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Biophys J, August 2002, p. 1098-1105, Vol. 83, No. 2

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

Steven J. Koch, Alla Shundrovsky, Benjamin C. Jantzen, and Michelle D. Wang

Department of Physics, Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York 14853 USA

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 ~25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant (KA) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of KA for EcoRI binding. The measured values are in good agreement with previous measurements of KA over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.

Biophys J, August 2002, p. 1098-1105, Vol. 83, No. 2
© 2002 by the Biophysical Society   0006-3495/02/08/1098/08  $2.00


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