F plasmid conjugative DNA transfer: the TraI helicase activity is essential for DNA strand transfer.
The product of the Escherichia coli F plasmid traI gene is required for DNA transfer via bacterial conjugation. This bifunctional protein catalyzes the unwinding of duplex DNA and is a sequence-specific DNA transesterase. The latter activity provides the site- and strand-specific nick required to initiate DNA transfer. To address the role of the TraI helicase activity in conjugative DNA transfer traI mutants were constructed and their function in DNA transfer was evaluated using genetic and biochemical methods. A traI deletion/insertion mutant was transfer-defective as expected. A traI C-terminal deletion that removed the helicase-associated motifs was also transfer-defective despite the fact that the region of traI encoding the transesterase activity was intact. Biochemical studies demonstrated that the N-terminal 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 activity was characterized. This protein catalyzed oriT-dependent transesterase activity in vitro and in vivo but failed to complement a traI deletion strain in conjugative DNA transfer assays. Thus, both the transesterase and helicase activities of TraI are essential for DNA strand transfer.
Matson
S W
SW
Department of Biology and the Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill 27599, USA. smatson@bio.uns.edu
Sampson
J K
JK
Byrd
D R
DR
D003227:Conjugation, Genetic; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D004950:Esterases / Q000378:metabolism; D005144:F Factor / Q000235:genetics*; D008957:Models, Genetic; D009154:Mutation; D018741:Replication Origin
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D004950:Esterases; C027093:TraI protein, E coli; D004265:DNA Helicases