Site- and strand-specific nicking at oriT of plasmid R100 in a purified system: Enhancement of the nicking activity of tral (helicase I) with traY and IHF

We developed a purified system for reproducing the nicking reaction at the site 59 base pairs upstream of the TraY protein binding site, sbyA, in the oriT region of plasmid R100. Nicking at oriT occurred efficiently in the presence of the plasmid-encoded proteins, Tral and TraY, integration host factor (IHF), and Mg2+, but inefficiently in the presence of the Tral protein and Mg2+. The products were complex DNA molecules with a protein covalent-ly linked with the 5' end of the nick in the strand, which is supposed to be transferred during conjugation. The same complex DNA molecules were formed in the presence of the Tral protein alone, indicating that the protein attached at the 5' end of the nick is the Tral protein. Stimulation of the nicking reaction by the TraY protein and by IHF, whose binding site has been mapped between the nicking site and sbyA, indicates that DNA bending is important in the formation of the complex including the Tral and TraY proteins at oriT.