Determination of the number of proteins bound non-specifically to DNA

• Type of Material: Article
• Authors: Paul Liebesny, Emory University; Sachin Goyal, Emory University; David Dunlap, Emory University; Fereydoon Family, Emory University; Laura Finzi, Emory University
• Language: English
• Date: 2010-10-20
• Publisher: Institute of Physics: Hybrid Open Access
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2010 IOP Publishing Ltd
• Final Published Version (URL): http://iopscience.iop.org/0953-8984/22/41/414104
• Title of Journal or Parent Work: Journal of Physics: Condensed Matter
• ISSN: 0953-8984
• Volume: 22
• Issue: 41
• Start Page: 414104
• End Page: 414104
• Grant/Funding Information: This work was supported by the Human Frontier Science Programme Organization (RGP0050/2002-C to LF); Emory University Research Council-2006 and NIH R01, RGM084070A to LF. FF would like to thank the Emory College of Arts and Sciences for a Seed Grant.
• Research Categories: Biology, Cell; Biology, Physiology

Abstract

We have determined the change in the number of proteins bound non-specifically to DNA as a function of applied force using force–extension measurements on tethered DNA. Using magnetic tweezers, single molecules of λ DNA were repeatedly stretched and relaxed in the absence and presence of 170 nM λ repressor protein (CI). CI binds to six specific sites of λ DNA with nanomolar affinity and also binds non-specifically with micromolar affinity. The force versus extension data were analyzed using a recently developed theoretical framework for quantitative determination of protein binding to the DNA. The results indicate that the non-specific binding of CI changes the force–extension relation significantly in comparison to that of naked DNA. The DNA tether used in our experiment would have about 640 bound repressors, if it was completely saturated with bound proteins. We find that as the pulling force on DNA is reduced from 4.81 to 0.13 pN, approximately 138 proteins bind to DNA, which is about 22% of the length of the tethered DNA. Our results show that 0.13 pN is not low enough to cause saturation of DNA by repressor and 4.81 pN is also not high enough to eliminate all the repressors bound to DNA. This demonstrates that the force–extension relation provides an effective approach for estimating the number of proteins bound non-specifically to a DNA molecule.