TrwC is required for conjugal DNA transfer of the broad host range plasmid R388. The purified protein showsin vitroDNA helicase activity. Here we report that it also hasin vitro oriT-endonulcease activity. TrwC specifically nicksoriT-containing supercoiled plasmid DNA in the presence of Mg 2+ , 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 R388oriTsequence. Both the 5′ and the 3′ ends of the nick were mapped. The 5′ end was not accessible to phosphorylation by T4 polynucleotide kinase, suggesting a covalent association with TrwC. Analysis of a collection of deletions inoriTindicated that the nucleotide sequences immediately surrounding thenicsite are important, but not the only essential feature, for the nicking reaction. Comparison of the R388nicsite with previously publishednicDNA sequence suggests that IncF, IncN and INcW plasmids form a family of relatednicsites. During the course of this work we have also demonstrated a terminal transferase activity of Sequence TM Version 2.0 DNA polymerase, as yet undocumented, which could account for some discrepancies in previously mappednicsites in other systems.