F plasmid conjugative DNA transfer - The TraI helicase activity is essential for DNA strand transfer

The product of the Escherichia coil F plasmid traI gene is required for DNA transfer via bacterial conjugation. This bifunctional protein catalyzes th e unwinding of duplex DNA and is a sequence-specific DNA transesterase. The latter activity provides the site- and strand-specific nick required to in itiate DNA transfer. To address the role of the TraI helicase activity in c onjugative DNA transfer traI mutants were constructed and their function in DNA transfer was evaluated using genetic and biochemical methods. A traI d eletion/insertion mutant was transfer-defective as expected. A traI C-termi nal deletion that removed the helicase-associated motifs was also transfer- defective despite the fact that the region of traI encoding the transestera se activity was intact. Biochemical studies demonstrated that the N-termina l domain was sufficient to catalyze oriT-dependent transesterase activity. Thus, a functional transesterase was not sufficient to support DNA transfer . Finally, a point mutant, TraI-K998M, that lacked detectable helicase acti vity was characterized. This protein catalyzed oriT-dependent transesterase activity in vitro and in vivo but failed to complement a traI deletion str ain in conjugative DNA transfer assays. Thus, both the transesterase and he licase activities of TraI are essential for DNA strand transfer.