Probing Protein-DNA Interactions by Unzipping a Single DNA Double Helix

Abstract

We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes (BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of approximately 25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant (K(A)) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K(A) for EcoRI binding. The measured values are in good agreement with previous measurements of K(A) over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.

DNA Looping Mediates Nucleosome Transfer

“…DNA tethers were formed in flow chambers and were unzipped using an optical trap with a loading rate clamp of 10 pN s À 1 ( Fig. 1 and Supplementary Figs 3 and 5) through the bound protein [10][11][12]15,16,[28][29][30][31]55,[57][58][59][60] . Briefly, chambers were first incubated with antidigoxygenin at 0.2 mg ml À 1 for 5 min, and then the surface was blocked by incubation with casein at 5 mg ml À 1 for 5 min.…”

“…The number of DNA base pairs unzipped as at each time point was calculated from the raw force and extension measurements 59,61,62 . We then applied an algorithm that defined peaks as a force rise Z20 pN, which increased at a rate 43 pN s À 1 .…”

Characterizing folding, structure, molecular interactions and ligand gated activation of single sodium/proton antiporters

“…Dynamic SMFS may be applied to explore the energy landscape of ligand binding and membrane protein activation. Similarly as SMFS experiments revealed the binding stochiometry and equilibrium binding constant of DNA-protein interactions (Koch et al 2002), we here assume that future developments may allow to spatially and temporally observe ligandbinding to native membrane proteins. SMFS may be also applied to directly probe and quantify interactions between a potent drug and a targeted protein (Edwardson and Henderson 2004).…”

A high throughput molecular force assay for protein–DNA interactions

“…In Fig. 5 two MFA titration curves for 12 The K D value obtained from EMSA is slightly higher than both MFA measurements, which might be due to different conditions caused by the gel in the EMSA.…”

“…12,38 In shear geometry, however, all bases in the DNA duplex are loaded simultaneously and the structure of the DNA duplex might change prior to rupture, e.g. by unwinding, which might detach the bound protein with a certain probability from the DNA before the duplex itself ruptures.…”