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J. Biol. Chem., Vol. 282, Issue 46, 33707-33713, November 16, 2007

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Roles of Active Site Residues and the HUH Motif of the F Plasmid TraI Relaxase^*

Christopher Larkin¹, Rembrandt J. F. Haft², Matthew J. Harley³, Beth Traxler, and Joel F. Schildbach⁴

From the Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 and the Department of Microbiology, University of Washington, Seattle, Washington 98195

Bacterial conjugation, transfer of a single strand of a conjugative plasmid between bacteria, requires sequence-specific single-stranded DNA endonucleases called relaxases or nickases. Relaxases contain an HUH (His-hydrophobe-His) motif, part of a three-His cluster that binds a divalent cation required for the cleavage reaction. Crystal structures of the F plasmid TraI relaxase domain, with and without bound single-stranded DNA, revealed an extensive network of interactions involving HUH and other residues. Here we study the roles of these residues in TraI function. Whereas substitutions for the three His residues alter metal-binding properties of the protein, the same substitution at each position elicits different effects, indicating that the residues contribute asymmetrically to metal binding. Substitutions for a conserved Asp that interacts with one HUH His demonstrate that the Asp modulates metal affinity despite its distance from the metal. The bound metal enhances binding of ssDNA to the protein, consistent with a role for the metal in positioning the scissile phosphate for cleavage. Most substitutions tested caused significantly reduced in vitro cleavage activities and in vivo transfer efficiencies. In summary, the results suggest that the metal-binding His cluster in TraI is a finely tuned structure that achieves a sufficient affinity for metal while avoiding the unfavorable electrostatics that would result from placing an acidic residue near the scissile phosphate of the bound ssDNA.

Received for publication, April 14, 2007 , and in revised form, September 13, 2007.

^* This work was supported in part by National Institutes of Health Grant GM61017 (to J. F. S.) and National Science Foundation Grant MCB-0345018 (to B. T.). 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.

¹ Present address: Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892.

² Supported in part by National Institutes of Health NIGMS Grant NRSA T32 GM07270.

³ Present address: The Harker School, 500 Saratoga Ave., San Jose, CA 95129.

⁴ To whom correspondence should be addressed: Mudd Hall 235, 3400 N. Charles St., Baltimore, MD 21218. Tel.: 410-516-0176; Fax: 410-516-5213; E-mail: joel{at}jhu.edu.

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				K. Hekman, K. Guja, C. Larkin, and J. F. Schildbach An intrastrand three-DNA-base interaction is a key specificity determinant of F transfer initiation and of F TraI relaxase DNA recognition and cleavage Nucleic Acids Res., August 1, 2008; 36(14): 4565 - 4572. [Abstract] [Full Text] [PDF]