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J. Biol. Chem., Vol. 282, Issue 39, 29002-29012, September 28, 2007

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Analysis of P Element Transposase Protein-DNA Interactions during the Early Stages of Transposition^*

Mei Tang, Ciro Cecconi, Carlos Bustamante^¶||**, and Donald C. Rio¹

From the Department of Molecular and Cell Biology, Division of Genetics, Genomics and Development and Division of Biochemistry and Molecular Biology, Center for Integrative Genomics, University of California, Berkeley, California 94720, CNR-INFM-S3 University of Modena e Reggio Emilia, 41100 Modena, Italy, the ^¶Department of Physics, University of California, Berkeley, California 94720, and the ^||Howard Hughes Medical Institute, ^**Lawrence Berkeley National Laboratory, Berkeley, California 94720

P elements are a family of transposable elements found in Drosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end.

Received for publication, May 17, 2007 , and in revised form, June 29, 2007.

This paper is dedicated to our friend and colleague, Nick Cozzarelli.

^* This work was supported by National Institutes of Health Grant R01GM61987. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence should be addressed: E-mail: don_rio{at}berkeley.edu.