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J. Biol. Chem., Vol. 279, Issue 39, 41058-41066, September 24, 2004

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Binding Modes of the Initiator and Inhibitor Forms of the Replication Protein to the ori Iteron of Plasmid R6K^*

Selvi Kunnimalaiyaan, Ricardo Krüger, Wilma Ross, Sheryl A. Rakowski, and Marcin Filutowicz¶

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

Discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of DNA replication initiation. In the origin of plasmid R6K, the Rep protein, , is distinctive in that it can bind the seven 22-bp iterons in two forms; monomers activate replication, whereas dimers act as inhibitors. In this work, we used wild type and variants of the protein with altered monomer/dimer ratios to study iteron/ interactions. High resolution contact mapping was conducted using multiple techniques (missing base contact probing, methylation protection, base modification, and hydroxyl radical footprinting), and the electrophoretic separation of nucleoprotein complexes allowed us to discriminate between contact patterns produced by monomers and dimers. We also isolated iteron mutants that affected the binding of monomers (only) or both monomers and dimers. The mutational studies and footprinting analyses revealed that, when binding DNA, monomers interact with nucleotides spanning the entire length of the iteron. In contrast, dimers interact with only the left half of the iteron; however, the retained interactions are strikingly similar to those seen with monomers. These results support a model in which Rep protein dimerization disturbs one of two DNA binding domains important for monomer/iteron interaction; the dimer/iteron interaction utilizes only one DNA binding domain.

Received for publication, March 22, 2004 , and in revised form, June 28, 2004.

^* This work was supported by funding from the Women in Science and Engineering Leadership Institute and National Institutes of Health (NIH) Grant GM40314 (to M. F.). 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 four additional figures.

Supported by CAPES/Brasilia/Brazil. Present address: Universidade Católica de Brasília, Campus II, SGAN 916, Módulo B, W5 Norte, Brasília, Brazil.

Supported by NIH Grant GM37048 (to Richard L. Gourse).

^¶ To whom correspondence should be addressed: Dept. of Bacteriology, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706. Tel.: 608-262-6947; Fax: 608-262-9865; E-mail: msfiluto{at}facstaff.wisc.edu.

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				S. Kunnimalaiyaan, S. A. Rakowski, and M. Filutowicz Structure-Based Functional Analysis of the Replication Protein of Plasmid R6K: Key Amino Acids at the {pi}/DNA Interface J. Bacteriol., July 1, 2007; 189(13): 4953 - 4956. [Abstract] [Full Text] [PDF]

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