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Nucleic Acids Research Advance Access published online on April 4, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm186
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Base-flipping dynamics in a DNA hairpin processing reaction

Julien Bischerour and Ronald Chalmers*

University of Oxford, Department of Biochemistry, South Parks Road, Oxford, OX1 3QU, UK

*To whom correspondence should be addressed. Tel: 44 1865 275307; Fax: 44 1865 275297

Received February 13, 2007. Revised March 14, 2007. Accepted March 15, 2007.

Many enzymes that repair or modify bases in double-stranded DNA gain access to their substrates by base flipping. Although crystal structures provide stunning snap shots, biochemical approaches addressing the dynamics have proven difficult, particularly in complicated multi-step reactions. Here, we use protein–DNA crosslinking and potassium permanganate reactivity to explore the base-flipping step in Tn5 transposition. We present a model to suggest that base flipping is driven by a combination of factors including DNA bending and the intrusion of a probe residue. The forces are postulated to act early in the reaction to create a state of tension, relieved by base flipping after cleavage of the first strand of DNA at the transposon end. Elimination of the probe residue retards the kinetics of nicking and reduces base flipping by 50%. Unexpectedly, the probe residue is even more important during the hairpin resolution step. Overall, base flipping is pivotal to the hairpin processing reaction because it performs two opposite but closely related functions. On one hand it disrupts the double helix, providing the necessary strand separation and steric freedom. While on the other, transposase appears to position the second DNA strand in the active site for cleavage using the flipped base as a handle.


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