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Originally published In Press as doi:10.1074/jbc.M111119200 on January 22, 2002

J. Biol. Chem., Vol. 277, Issue 13, 11284-11291, March 29, 2002
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Mutational Analysis of the Base Flipping Event Found in Tn5 Transposition*

Brandon Ason and William S. ReznikoffDagger

From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

This work identifies novel structure-function relationships between Tn5 transposase (Tnp) and its DNA recognition sequence. The Tn5 Tnp-DNA co-crystal structure revealed the protein-DNA contacts of the post-cleavage complex (Davies, D. R., Goryshin, I. Y., Reznikoff, W. S., and Rayment, I. (2000) Science 289, 77-85). One of the most striking features of this complex is the rotation of thymine 2 (T2) away from the DNA helix and into a pocket within the Tnp. This interaction appears similar to the "base flipping" phenomenon found in many DNA repair enzymes such as T4 endonuclease V and uracil DNA glycosylase (Roberts, R. J., and Cheng, X. (1998) Annu. Rev. Biochem. 67, 181-198). To study the biochemical significance of this phenomenon, we mutated the Tnp residues proposed to be involved in stabilizing this interaction and removed the T2 nucleotide to examine which steps in the transposition reaction require T2-Tnp interactions. From this work, we have determined that stacking interactions between T2 and Tnp are critical for efficient transposition in vitro. In addition, our results suggested that T2-Tnp interactions facilitate hairpin formation and hairpin resolution primarily through base stacking and that T2 plays a role in the alignment of the transposon DNA for strand transfer.

* This work was supported by the National Institute of Health Grant GM50692.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Madison, WI 53706. Tel.: 608-262-3608; Fax: 608-262-3453; E-mail: reznikoff@biochem.wisc.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.