NICKING ACTIVITY OF TRWC DIRECTED AGAINST THE ORIGIN OF TRANSFER OF THE INCW PLASMID R388

TrwC is required for conjugal DNA transfer of the broad host range pla smid R388. The purified protein shows in vitro DNA helicase activity H ere we report that it also has in vitro oriT-endonuclease activity: Tr wC specifically nicks oriT-containing supercoiled plasmid DNA in the p resence of Mg2+, and the nicked DNA can be visualized after treatment with SDS. Sequencing of the nicked DNA showed a specific interruption of the lower DNA strand on the R388 oriT sequence. Both the 5' and the 3' ends of the nick were mapped. The 5' end was not accesible to phos phorylation by T4 polynucleotyde kinase, suggesting a covalent associa tion with TrwC. Analysis of a collection of deletions in oriT indicate d that the nucleotide sequences immediately surrounding the nic site a re important, but not the only essential feature, for the nicking reac tion. Comparison of the R388 nic site with previously published nic DN A sequences suggests that IncF, IncN and IncW plasmids form a family o f related nic sites. During the course of this work we have also demon strated a terminal transferase activity of Sequenase(TM) Version 2.0 D NA polymerase, as yet undocumented, which could account for some discr epancies in previously mapped nic sites in other systems.