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J. Biol. Chem., Vol. 280, Issue 4, 2503-2511, January 28, 2005

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Identification of the Structural and Functional Domains of the Large Serine Recombinase TnpX from Clostridium perfringens^*

Isabelle S. Lucet¶, Fleur E. Tynan||, Vicki Adams¶, Jamie Rossjohn||**, Dena Lyras, and Julian I. Rood**

From the Department of Microbiology, Australian Bacterial Pathogenesis Program, Monash University, Victoria 3800, Australia, ^||Department of Biochemistry and Molecular Biology, Protein Crystallography Unit, Monash University, Victoria 3800, Australia, and ^**Australian Research Council Centre for Structural and Functional Microbial Genomics, Monash University, Victoria 3800, Australia

Members of the large serine resolvase family of site-specific recombinases are responsible for the movement of several mobile genetic elements; however, little is known regarding the structure or function of these proteins. TnpX is a serine recombinase that is responsible for the movement of the chloramphenicol resistance elements of the Tn4451/3 family. We have shown that TnpX binds differentially to its transposon and target sites, suggesting that resolvase-like excision and insertion were two distinct processes. To analyze the structural and functional domains of TnpX and, more specifically, to define the domains involved in protein-DNA and protein-protein interactions, we conducted limited proteolysis studies on the wild-type dimeric TnpX_1–707 protein and its functional truncation mutant, TnpX_1–597. The results showed that TnpX was organized into three major domains: domain I (amino acids (aa) 1–170), which included the resolvase catalytic domain; domain II (aa 170–266); and domain III (aa 267–707), which contained the dimerization region and two separate regions involved in binding to the DNA target. A small polypeptide (aa 533–587) was shown to bind specifically to the TnpX binding sites providing further evidence that it was the primary DNA binding region. In addition, a previously unidentified DNA binding site was shown to be located between residues 583 and 707. Finally, the DNA binding and multerimization but not the catalytic functions of TnpX could be reconstituted by recombining separate polypeptides that contain the N- and C-terminal regions of the protein. These data provide evidence that TnpX has separate catalytic, DNA binding, and multimerization domains.

Received for publication, August 24, 2004 , and in revised form, November 12, 2004.

^* This research was supported by grants from the Australian National Health and Medical Research Council and the Australian Research Council. 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.

Present address: Dept. of Biochemistry and Molecular Biology, Protein Crystallography Unit, Monash University, Victoria 3800, Australia.

^¶ Recipients of a Monash University Faculty of Medicine, Nursing, and Health Sciences Postdoctoral Fellowship and Postgraduate Scholarship, respectively.

To whom correspondence should be addressed: Dept. of Microbiology, Monash University, Victoria 3800, Australia. Tel.: 61-3-9905-4821; Fax: 61-3-9905-4811; E-mail: julian.rood{at}med.monash.edu.au.

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