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J. Biol. Chem., Vol. 279, Issue 24, 25359-25363, June 11, 2004

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Three-dimensional Reconstruction of Agrobacterium VirE2 Protein with Single-stranded DNA^*

Asmahan Abu-Arish, Daphna Frenkiel-Krispin, Tobin Fricke, Tzvi Tzfira, Vitaly Citovsky, Sharon Grayer Wolf¶, and Michael Elbaum||

From the Department of Materials and Interfaces and ^¶Electron Microscopy Unit, Weizmann Institute of Science, Rehovot 76100, Israel and Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794-5215

Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5'-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.

Received for publication, February 18, 2004 , and in revised form, March 29, 2004.

^* This work was supported in part by the Minerva Research Foundation, which is funded through the German Federal Ministry for Education and Research (BMBF), by the United States-Israel Binational Agricultural Research and Development Fund, by the Gerhardt M. J. Schmidt Center for Supramolecular Architecture, and by the Jean and Jula Goldwurm Foundation. 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.

^|| To whom correspondence should be addressed. Tel.: 972-8-9343537; Fax: 972-8-9344138; E-mail: michael.elbaum{at}weizmann.ac.il.

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