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Nucleic Acids Research 2006 34(1):167-174; doi:10.1093/nar/gkj432
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Published online 10 January 2006

© The Author 2006. 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

Article

Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI

Bram van den Broek, Francesco Vanzi1, Davide Normanno1, Francesco S. Pavone1 and Gijs J.L. Wuite*

Faculty of Sciences, Department of Physics and Astronomy and Laser Centre De Boelelaan 1081 Vrije Universiteit Amsterdam, 1081 HV, The Netherlands 1European Laboratory for Non-linear Spectroscopy (LENS), Via Nello Carrara 1 50019 Sesto Fiorentino (Firenze), Italy

*To whom correspondence should be addressed. Tel: +31205987987; Fax: +31205987991; Email: gwuite{at}nat.vu.nl

Received October 3, 2005. Revised December 7, 2005. Accepted December 21, 2005.

Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein–DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates non-specific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca2+ increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific binding by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indicating that looping is dominated by (re)binding the second recognition site.


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