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J. Biol. Chem., Vol. 278, Issue 49, 48696-48703, December 5, 2003

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Extent of Single-stranded DNA Required for Efficient TraI Helicase Activity in Vitro^*

Vanessa C. Csitkovits and Ellen L. Zechner

From the Institut für Molekularbiologie, Biochemie und Mikrobiologie, Karl-Franzens Universität Graz, Universitätsplatz 2, 8010 Graz, Austria

The IncF plasmid protein TraI functions during bacterial conjugation as a site- and strand-specific DNA transesterase and a highly processive 5' to 3' DNA helicase. The N-terminal DNA transesterase domain of TraI localizes the protein to nic and cleaves this site within the plasmid transfer origin. In the cell the C-terminal DNA helicase domain of TraI is essential for driving the 5' to 3' unwinding of plasmid DNA from nic to provide the strand destined for transfer. In vitro, however, purified TraI protein cannot enter and unwind nicked plasmid DNA and instead requires a 5' tail of single-stranded DNA at the duplex junction. In this study we evaluate the extent of single-stranded DNA adjacent to the duplex that is required for efficient TraI-catalyzed DNA unwinding in vitro. A series of linear partial duplex DNA substrates containing a central stretch of single-stranded DNA of defined length was created and its structure verified. We found that substrates containing 27 nucleotides of single-stranded DNA 5' to the duplex were unwound efficiently by TraI, whereas substrates containing 20 or fewer nucleotides were not. These results imply that during conjugation localized unwinding of >20 nucleotides at nic is necessary to initiate unwinding of plasmid DNA strands.

Received for publication, September 9, 2003

^* This work was financed through a direct grant from the Austrian Bundesministerium für Bildung, Wissenschaft und Kultur, Fonds zur Förderung der Wissenschaftlichen Forschung Projects 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.

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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