Dynamic Force Spectroscopy of Protein-DNA Interactions by Unzipping DNA

Steven J. Koch and Michelle D. Wang

Phys. Rev. Lett. 91, 028103 – Published 10 July 2003; Erratum Phys. Rev. Lett. 91, 049903 (2003)

Abstract

We demonstrate the first site-specific single-molecule characterization of the prominent activation barrier for the disruption of a protein-DNA binding complex. We achieved this new capability by combining dynamic force spectroscopy with unzipping force analysis of protein association and used the combination to investigate restriction enzyme binding to specific DNA sites. Analysis revealed lifetimes and interaction distances for three protein-DNA interactions. This new method is able to distinguish protein-DNA binding complexes on a site-specific, single-molecule basis.

Received 18 December 2002
Publisher error corrected 17 July 2003

DOI:https://doi.org/10.1103/PhysRevLett.91.028103

Corrections

17 July 2003

Erratum

Publisher’s Note: Dynamic Force Spectroscopy of Protein-DNA Interactions by Unzipping DNA [Phys. Rev. Lett.PRLTAO0031-9007 91, 028103 (2001)]

Steven J. Koch and Michelle D. Wang

Phys. Rev. Lett. 91, 049903 (2003)

Authors & Affiliations

Steven J. Koch and Michelle D. Wang^*

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

^*Corresponding author. Email address: mwang@physics.cornell.edu

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Vol. 91, Iss. 2 — 11 July 2003

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Images

Figure 1
Schematic of the unzipping configuration (not to scale). Applied tension unzips the two strands of the DNA molecule. The location of the unzipping fork is indicated by an unzipping index $j$ . The presence of a DNA-binding protein at the unzipping fork is observed as an increase in the force required to separate the strands.Reuse & Permissions
Figure 2
Example of unzipping data taken using loading rate clamp ( $59 p N / s$ ). (a) Force versus time. The graph demonstrates the uniform force loading rate for forces greater than 15 pN. One of the eight events from the automated event detection is highlighted. The dotted line shows the loading rate fit for the event. (b) Calculated unzipping index, $j$ , vs time. Each horizontal step represents data where a restriction enzyme pins the unzipping index at a certain value until the complex disrupts. The same event as in (a) is highlighted. Horizontal dashed lines mark unzipping indices which are predicted $B s o B I$ binding sites, of types $α$ (ttcCTCGGGaat) and $β$ (aaaCTCGAGact).Reuse & Permissions
Figure 3
Dynamic force spectroscopy for $B s o B I$ unbinding from $α$ sites ( $N = 449$ events total). Each histogram shows unbinding force distribution with bins of 2.5 pN width at a given force loading rate. Dashed curves represent predicted force probability density functions resulting from the determination of local values of $d$ and $t_{o f f}$ (see text). Solid curves represent predicted force probability density functions resulting from the determination of global values of $d$ and $t_{o f f}$ (also see text). Vertical dashed lines designate the experimentally accessible force range of the current implementation of UFAPA.Reuse & Permissions
Figure 4
Dynamic force spectroscopy for three binding species. Data points represent the most-probable unbinding force for a given force-loading rate, obtained from the maximum likelihood method described in the text. Error bars were determined using a Monte Carlo method [12]. Open circles represent $B s o B I$ unbinding from $α$ sites, open squares represent $B s o B I$ unbinding from $β$ sites, and filled squares represent $X h o I$ unbinding from $β$ sites. Solid lines are linear fits of Eq. (2) to the data. Parameters $d$ and $t_{o f f}$ obtained from the fits are listed in Table .Reuse & Permissions
Figure 5
Distinct unbinding force distributions. Solid gray bars represent $B s o B I$ unbinding from $α$ sites ( $N = 141$ ), while lined bars represent $B s o B I$ unbinding from $β$ sites ( $N = 35$ ). Solid lines are predicted distributions based on the global fit parameters from Table . All data are for a force loading rate of $\sim 60 p N / s$ . At this particular stretch rate, the two sites produce highly distinct unbinding signatures, as shown by both the data and the predicted PDF.Reuse & Permissions

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