PDF version of paper (434KB)|