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Nucleic Acids Research 2005 33(13):4322-4334; doi:10.1093/nar/gki708
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Published online 1 August 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Article

Determination of protein–DNA binding constants and specificities from statistical analyses of single molecules: MutS–DNA interactions

Yong Yang1, Lauryn E. Sass1, Chunwei Du3, Peggy Hsieh3 and Dorothy A. Erie1,2,*

1Department of Chemistry, University of North Carolina at Chapel Hill Chapel Hill, NC 27599-3290, USA 2Curriculum in Applied and Materials Sciences, University of North Carolina at Chapel Hill Chapel Hill, NC 27599-3290, USA 3Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Bethesda, MD 20892, USA

*To whom correspondence should be addressed. Tel: +1 919 962 6370; Fax: +1 919 966 3675; Email: derie{at}unc.edu

Received May 4, 2005. Revised June 13, 2005. Accepted June 29, 2005.

Atomic force microscopy (AFM) is a powerful technique for examining the conformations of protein–DNA complexes and determining the stoichiometries and affinities of protein–protein complexes. We extend the capabilities of AFM to the determination of protein–DNA binding constants and specificities. The distribution of positions of the protein on the DNA fragments provides a direct measure of specificity and requires no knowledge of the absolute binding constants. The fractional occupancies of the protein at a given position in conjunction with the protein and DNA concentrations permit the determination of the absolute binding constants. We present the theoretical basis for this analysis and demonstrate its utility by characterizing the interaction of MutS with DNA fragments containing either no mismatch or a single mismatch. We show that MutS has significantly higher specificities for mismatches than was previously suggested from bulk studies and that the apparent low specificities are the result of high affinity binding to DNA ends. These results resolve the puzzle of the apparent low binding specificity of MutS with the expected high repair specificities. In conclusion, from a single set of AFM experiments, it is possible to determine the binding affinity, specificity and stoichiometry, as well as the conformational properties of the protein–DNA complexes.


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