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J. Biol. Chem., Vol. 279, Issue 44, 45477-45484, October 29, 2004

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Concomitant Reconstitution of TraI-catalyzed DNA Transesterase and DNA Helicase Activity in Vitro^*

Vanessa C. Csitkovits, Damir Ðermi¶, and Ellen L. Zechner||

From the Institut für Molekulare Biowissenschaften, Karl-Franzens Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria and Department of Molecular Biology, Rudjer Bokovi Institute, 10002 Zagreb, Croatia

TraI protein of plasmid R1 possesses two activities, a DNA transesterase and a highly processive 5'-3' DNA helicase, which are essential for bacterial conjugation. Regulation of the functional domains of the enzyme is poorly understood. TraI cleaves supercoiled oriT DNA with site and strand specificity in vitro but fails to initiate unwinding from this site (nic). The helicase requires an extended region of adjacent single-stranded DNA to enter the duplex, yet interaction of purified TraI with oriT DNA alone or as an integral part of the IncF relaxosome does not melt sufficient duplex to load the helicase. This study aims to gain insights into the controlled initiation of both TraI-catalyzed activities. Linear double-stranded DNA substrates with a central region of sequence heterogeneity were used to trap defined lengths of R1 oriT sequence in unwound conformation. Concomitant reconstitution of TraI DNA transesterase and helicase activities was observed. Efficient helicase activity was measured on substrates containing 60 bases of open duplex but not on substrates containing 30 bases in open conformation. The additional presence of auxiliary DNA-binding proteins TraY and Escherichia coli integration host factor did not stimulate TraI activities on these substrates. This model system offers a novel approach to investigate factors controlling helicase loading and the directionality of DNA unwinding from nic.

Received for publication, July 14, 2004 , and in revised form, August 16, 2004.

^* This work was financed by the Austrian Bundesministerium für Bildung, Wissenschaft und Kultur, Fonds zur Förderung der Wissenschaftlichen Forschung Grants P13227GEN and P16722-B12, and European Union Grant QLK2-2000-01624. 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.

^¶ Supported by an Ernst Mach stipend (Österreichischer Austauschdienst).

^|| To whom correspondence should be addressed. Tel.: 43-316-3805624; Fax: 43-316-3809898; E-mail: ellen.zechner{at}uni-graz.at.

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