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Nucleic Acids Research 2005 33(16):5145-5155; doi:10.1093/nar/gki808
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Published online 9 September 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}oxfordjournals.org

Molecular Biology

Trapping DNA–protein binding reactions with neutral osmolytes for the analysis by gel mobility shift and self-cleavage assays

Nina Y. Sidorova*, Shakir Muradymov and Donald C. Rau

Laboratory of Physical and Structural Biology, NICHD, National Institutes of Health Bethesda, MD 20892, USA

*To whom correspondence should be addressed at NIH/NICHD, Bldg 9, Rm 1E-108, Bethesda, MD 20892; Tel: +1 301 402 4698; Fax: +1 301 402 9462; Email: sidorova{at}mail.nih.gov

Received July 14, 2005. Accepted August 15, 2005.

We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA–protein complex dissociation to develop a method that uses osmotic stress to ‘freeze’ mixtures of DNA–protein complexes and prevent further reaction enabling analysis of the products. We apply this approach to the gel mobility shift assay and use it to modify a self-cleavage assay that uses the nuclease activity of the restriction endonucleases to measure sensitively their specific binding to DNA. At sufficiently high concentrations of neutral osmolytes the cleavage reaction can be triggered at only those DNA fragments with initially bound enzyme. The self-cleavage assay allows measurement of binding equilibrium and kinetics directly in solution avoiding the intrinsic problems of gel mobility shift and filter binding assays while providing the same sensitivity level. Here we compare the self-cleavage and gel mobility shift assays applied to the DNA binding of EcoRI and BamHI restriction endonucleases. Initial results indicate that BamHI dissociation from its specific DNA sequence is strongly linked to water activity with the half-life time of the specific complex increasing ~20-fold from 0 to 1 osmolal betaine.


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