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Nucleic Acids Research 2006 34(9):2820-2832; doi:10.1093/nar/gkl179
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Published online 22 May 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@oxfordjournals.org

Article

Defining characteristics of Tn5 Transposase non-specific DNA binding

Mindy Steiniger, Christian D. Adams, John F. Marko1 and William S. Reznikoff*

Department of Biochemistry, University of Wisconsin-Madison Madison, WI 53706, USA 1 Department of Physics, University of Illinois at Chicago Chicago, IL 60607, USA

*To whom correspondence should be addressed. Tel: +1 608 262 3608; Fax: +1 608 265 2603; Email: reznikoff{at}biochem.wisc.edu

Received December 6, 2005. Revised February 14, 2006. Accepted March 21, 2006.

While non-specific DNA plays a role in target localization for many recombinases, transcription factors and restriction enzymes, the importance of non-specific DNA interactions for transposases has not been investigated. Here, we discuss non-specific DNA-Tn5 Transposase (Tnp) interactions and suggest how they stabilize the Tnp and modulate Tnp localization of the 19 bp Tnp recognition end sequences (ESes). DNA protection assays indicate that full-length Tnp interacts efficiently with supercoiled DNA that does not contain ESes. These interactions significantly prolong the lifetime of Tnp, in vitro. The balance between non-specific DNA bound and free Tnp is affected by DNA topology, yet, intermolecular transfer of active Tnp occurs with both supercoiled and linear non-specific DNA. Experiments with substrates of varying lengths show that Tn5 Tnp can utilize non-specific DNA to facilitate localization of an intramolecular ES over distances less than 464 bp. Finally, synaptic complex formation is inhibited in the presence of increasing concentrations of supercoiled and linear pUC19. These experiments strongly suggest that Tn5 Tnp has a robust non-specific DNA binding activity, that non-specific DNA modulates ES sequence localization within the global DNA, most likely through a direct transfer mechanism, and that non-specific DNA binding may play a role in the cis bias manifested by Tn5 transposition.


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