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J. Biol. Chem., Vol. 282, Issue 35, 25569-25576, August 31, 2007

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The ATPase Activity of the DNA Transporter TrwB Is Modulated by Protein TrwA

IMPLICATIONS FOR A COMMON ASSEMBLY MECHANISM OF DNA TRANSLOCATING MOTORS^*

From the Departamento de Biología Molecular, Universidad de Cantabria (UC) e Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC (CSIC-UC-IDICAN), 39011 Santander, Spain

Conjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F₁-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwBN70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwBN70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwBN70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly.

Received for publication, April 25, 2007 , and in revised form, June 20, 2007.

^* This work was supported in part by Grant BFU2005-02718 (Spanish Ministry of Education) (to E. C.) and Grants BFU2005-03477/BMC (Spanish Ministry of Education) and LSHM-CT-2005_019023 (European VI Framework Program) (to F. C.). 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 Supported by a fellowship from the Basque Government.

² To whom correspondence may be addressed. E-mail: delacruz{at}unican.es. ³ To whom correspondence may be addressed. E-mail: cabezone{at}unican.es.

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